James Spudich

All Publications

• One must reconstitute the functions of interest from purified proteins. Frontiers in physiology Spudich, J. A. 2024; 15: 1390186

  Abstract

  I am often asked by students and younger colleagues and now by the editors of this issue to tell the history of the development of the in vitro motility assay and the dual-beam single-molecule laser trap assay for myosin-driven actin filament movement, used widely as key assays for understanding how both muscle and nonmuscle myosin molecular motors work. As for all discoveries, the history of the development of the myosin assays involves many people who are not authors of the final publications, but without whom the assays would not have been developed as they are. Also, early experiences shape how one develops ideas and experiments, and influence future discoveries in major ways. I am pleased here to trace my own path and acknowledge the many individuals involved and my early science experiences that led to the work I and my students, postdoctoral fellows, and sabbatical visitors did to develop these assays. Mentors are too often overlooked in historical descriptions of discoveries, and my story starts with those who mentored me.

  View details for DOI 10.3389/fphys.2024.1390186

  View details for PubMedID 38827995

  View details for PubMedCentralID PMC11140241

• Mavacamten, a precision medicine for hypertrophic cardiomyopathy: From a motor protein to patients. Science advances Nag, S., Gollapudi, S. K., Del Rio, C. L., Spudich, J. A., McDowell, R. 2023; 9 (30): eabo7622

  Abstract

  Hypertrophic cardiomyopathy (HCM) is a primary myocardial disorder characterized by left ventricular hypertrophy, hyperdynamic contraction, and impaired relaxation of the heart. These functional derangements arise directly from altered sarcomeric function due to either mutations in genes encoding sarcomere proteins, or other defects such as abnormal energetics. Current treatment options do not directly address this causal biology but focus on surgical and extra-sarcomeric (sarcolemmal) pharmacological symptomatic relief. Mavacamten (formerly known as MYK-461), is a small molecule designed to regulate cardiac function at the sarcomere level by selectively but reversibly inhibiting the enzymatic activity of myosin, the fundamental motor of the sarcomere. This review summarizes the mechanism and translational progress of mavacamten from proteins to patients, describing how the mechanism of action and pharmacological characteristics, involving both systolic and diastolic effects, can directly target pathophysiological derangements within the cardiac sarcomere to improve cardiac structure and function in HCM. Mavacamten was approved by the Food and Drug Administration in April 2022 for the treatment of obstructive HCM and now goes by the commercial name of Camzyos. Full information about the risks, limitations, and side effects can be found at www.accessdata.fda.gov/drugsatfda_docs/label/2022/214998s000lbl.pdf.

  View details for DOI 10.1126/sciadv.abo7622

  View details for PubMedID 37506209

• Cryo-EM structure of the folded-back state of human beta-cardiac myosin Grinzato, A., Auguin, D., Kikuti, C., Nandwani, N., Moussaoui, D., Pathak, D., Kandiah, E., Ruppel, K., Spudich, J. A., Houdusse, A. M., Robert-Paganin, J. CELL PRESS. 2023: 258A-259A

  View details for Web of Science ID 000989629701377

• Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function. eLife Morck, M. M., Bhowmik, D., Pathak, D., Dawood, A., Spudich, J., Ruppel, K. M. 2022; 11

  Abstract

  Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition. The pliant region mutations had varying effects on the motor function of a myosin construct lacking the S2 tail: overall, D778V increased power output, L781P reduced power output, and S782N had little effect on power output, while all three reduced the external force sensitivity of the actin detachment rate. With a myosin containing the motor domain and the proximal S2 tail, the pliant region mutations also attenuated autoinhibition in the presence of filamentous actin but had no impact in the absence of actin. By contrast, the light chain-binding region mutations had little effect on motor activity but produced marked reductions in autoinhibition in both the presence and absence of actin. Thus, mutations in the lever arm of β-cardiac myosin have divergent allosteric effects on myosin function, depending on whether they are in the pliant or light chain-binding regions.

  View details for DOI 10.7554/eLife.76805

  View details for PubMedID 35767336

• Hypertrophic cardiomyopathy: Mutations to mechanisms to therapies. Frontiers in physiology Kawana, M., Spudich, J. A., Ruppel, K. M. 2022; 13: 975076

  Abstract

  Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 people in the general population with an extensive burden of morbidity in the form of arrhythmia, heart failure, and sudden death. More than 25years since the discovery of the genetic underpinnings of HCM, the field has unveiled significant insights into the primary effects of these genetic mutations, especially for the myosin heavy chain gene, which is one of the most commonly mutated genes. Our group has studied the molecular effects of HCM mutations on human beta-cardiac myosin heavy chain using state-of-the-art biochemical and biophysical tools for the past 10years, combining insights from clinical genetics and structural analyses of cardiac myosin. The overarching hypothesis is that HCM-causing mutations in sarcomere proteins cause hypercontractility at the sarcomere level, and we have shown that an increase in the number of myosin molecules available for interaction with actin is a primary driver. Recently, two pharmaceutical companies have developed small molecule inhibitors of human cardiac myosin to counteract the molecular consequences of HCM pathogenesis. One of these inhibitors (mavacamten) has recently been approved by the FDA after completing a successful phase III trial in HCM patients, and the other (aficamten) is currently being evaluated in a phase III trial. Myosin inhibitors will be the first class of medication used to treat HCM that has both robust clinical trial evidence of efficacy and that targets the fundamental mechanism of HCM pathogenesis. The success of myosin inhibitors in HCM opens the door to finding other new drugs that target the sarcomere directly, as we learn more about the genetics and fundamental mechanisms of this disease.

  View details for DOI 10.3389/fphys.2022.975076

  View details for PubMedID 36225299

• Hypertrophic cardiomyopathy beta-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state. Proceedings of the National Academy of Sciences of the United States of America Vander Roest, A. S., Liu, C., Morck, M. M., Kooiker, K. B., Jung, G., Song, D., Dawood, A., Jhingran, A., Pardon, G., Ranjbarvaziri, S., Fajardo, G., Zhao, M., Campbell, K. S., Pruitt, B. L., Spudich, J. A., Ruppel, K. M., Bernstein, D. 2021; 118 (24)

  Abstract

  Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in beta-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.

  View details for DOI 10.1073/pnas.2025030118

  View details for PubMedID 34117120

• Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness. Cell Gyimesi, M., Horvath, A. I., Turos, D., Suthar, S. K., Penzes, M., Kurdi, C., Canon, L., Kikuti, C., Ruppel, K. M., Trivedi, D. V., Spudich, J. A., Lorincz, I., Rauscher, A. A., Kovacs, M., Pal, E., Komoly, S., Houdusse, A., Malnasi-Csizmadia, A. 2020

  Abstract

  Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.

  View details for DOI 10.1016/j.cell.2020.08.050

  View details for PubMedID 33035452

• The hypertrophic cardiomyopathy mutations R403Q and R663H increase the number of myosin heads available to interact with actin SCIENCE ADVANCES Sarkar, S. S., Trivedi, D., Morck, M. M., Adhikari, A. S., Pasha, S. N., Ruppel, K. M., Spudich, J. A. 2020; 6 (14): eaax0069

  Abstract

  Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin and myosin binding protein-C (MyBP-C) lead to hypercontractility of the heart, an early hallmark of HCM. We show that hypercontractility caused by the HCM-causing mutation R663H cannot be explained by changes in fundamental myosin contractile parameters, much like the HCM-causing mutation R403Q. Using enzymatic assays with purified human β-cardiac myosin, we provide evidence that both mutations cause hypercontractility by increasing the number of functionally accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of MyBP-C. Furthermore, addition of C0-C7 decreases the wild-type myosin basal ATPase single turnover rate, while the mutants do not show a similar reduction. These data suggest that a primary mechanism of action for these mutations is to increase the number of myosin heads functionally available for interaction with actin, which could contribute to hypercontractility.

  View details for DOI 10.1126/sciadv.aax0069

  View details for Web of Science ID 000523302400002

  View details for PubMedID 32284968

  View details for PubMedCentralID PMC7124958

• The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches. Annual review of biochemistry Trivedi, D. V., Nag, S., Spudich, A., Ruppel, K. M., Spudich, J. A. 2020

  Abstract

  Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human beta-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human beta-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts. Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-biochem-011520-105234

  View details for PubMedID 32169021

• beta-Cardiac myosin hypertrophic cardiomyopathy mutations release sequestered heads and increase enzymatic activity. Nature communications Adhikari, A. S., Trivedi, D. V., Sarkar, S. S., Song, D., Kooiker, K. B., Bernstein, D., Spudich, J. A., Ruppel, K. M. 2019; 10 (1): 2685

  Abstract

  Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human beta-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human beta-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif (IHM) lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, which could lead to hypercontractility at the molecular level.

  View details for DOI 10.1038/s41467-019-10555-9

  View details for PubMedID 31213605

• Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY Spudich, J. A. 2019; 471 (5): 701–17

  View details for DOI 10.1007/s00424-019-02259-2

  View details for Web of Science ID 000468531500005

• Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Pflugers Archiv : European journal of physiology Spudich, J. A. 2019

  Abstract

  Several lines of evidence suggest that the primary effect of hypertrophic cardiomyopathy mutations in human beta-cardiac myosin is hypercontractility of the heart, which leads to subsequent hypertrophy, fibrosis, and myofilament disarray. Here, I describe three perspectives on the molecular basis of this hypercontractility. The first is that hypercontractility results from changes in the fundamental parameters of the actin-activated beta-cardiac myosin chemo-mechanical ATPase cycle. The second considers that hypercontractility results from an increase in the number of functionally accessible heads in the sarcomere for interaction with actin. The final and third perspective is that load dependence of contractility is affected by cardiomyopathy mutations and small-molecule effectors in a manner that changes the power output of cardiac contraction. Experimental approaches associated with each perspective are described along with concepts of therapeutic approaches that could prove valuable in treating hypertrophic cardiomyopathy.

  View details for PubMedID 30767072

• The Myosin Mesa and Hypertrophic Cardiomyopathy: Mutations to Mechanisms to Therapies Spudich, J. CELL PRESS. 2019: 35A

  View details for DOI 10.1016/j.bpj.2018.11.233

  View details for Web of Science ID 000460779800178

• On the Functional Assessment of Hypertrophic Cardiomyopathy-Causing Mutations in Human beta-Cardiac Myosin and the Role of Myosin Binding Protein-C Trivedi, D. V., Sarkar, S. S., Adhikari, A. S., Morck, M. M., Kooiker, K. B., Bernstein, D., Ruppel, K. M., Spudich, J. A. CELL PRESS. 2019: 466A–467A

  View details for DOI 10.1016/j.bpj.2018.11.2520

  View details for Web of Science ID 000460779802344

• Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, R. L., Trivedi, D. V., Sarkar, S. S., Henze, M., Ma, W., Gong, H., Rogers, C. S., Gorham, J. M., Wong, F. L., Morck, M. M., Seidman, J. G., Ruppel, K. M., Irving, T. C., Cooke, R., Green, E. M., Spudich, J. A. 2018; 115 (35): EB143–EB152

  Abstract

  Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

  View details for PubMedID 30104387

• Controlling load-dependent kinetics of beta-cardiac myosin at the single-molecule level. Nature structural & molecular biology Liu, C., Kawana, M., Song, D., Ruppel, K. M., Spudich, J. A. 2018; 25 (6): 505–14

  Abstract

  Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate of myosin from actin, which determines the time that myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which myosin works. Here we measure the detachment rate of single molecules of human beta-cardiac myosin and its load dependence. We find that both can be modulated by both small-molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating versus inhibitory perturbations of cardiac myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and suggest that cardiac contractility can be controlled by tuning the load-dependent kinetics of single myosin molecules.

  View details for PubMedID 29867217

• The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations. Nature structural & molecular biology Nag, S., Trivedi, D. V., Sarkar, S. S., Adhikari, A. S., Sunitha, M. S., Sutton, S., Ruppel, K. M., Spudich, J. A. 2017; 24 (6): 525-533

  Abstract

  Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

  View details for DOI 10.1038/nsmb.3408

  View details for PubMedID 28481356

• Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light. Biophysical reviews Trivedi, D. V., Adhikari, A. S., Sarkar, S. S., Ruppel, K. M., Spudich, J. A. 2017

  Abstract

  The sarcomere is an exquisitely designed apparatus that is capable of generating force, which in the case of the heart results in the pumping of blood throughout the body. At the molecular level, an ATP-dependent interaction of myosin with actin drives the contraction and force generation of the sarcomere. Over the past six decades, work on muscle has yielded tremendous insights into the workings of the sarcomeric system. We now stand on the cusp where the acquired knowledge of how the sarcomere contracts and how that contraction is regulated can be extended to an understanding of the molecular mechanisms of sarcomeric diseases, such as hypertrophic cardiomyopathy (HCM). In this review we present a picture that combines current knowledge of the myosin mesa, the sequestered state of myosin heads on the thick filament, known as the interacting-heads motif (IHM), their possible interaction with myosin binding protein C (MyBP-C) and how these interactions can be abrogated leading to hyper-contractility, a key clinical manifestation of HCM. We discuss the structural and functional basis of the IHM state of the myosin heads and identify HCM-causing mutations that can directly impact the equilibrium between the 'on state' of the myosin heads (the open state) and the IHM 'off state'. We also hypothesize a role of MyBP-C in helping to maintain myosin heads in the IHM state on the thick filament, allowing release in a graded manner upon adrenergic stimulation. By viewing clinical hyper-contractility as the result of the destabilization of the IHM state, our aim is to view an old disease in a new light.

  View details for PubMedID 28717924

• Multidimensional structure-function relationships in human beta-cardiac myosin from population-scale genetic variation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Homburger, J. R., Green, E. M., Caleshu, C., Sunitha, M. S., Taylor, R. E., Ruppel, K. M., Metpally, R. P., Colan, S. D., Michels, M., Day, S. M., Olivotto, I., Bustamante, C. D., Dewey, F. E., Ho, C. Y., Spudich, J. A., Ashley, E. A. 2016; 113 (24): 6701-6706

  Abstract

  Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.

  View details for DOI 10.1073/pnas.1606950113

  View details for Web of Science ID 000377948800046

  View details for PubMedID 27247418

  View details for PubMedCentralID PMC4914177

• A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice SCIENCE Green, E. M., Wakimoto, H., Anderson, R. L., Evanchik, M. J., Gorham, J. M., Harrison, B. C., Henze, M., Kawas, R., Oslob, J. D., Rodriguez, H. M., Song, Y., Wan, W., Leinwand, L. A., Spudich, J. A., McDowell, R. S., Seidman, J. G., Seidman, C. E. 2016; 351 (6273): 617-621

  Abstract

  Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.

  View details for DOI 10.1126/science.aad3456

  View details for Web of Science ID 000369291600044

  View details for PubMedID 26912705

  View details for PubMedCentralID PMC4784435

• Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human beta-cardiac myosin JOURNAL OF EXPERIMENTAL BIOLOGY Spudich, J. A., Aksel, T., Bartholomew, S. R., Nag, S., Kawana, M., Yu, E. C., Sarkar, S. S., Sung, J., Sommese, R. F., Sutton, S., Cho, C., Adhikari, A. S., Taylor, R., Liu, C., Trivedi, D., Ruppel, K. M. 2016; 219 (2): 161-167

  Abstract

  Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

  View details for DOI 10.1242/jeb.125930

  View details for Web of Science ID 000368546300006

• Ensemble force changes that result from human cardiac myosin mutations and a small-molecule effector. Cell reports Aksel, T., Choe Yu, E., Sutton, S., Ruppel, K. M., Spudich, J. A. 2015; 11 (6): 910-920

  Abstract

  Cardiomyopathies due to mutations in human β-cardiac myosin are a significant cause of heart failure, sudden death, and arrhythmia. To understand the underlying molecular basis of changes in the contractile system's force production due to such mutations and search for potential drugs that restore force generation, an in vitro assay is necessary to evaluate cardiac myosin's ensemble force using purified proteins. Here, we characterize the ensemble force of human α- and β-cardiac myosin isoforms and those of β-cardiac myosins carrying left ventricular non-compaction (M531R) and dilated cardiomyopathy (S532P) mutations using a utrophin-based loaded in vitro motility assay and new filament-tracking software. Our results show that human α- and β-cardiac myosin, as well as the mutants, show opposite mechanical and enzymatic phenotypes with respect to each other. We also show that omecamtiv mecarbil, a previously discovered cardiac-specific myosin activator, increases β-cardiac myosin force generation.

  View details for DOI 10.1016/j.celrep.2015.04.006

  View details for PubMedID 25937279

  View details for PubMedCentralID PMC4431957

• The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy BIOCHEMICAL SOCIETY TRANSACTIONS Spudich, J. A. 2015; 43: 64-72

  Abstract

  No matter how many times one explores the structure of the myosin molecule, there is always something new to discover. Here, I describe the myosin mesa, a structural feature of the motor domain that has the characteristics of a binding domain for another protein, possibly myosin-binding protein C (MyBP-C). Interestingly, many well-known hypertrophic cardiomyopathy (HCM) mutations lie along this surface and may affect the putative interactions proposed here. A potential unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy is discussed here. It involves increased power output of the cardiac muscle as a result of HCM mutations causing the release of inhibition by myosin binding protein C.

  View details for DOI 10.1042/BST20140324

  View details for Web of Science ID 000350741200009

  View details for PubMedID 25619247

  View details for PubMedCentralID PMC4349527

• Harmonic force spectroscopy measures load-dependent kinetics of individual human ß-cardiac myosin molecules. Nature communications Sung, J., Nag, S., Mortensen, K. I., Vestergaard, C. L., Sutton, S., Ruppel, K., Flyvbjerg, H., Spudich, J. A. 2015; 6: 7931-?

  Abstract

  Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.

  View details for DOI 10.1038/ncomms8931

  View details for PubMedID 26239258

  View details for PubMedCentralID PMC4532873

• Hypertrophic and Dilated Cardiomyopathy: Four Decades of Basic Research on Muscle Lead to Potential Therapeutic Approaches to These Devastating Genetic Diseases BIOPHYSICAL JOURNAL Spudich, J. A. 2014; 106 (6): 1236-1249

  Abstract

  With the advent of technologies to obtain the complete sequence of the human genome in a cost-effective manner, this decade and those to come will see an exponential increase in our understanding of the underlying genetics that lead to human disease. And where we have a deep understanding of the biochemical and biophysical basis of the machineries and pathways involved in those genetic changes, there are great hopes for the development of modern therapeutics that specifically target the actual machinery and pathways altered by individual mutations. Prime examples of such a genetic disease are those classes of hypertrophic and dilated cardiomyopathy that result from single amino-acid substitutions in one of several of the proteins that make up the cardiac sarcomere or from the truncation of myosin binding protein C. Hypertrophic cardiomyopathy alone affects ∼1 in 500 individuals, and it is the leading cause of sudden cardiac death in young adults. Here I describe approaches to understand the molecular basis of the alterations in power output that result from these mutations. Small molecules binding to the mutant sarcomeric protein complex should be able to mitigate the effects of hypertrophic and dilated cardiomyopathy mutations at their sources, leading to possible new therapeutic approaches for these genetic diseases.

  View details for DOI 10.1016/j.bpj.2014.02.011

  View details for Web of Science ID 000333226900006

  View details for PubMedID 24655499

  View details for PubMedCentralID PMC3985504

• One path to understanding energy transduction in biological systems NATURE MEDICINE Spudich, J. A. 2012; 18 (10): 1478-1482

  View details for DOI 10.1038/nm.2924

  View details for Web of Science ID 000309587500021

  View details for PubMedID 23042356

• The power stroke of myosin VI and the basis of reverse directionality PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bryant, Z., Altman, D., Spudich, J. A. 2007; 104 (3): 772-777

  Abstract

  Myosin VI supports movement toward the (-) end of actin filaments, despite sharing extensive sequence and structural homology with (+)-end-directed myosins. A class-specific stretch of amino acids inserted between the converter domain and the lever arm was proposed to provide the structural basis of directionality reversal. Indeed, the unique insert mediates a 120 degrees redirection of the lever arm in a crystal structure of the presumed poststroke conformation of myosin VI [Ménétrey J, Bahloul A, Wells AL, Yengo CM, Morris CA, Sweeney HL, Houdusse A (2005) Nature 435:779-785]. However, this redirection alone is insufficient to account for the large (-)-end-directed stroke of a monomeric myosin VI construct. The underlying motion of the myosin VI converter domain must therefore differ substantially from the power stroke of (+)-end-directed myosins. To experimentally map out the motion of the converter domain and lever arm, we have generated a series of truncated myosin VI constructs and characterized the size and direction of the power stroke for each construct using dual-labeled gliding filament assays and optical trapping. Motors truncated near the end of the converter domain generate (+)-end-directed motion, whereas longer constructs move toward the (-) end. Our results directly demonstrate that the unique insert is required for directionality reversal, ruling out a large class of models in which the converter domain moves toward the (-) end. We suggest that the lever arm rotates approximately 180 degrees between pre- and poststroke conformations.

  View details for DOI 10.1073/pnas.0610144104

  View details for Web of Science ID 000243761100018

  View details for PubMedID 17182734

  View details for PubMedCentralID PMC1713167

• Characterization of the one-head bound intermediate that occurs as myosin V walks on actin 51st Annual Meeting of the Biophysical-Society Dunn, A. R., Spudich, J. A. CELL PRESS. 2007: 190A–190A

  View details for Web of Science ID 000243972401080

• Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Churchman, L. S., Okten, Z., Rock, R. S., Dawson, J. F., Spudich, J. A. 2005; 102 (5): 1419-1423

  Abstract

  Here we present a technique called single-molecule high-resolution colocalization (SHREC) of fluorescent dyes that allows the measurement of interfluorophore distances in macromolecules and macromolecular complexes with better than 10-nm resolution. By using two chromatically differing fluorescent molecules as probes, we are able to circumvent the Rayleigh criterion and measure distances much smaller than 250 nm. The probes are imaged separately and localized individually with high precision. The registration between the two imaging channels is measured by using fiduciary markers, and the centers of the two probes are mapped onto the same space. Multiple measurements can be made before the fluorophores photobleach, allowing intramolecular and intermolecular distances to be tracked through time. This technique's lower resolution limit lies at the upper resolution limit of single molecule FRET (smFRET) microscopy. The instrumentation and fluorophores used for SHREC can also be used for smFRET, allowing the two types of measurements to be made interchangeably, covering a wide range of interfluorophore distances. A dual-labeled duplex DNA molecule (30 bp) was used as a 10-nm molecular ruler to confirm the validity of the method. We also used SHREC to study the motion of myosin V. We directly observed myosin V's alternating heads while it walked hand-over-hand along an actin filament.

  View details for DOI 10.1073/pnas.0409487102

  View details for Web of Science ID 000226877300032

  View details for PubMedID 15668396

  View details for PubMedCentralID PMC545495

• A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin CELL Shih, W. M., Gryczynski, Z., Lakowicz, J. R., Spudich, J. A. 2000; 102 (5): 683-694

  Abstract

  The molecular motor myosin is proposed to bind to actin and swing its light-chain binding region through a large angle to produce an approximately 10 nm step in motion coupled to changes in the nucleotide state at the active site. To date, however, direct dynamic measurements have largely failed to show changes of that magnitude. Here, we use a cysteine engineering approach to create a high resolution, FRET-based sensor that reports a large, approximately 70 degree nucleotide-dependent angle change of the light-chain binding region. The combination of steady-state and time-resolved fluorescence resonance energy transfer measurements unexpectedly reveals two distinct prestroke states. The measurements also show that bound Mg.ADP.Pi, and not bound Mg.ATP, induces the myosin to adopt the prestroke states.

  View details for Web of Science ID 000089105200016

  View details for PubMedID 11007486

• The neck region of the myosin motor domain acts as a lever arm to generate movement PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Uyeda, T. Q., ABRAMSON, P. D., Spudich, J. A. 1996; 93 (9): 4459-4464

  Abstract

  The myosin head consists of a globular catalytic domain that binds actin and hydrolyzes ATP and a neck domain that consists of essential and regulatory light chains bound to a long alpha-helical portion of the heavy chain. The swinging neck-level model assumes that a swinging motion of the neck relative to the catalytic domain is the origin of movement. This model predicts that the step size, and consequently the sliding velocity, are linearly related to the length of the neck. We have tested this point by characterizing a series of mutant Dictyostelium myosins that have different neck lengths. The 2xELCBS mutant has an extra binding site for essential light chain. The delta RLCBS mutant myosin has an internal deletion that removes the regulatory light chain binding site. The delta BLCBS mutant lacks both light chain binding sites. Wild-type myosin and these mutant myosins were subjected to the sliding filament in vitro motility assay. As expected, mutants with shorter necks move slower than wild-type myosin in vitro. Most significantly, a mutant with a longer neck moves faster than the wild type, and the sliding velocities of these myosins are linearly related to the neck length, as predicted by the swinging neck-lever model. A simple extrapolation to zero speed predicts that the fulcrum point is in the vicinity of the SH1-SH2 region in the catalytic domain.

  View details for Web of Science ID A1996UK55700134

  View details for PubMedID 8633089

• SINGLE MYOSIN MOLECULE MECHANICS - PICONEWTON FORCES AND NANOMETER STEPS NATURE Finer, J. T., Simmons, R. M., Spudich, J. A. 1994; 368 (6467): 113-119

  Abstract

  A new in vitro assay using a feedback enhanced laser trap system allows direct measurement of force and displacement that results from the interaction of a single myosin molecule with a single suspended actin filament. Discrete stepwise movements averaging 11 nm were seen under conditions of low load, and single force transients averaging 3-4 pN were measured under isometric conditions. The magnitudes of the single forces and displacements are consistent with predictions of the conventional swinging-crossbridge model of muscle contraction.

  View details for Web of Science ID A1994NA03000055

  View details for PubMedID 8139653

• MYOSIN SUBFRAGMENT-1 IS SUFFICIENT TO MOVE ACTIN-FILAMENTS INVITRO NATURE Toyoshima, Y. Y., Kron, S. J., McNally, E. M., NIEBLING, K. R., Toyoshima, C., Spudich, J. A. 1987; 328 (6130): 536-539

  Abstract

  The rotating crossbridge model for muscle contraction proposes that force is produced by a change in angle of the crossbridge between the overlapping thick and thin filaments. Myosin, the major component of the thick filament, is comprised of two heavy chains and two pairs of light chains. Together they form two globular heads, which give rise to the crossbridge in muscle, and a coiled-coil rod, which forms the shaft of the thick filament. The isolated head fragment, subfragment-1 (S1), contains the ATPase and actin-binding activities of myosin (Fig. 1). Although S1 seems to have the requisite enzymatic activity, direct evidence that S1 is sufficient to drive actin movement has been lacking. It has long been recognized that in vitro movement assays are an important approach for identifying the elements in muscle responsible for force generation. Hynes et al. showed that beads coated with heavy meromyosin (HMM), a soluble proteolytic fragment of myosin consisting of a part of the rod and the two heads, can move on Nitella actin filaments. Using the myosin-coated surface assay of Kron and Spudich, Harada et al. showed that single-headed myosin filaments bound to glass support movement of actin at nearly the same speed as intact myosin filaments. These studies show that the terminal portion of the rod and the two-headed nature of myosin are not required for movement. To restrict the region responsible for movement further, we have modified the myosin-coated surface assay by replacing the glass surface with a nitrocellulose film. Here we report that myosin filaments, soluble myosin, HMM or S1, when bound to a nitrocellulose film, support actin sliding movement (Fig. 2). That S1 is sufficient to cause sliding movement of actin filaments in vitro gives strong support to models of contraction that place the site of active movement in muscle within the myosin head.

  View details for Web of Science ID A1987J481200067

  View details for PubMedID 2956522

• DISRUPTION OF THE DICTYOSTELIUM MYOSIN HEAVY-CHAIN GENE BY HOMOLOGOUS RECOMBINATION SCIENCE DeLozanne, A., Spudich, J. A. 1987; 236 (4805): 1086-1091

  Abstract

  The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.

  View details for Web of Science ID A1987H469300026

  View details for PubMedID 3576222

• Myosin Subfragment-1 is Sufficient to Move Actin Filaments In Vitro Myosin Subfragment-1 is Sufficient to Move Actin Filaments In Vitro Toyoshima, Y. Y., et al 1987; 328: 536-539
• FLUORESCENT ACTIN-FILAMENTS MOVE ON MYOSIN FIXED TO A GLASS-SURFACE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kron, S. J., Spudich, J. A. 1986; 83 (17): 6272-6276

  Abstract

  Single actin filaments stabilized with fluorescent phalloidin exhibit ATP-dependent movement on myosin filaments fixed to a surface. At pH 7.4 and 24 degrees C, the rates of movement average 3-4 micron/s with skeletal muscle myosin and 1-2 micron/s with Dictyostelium myosin. These rates are very similar to those measured in our previous myosin movement assays. The rates of movement are relatively independent of the type of actin used. The filament velocity shows a broad pH optimum between 7.0 and 9.0, and the concentration of ATP required for half-maximal velocity is 50 microM. Evidence was obtained to suggest that movement of actin over myosin requires at most the number of heads in a single thick filament. This system provides a practical, quantitative myosin-movement assay with purified proteins.

  View details for Web of Science ID A1986D837900010

  View details for PubMedID 3462694

• MOVEMENT OF MYOSIN-COATED BEADS ON ORIENTED FILAMENTS RECONSTITUTED FROM PURIFIED ACTIN NATURE Spudich, J. A., Kron, S. J., Sheetz, M. P. 1985; 315 (6020): 584-586

  Abstract

  Although the biochemical properties of the actin/myosin interaction have been studied extensively using actin activation of myosin ATPase as an assay, until recently no well-defined assay has been available to measure the mechanical properties of ATP-dependent movement of myosin along actin filaments. The first direct measurements of the rate of myosin movement in vitro used a naturally occurring, biochemically ill-defined array of actin filaments from the alga Nitella. We report here the construction of an oriented array of filaments reconstituted from purified muscle actin and the use of this array in a biochemically defined quantitative assay for the directed movement of myosin-coated polystyrene beads. We demonstrate for the first time that actin alone, linked to a substratum by a protein anchor, is sufficient to support movement of myosin at rates consistent with the speeds of muscle contraction and other forms of cell motility.

  View details for Web of Science ID A1985AKB6600045

  View details for PubMedID 3925346

• CYTOSKELETAL ELEMENTS OF CHICK-EMBRYO FIBROBLASTS REVEALED BY DETERGENT EXTRACTION JOURNAL OF SUPRAMOLECULAR STRUCTURE Brown, S., Levinson, W., Spudich, J. A. 1976; 5 (2): 119-130

  Abstract

  Treatment of chick embryo fibroblasts with 0.5% Triton X-100 extracts most of the cell protein, leaving an organized part of the cell structure attached to the tissue culture dish. This "Triton cytoskeleton" consists largely of intermediate-sized filaments and bundles of microfilaments. SDS polyacrylamide gel electrophoresis reveals that this cytoskeleton is made up of three main proteins. One protein component is 42,000 daltons and co-migrates with muscle actin. The other two components are 52,000 and 230,000 daltons and remain quantitatively associated with the cytoskeleton during the detergent extraction. The possible identity of these three protein components and their organization into a supramolecular structure is discussed.

  View details for Web of Science ID A1976CT02900002

  View details for PubMedID 1034175

• Motility Assay to Probe the Calcium Sensitivity of Myosin and Regulated Thin Filaments. Methods in molecular biology (Clifton, N.J.) Liu, C., Ruppel, K. M., Spudich, J. A. 2024; 2735: 169-189

  Abstract

  Calcium-dependent activation of the thin filament mediated by the troponin-tropomyosin complex is key in the regulation of actin-myosin based muscle contraction. Perturbations to this system, either physiological (e.g., phosphorylation of myosin light chains) or pathological (e.g., mutations that cause familial cardiomyopathies), can alter calcium sensitivity and thus have important implications in human health and disease. The in vitro motility assay provides a quantitative and precise method to study the calcium sensitivity of the reconstituted myosin-thin filament motile system. Here we present a simple and robust protocol to perform calcium-dependent motility of β-cardiac myosin and regulated thin filaments. The experiment is done on a multichannel microfluidic slide requiring minimal amounts of proteins. A complete velocity vs. calcium concentration curve is produced from one experiment in under 1 h.

  View details for DOI 10.1007/978-1-0716-3527-8_10

  View details for PubMedID 38038849

  View details for PubMedCentralID 386485

• Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration. Proceedings of the National Academy of Sciences of the United States of America Lee, S., Vander Roest, A. S., Blair, C. A., Kao, K., Bremner, S. B., Childers, M. C., Pathak, D., Heinrich, P., Lee, D., Chirikian, O., Mohran, S. E., Roberts, B., Smith, J. E., Jahng, J. W., Paik, D. T., Wu, J. C., Gunawardane, R. N., Ruppel, K. M., Mack, D. L., Pruitt, B. L., Regnier, M., Wu, S. M., Spudich, J. A., Bernstein, D. 2024; 121 (19): e2318413121

  Abstract

  Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

  View details for DOI 10.1073/pnas.2318413121

  View details for PubMedID 38683993

• Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation. Proceedings of the National Academy of Sciences of the United States of America Liu, C., Karabina, A., Meller, A., Bhattacharjee, A., Agostino, C. J., Bowman, G. R., Ruppel, K. M., Spudich, J. A., Leinwand, L. A. 2024; 121 (9): e2315472121

  Abstract

  Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature.

  View details for DOI 10.1073/pnas.2315472121

  View details for PubMedID 38377203

• Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context. bioRxiv : the preprint server for biology Woldeyes, R. A., Nishiga, M., Vander Roest, A. S., Engel, L., Giri, P., Montenegro, G. C., Wu, A. C., Dunn, A. R., Spudich, J. A., Bernstein, D., Schmid, M. F., Wu, J. C., Chiu, W. 2023

  Abstract

  Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

  View details for DOI 10.1101/2023.10.26.564098

  View details for PubMedID 37961228

  View details for PubMedCentralID PMC10634850

• Homologous mutations in β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation. bioRxiv : the preprint server for biology Liu, C., Karabina, A., Meller, A., Bhattacharjee, A., Agostino, C. J., Bowman, G. R., Ruppel, K. M., Spudich, J. A., Leinwand, L. A. 2023

  Abstract

  Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins, with the most dramatic in perinatal, but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing ATPase cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents the first direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are yet another testament to myosin's highly allosteric nature.

  View details for DOI 10.1101/2023.07.02.547385

  View details for PubMedID 37425764

  View details for PubMedCentralID PMC10327197

• Cryo-EM structure of the folded-back state of human β-cardiac myosin. Nature communications Grinzato, A., Auguin, D., Kikuti, C., Nandwani, N., Moussaoui, D., Pathak, D., Kandiah, E., Ruppel, K. M., Spudich, J. A., Houdusse, A., Robert-Paganin, J. 2023; 14 (1): 3166

  Abstract

  To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human β-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.6 Å resolution, providing details of all the interfaces stabilizing the 'off' state. The structure shows that these interfaces are hot spots of hypertrophic cardiomyopathy mutations that are thought to cause hypercontractility by destabilizing the 'off' state. Importantly, the cardiac and smooth muscle myosin IHM structures dramatically differ, providing structural evidence for the divergent physiological regulation of these muscle types. The cardiac IHM structure will facilitate development of clinically useful new molecules that modulate IHM stability.

  View details for DOI 10.1038/s41467-023-38698-w

  View details for PubMedID 37258552

  View details for PubMedCentralID 3156359

• Cryo-EM structure of the folded-back state of human β-cardiac myosin. bioRxiv : the preprint server for biology Grinzato, A., Auguin, D., Kikuti, C., Nandwani, N., Moussaoui, D., Pathak, D., Kandiah, E., Ruppel, K. M., Spudich, J. A., Houdusse, A., Robert-Paganin, J. 2023

  Abstract

  During normal levels of exertion, many cardiac muscle myosin heads are sequestered in an off-state even during systolic contraction to save energy and for precise regulation. They can be converted to an on-state when exertion is increased. Hypercontractility caused by hypertrophic cardiomyopathy (HCM) myosin mutations is often the result of shifting the equilibrium toward more heads in the on-state. The off-state is equated with a folded-back structure known as the interacting head motif (IHM), which is a regulatory feature of all muscle myosins and class-2 non-muscle myosins. We report here the human β-cardiac myosin IHM structure to 3.6 Å resolution. The structure shows that the interfaces are hot spots of HCM mutations and reveals details of the significant interactions. Importantly, the structures of cardiac and smooth muscle myosin IHMs are dramatically different. This challenges the concept that the IHM structure is conserved in all muscle types and opens new perspectives in the understanding of muscle physiology. The cardiac IHM structure has been the missing puzzle piece to fully understand the development of inherited cardiomyopathies. This work will pave the way for the development of new molecules able to stabilize or destabilize the IHM in a personalized medicine approach. *This manuscript was submitted to Nature Communications in August 2022 and dealt efficiently by the editors. All reviewers received this version of the manuscript before 9 208 August 2022. They also received coordinates and maps of our high resolution structure on the 18 208 August 2022. Due to slowness of at least one reviewer, this contribution was delayed for acceptance by Nature Communications and we are now depositing in bioRxiv the originally submitted version written in July 2022 for everyone to see. Indeed, two bioRxiv contributions at lower resolution but adding similar concepts on thick filament regulation were deposited this week in bioRxiv, one of the contributions having had access to our coordinates. We hope that our data at high resolution will be helpful for all readers that appreciate that high resolution information is required to build accurate atomic models and discuss implications for sarcomere regulation and the effects of cardiomyopathy mutations on heart muscle function.

  View details for DOI 10.1101/2023.04.15.536999

  View details for PubMedID 37131793

  View details for PubMedCentralID PMC10153137

• Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy Kawana, M., Goluguri, R., Dawood, A., Spudich, J. A., Ruppel, K. CELL PRESS. 2023: 258A

  View details for Web of Science ID 000989629701376

• Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction. Biophysical journal Pathak, D., Nandwani, N., Spudich, J. A., Ruppel, K. 2023; 122 (3S1): 147a

  View details for DOI 10.1016/j.bpj.2022.11.1011

  View details for PubMedID 36782676

• Cryo-EM structure of the folded-back state of human beta-cardiac myosin. Biophysical journal Grinzato, A., Auguin, D., Kikuti, C., Nandwani, N., Moussaoui, D., Pathak, D., Kandiah, E., Ruppel, K., Spudich, J. A., Houdusse, A. M., Robert-Paganin, J. 2023; 122 (3S1): 258a-259a

  View details for DOI 10.1016/j.bpj.2022.11.1489

  View details for PubMedID 36783267

• Molecular characterization of a novel MYH7 mutation Q222H in a patient with severe dilated cardiomyopathy. Biophysical journal Kawana, M., Reddy Goluguri, R., Dawood, A., Spudich, J. A., Ruppel, K. 2023; 122 (3S1): 258a

  View details for DOI 10.1016/j.bpj.2022.11.1488

  View details for PubMedID 36783268

• Sarcomere dynamics simulations to uncover mechanisms in hypertrophic cardiomyopathy. Biophysical journal Roest, A. S., Spudich, J. A., Ruppel, K., Regnier, M., Bernstein, D. 2023; 122 (3S1): 148a-149a

  View details for DOI 10.1016/j.bpj.2022.11.1017

  View details for PubMedID 36782679

• Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes. Biophysical journal Bernstein, D., Vander Roest, A. S., Wu, S., Pruitt, B., Zhao, M., Fajardo, G., Ruppel, K., Spudich, J. A. 2023; 122 (3S1): 148a

  View details for DOI 10.1016/j.bpj.2022.11.1014

  View details for PubMedID 36782680

• Sarcomere dynamics simulations to uncover mechanisms in hypertrophic cardiomyopathy Vander Roest, A. S., Spudich, J. A., Ruppel, K., Regnier, M., Bernstein, D. CELL PRESS. 2023: 148A-149A

  View details for Web of Science ID 000989629700725

• Changes in myosin biomechanics influence growth and maturation of iPSC-cardiomyocytes Bernstein, D., Vander Roest, A. S., Wu, S., Pruitt, B., Zhao, M., Fajardo, G., Ruppel, K., Spudich, J. A. CELL PRESS. 2023: 148A

  View details for Web of Science ID 000989629700722

• Structural changes in myosin affect chemo-mechanical properties of the myosin-actin interaction Pathak, D., Nandwani, N., Spudich, J. A., Ruppel, K. CELL PRESS. 2023: 147A

  View details for Web of Science ID 000989629700719

• First-in-class drug candidate (MPH-220) efficiently improves spastic gait disorders by selective inhibition of fast skeletal muscle myosin-2 Gyimesi, M., Horvath, A. I., Turos, D., Suthar, S., Penzes, M., Canon, L., Kikuti, C., Ruppel, K., Trivedi, D. V., Spudich, J. A., Rauscher, A. A., Kovacs, M., Komoly, S., Houdusse, A. M., Malnasi-Csizmadia, A. CELL PRESS. 2022: 291A

  View details for Web of Science ID 000759523001678

• Understanding the molecular basis of HCM-causing mutations in cardiac myosin and cardiac myosin binding protein-C Pathak, D., Nandwani, N., Ruppel, K., Spudich, J. A. CELL PRESS. 2022: 255A

  View details for Web of Science ID 000759523001505

• Allosteric destabilization of the super-relaxed state of cardiac myosin by hypertrophic cardiomyopathy-causing mutations Nandwani, N., Bhowmik, D., Dawood, A., Ruppel, K., Spudich, J. A. CELL PRESS. 2022: 292A

  View details for Web of Science ID 000759523001682

• Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy. Circulation Ranjbarvaziri, S., Kooiker, K. B., Ellenberger, M., Fajardo, G., Zhao, M., Vander Roest, A. S., Woldeyes, R. A., Koyano, T. T., Fong, R., Ma, N., Tian, L., Traber, G. M., Chan, F., Perrino, J., Reddy, S., Chiu, W., Wu, J. C., Woo, J. Y., Ruppel, K. M., Spudich, J. A., Snyder, M. P., Contrepois, K., Bernstein, D. 2021

  Abstract

  Background: Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes. In this work, we test the hypothesis that changes in cardiac energetics represent a common pathophysiologic pathway in HCM. Methods: We performed a comprehensive multi-omics profile of the molecular (transcripts, metabolites, and complex lipids), ultrastructural, and functional components of HCM energetics using myocardial samples from 27 HCM patients and 13 normal controls (donor hearts). Results: Integrated omics analysis revealed alterations in a wide array of biochemical pathways with major dysregulation in fatty acid metabolism, reduction of acylcarnitines, and accumulation of free fatty acids. HCM hearts showed evidence of global energetic decompensation manifested by a decrease in high energy phosphate metabolites [ATP, ADP, and phosphocreatine (PCr)] and a reduction in mitochondrial genes involved in creatine kinase and ATP synthesis. Accompanying these metabolic derangements, electron microscopy showed an increased fraction of severely damaged mitochondria with reduced cristae density, coinciding with reduced citrate synthase (CS) activity and mitochondrial oxidative respiration. These mitochondrial abnormalities were associated with elevated reactive oxygen species (ROS) and reduced antioxidant defenses. However, despite significant mitochondrial injury, HCM hearts failed to upregulate mitophagic clearance. Conclusions: Overall, our findings suggest that perturbed metabolic signaling and mitochondrial dysfunction are common pathogenic mechanisms in patients with HCM. These results highlight potential new drug targets for attenuation of the clinical disease through improving metabolic function and reducing mitochondrial injury.

  View details for DOI 10.1161/CIRCULATIONAHA.121.053575

  View details for PubMedID 34672721

• Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy. ACS nano Suay-Corredera, C., Pricolo, M. R., Velazquez-Carreras, D., Pathak, D., Nandwani, N., Pimenta-Lopes, C., Sanchez-Ortiz, D., Urrutia-Irazabal, I., Vilches, S., Dominguez, F., Frisso, G., Monserrat, L., Garcia-Pavia, P., de Sancho, D., Spudich, J. A., Ruppel, K. M., Herrero-Galan, E., Alegre-Cebollada, J. 2021

  Abstract

  Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

  View details for DOI 10.1021/acsnano.1c02242

  View details for PubMedID 34060810

• Molecular Mechanisms and Cellular Models of Hypertrophic Cardiomyopathy: Insights from a Surprising Mutation Vander Roest, A. S., Liu, C., Kooiker, K. B., Morck, M. M., Pruitt, B., Campbell, K. S., Ruppel, K., Spudich, J. A., Bernstein, D. CELL PRESS. 2021: 253A

  View details for Web of Science ID 000629601401473

• Hypertrophic Cardiomyopathy, a Disease of Altered Cardiac Energetics Ranjbarvaziri, S., Ellenberger, M., Kooiker, K., Fajardo, G., Zhao, M., Schroer, A., Woo, Y. Y., Ruppel, K. M., Spudich, J. A., Snyder, M., Contrepois, K., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2020

  View details for DOI 10.1161/res.127.suppl_1.235

  View details for Web of Science ID 000606541500028

• Study of Hcm Causing beta-Cardiac Myosin Mutations Located at Different Structurally Significant Regions of the Myosin-Head Bhowmik, D., Nandwani, N., Ruppel, K., Liu, C., Spudich, J. A. CELL PRESS. 2020: 435A

  View details for Web of Science ID 000513023202668

• Functional Comparison of Homologous Mutations in Human Beta, Perinatal, and Embryonic Muscle Myosin Isoforms Karabina, A., Liu, C., Spudich, J. A., Leinwand, L. A. CELL PRESS. 2020: 433A

  View details for Web of Science ID 000513023202659

• Uncovering the Molecular and Structural Basis of Hypertrophic Cardiomyopathy-Causing Mutations in Myosin and Myosin Binding Protein-C Nandwani, N., Trivedi, D. V., Sarkar, S. S., Morck, M., Ruppel, K., Spudich, J. A. CELL PRESS. 2020: 435A

  View details for Web of Science ID 000513023202667

• Hypertrophic Cardiomyopathy Mutations With Opposite Effects on [latin sharp s]-myosin Biomechanics Show Similar Structural and Biomechanical Phenotypes in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hipsc-cms) Schroer, A., Jung, G., Kooiker, K., Adhikari, A., Song Linda, Liu Chao, Ruppel, K., Wu Sean, Pruitt, B., Spudich, J., Bernstein, D. LIPPINCOTT WILLIAMS & WILKINS. 2019

  View details for Web of Science ID 000511467800091

• Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties. The Journal of biological chemistry Vera, C. D., Johnson, C. A., Walklate, J. n., Adhikari, A. n., Svicevic, M. n., Mijailovich, S. M., Combs, A. C., Langer, S. J., Ruppel, K. M., Spudich, J. A., Geeves, M. A., Leinwand, L. A. 2019

  Abstract

  Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, since HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (DR; the fraction of myosin in strongly attached force-holding states) and the measured ATPases all changed in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.

  View details for DOI 10.1074/jbc.RA119.010563

  View details for PubMedID 31582565

• Dilated cardiomyopathy myosin mutants have reduced force-generating capacity JOURNAL OF BIOLOGICAL CHEMISTRY Ujfalusi, Z., Vera, C. D., Mijailovich, S. M., Svicevic, M., Yu, E., Kawana, M., Ruppel, K. M., Spudich, J. A., Geeves, M. A., Leinwand, L. A. 2018; 293 (23): 9017–29

  Abstract

  Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human β-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M·D complex in the steady state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force-holding capacity due to the reduced occupancy of the force-holding state.

  View details for PubMedID 29666183

  View details for PubMedCentralID PMC5995530

• Mechanobiology of Myosin Mutations and Myofibril Remodeling in iPSC-Cardiomyocytes Schroer, A., Kooiker, K., Adhikari, A., Ruppel, K., Bernstein, D., Spudich, J., Pruitt, B. CELL PRESS. 2018: 496A–497A

  View details for Web of Science ID 000430563200233

• A Molecular Approach to Understand the Super-Relaxed State of Myosin Observed in Cardiac Muscle Sarkar, S. S., Trivedi, D. V., Morck, M. M., Adhikari, A. S., Ruppel, K. M., Spudich, J. A. CELL PRESS. 2018: 141A

  View details for Web of Science ID 000429315800710

• Impact of Hypertrophic Cardiomyopathy Mutations and the Role of Myosin Binding Protein-C on the Sequestered State of Myosin Trivedi, D. V., Sarkar, S. S., Morck, M. M., Adhikari, A. A., Ruppel, K. M., Spudich, J. A. CELL PRESS. 2018: 317A

  View details for Web of Science ID 000430450000089

• Controlling Cardiac Contractility at the Single Molecule Level Liu, C., Song, D. L., Kawana, M., Ruppel, K. M., Spudich, J. A. CELL PRESS. 2018: 37A

  View details for Web of Science ID 000429315800195

• SETD3 is an actin histidine methyltransferase that prevents primary dystocia. Nature Wilkinson, A. W., Diep, J. n., Dai, S. n., Liu, S. n., Ooi, Y. S., Song, D. n., Li, T. M., Horton, J. R., Zhang, X. n., Liu, C. n., Trivedi, D. V., Ruppel, K. M., Vilches-Moure, J. G., Casey, K. M., Mak, J. n., Cowan, T. n., Elias, J. E., Nagamine, C. M., Spudich, J. A., Cheng, X. n., Carette, J. E., Gozani, O. n. 2018

  Abstract

  For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur1. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.

  View details for PubMedID 30626964

• Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gangadharan, B., Sunitha, M. S., Mukherjee, S., Chowdhury, R., Haque, F., Sekar, N., Sowdhamini, R., Spudich, J. A., Mercer, J. A. 2017; 114 (42): 11115–20

  Abstract

  Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the TNNT2 gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca2+ in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca2+ sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these TNNT2 mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.

  View details for PubMedID 28973951

• Biophysical properties of human ß-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy. Science advances Kawana, M., Sarkar, S. S., Sutton, S., Ruppel, K. M., Spudich, J. A. 2017; 3 (2)

  Abstract

  Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the β-cardiac myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human β-cardiac myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.

  View details for DOI 10.1126/sciadv.1601959

  View details for PubMedID 28246639

  View details for PubMedCentralID PMC5302870

• How to Measure Load-Dependent Kinetics of Individual Motor Molecules Without a Force-Clamp. Methods in enzymology Sung, J., Mortensen, K. I., Spudich, J. A., Flyvbjerg, H. 2017; 582: 1-29

  Abstract

  Single-molecule force spectroscopy techniques, including optical trapping, magnetic trapping, and atomic force microscopy, have provided unprecedented opportunities to understand biological processes at the smallest biological length scales. For example, they have been used to elucidate the molecular basis of muscle contraction and intracellular cargo transport along cytoskeletal filamentous proteins. Optical trapping is among the most sophisticated single-molecule techniques. With exceptionally high spatial and temporal resolutions, it has been extensively utilized to understand biological functions at the single molecule level, such as conformational changes and force-generation of individual motor proteins or force-dependent kinetics in molecular interactions. Here, we describe a new method, "Harmonic Force Spectroscopy (HFS)." With a conventional dual-beam optical trap and a simple harmonic oscillation of the sample stage, HFS can measure the load-dependent kinetics of transient molecular interactions, such as a human β-cardiac myosin II interacting with an actin filament. We demonstrate that the ADP release rate of an individual human β-cardiac myosin II molecule depends exponentially on the applied load, which provides a clue to understanding the molecular mechanism behind the force-velocity curve of a contracting cardiac muscle. The experimental protocol and the data analysis are simple, fast, and efficient. This chapter provides a practical guide to the method: basic concepts, experimental setup, step-by-step experimental protocol, theory, data analysis, and results.

  View details for DOI 10.1016/bs.mie.2016.08.002

  View details for PubMedID 28062031

• Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human beta-Cardiac Myosin CELL REPORTS Adhikari, A. S., Kooiker, K. B., Sarkar, S. S., Liu, C., Bernstein, D., Spudich, J. A., Ruppel, K. M. 2016; 17 (11): 2857-2864

  Abstract

  Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%-90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

  View details for DOI 10.1016/j.celrep.2016.11.040

  View details for Web of Science ID 000390894700007

  View details for PubMedID 27974200

• How to Measure Separations and Angles Between Intramolecular Fluorescent Markers. Methods in enzymology Mortensen, K. I., Sung, J., Spudich, J. A., Flyvbjerg, H. 2016; 581: 147-185

  Abstract

  Structure and function of an individual biomolecule can be explored with minimum two fluorescent markers of different colors. Since the light of such markers can be spectrally separated and imaged simultaneously, the markers can be colocalized. Here, we describe the method used for such two-color colocalization microscopy. Then we extend it to fluorescent markers with fixed orientations and in intramolecular proximity. Our benchmarking of this extension produced two extra results: (a) we established short double-labeled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly; (b) we established how to map with super-resolution between color-separated channels, which should be useful for all dual-color colocalization measurements with either fixed or freely rotating fluorescent molecules. Throughout, we use only simple means: from each color-separated microscope image in a time-lapse movie, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space, both with accuracy and precision. The relative positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA (dsDNA) molecules internally labeled with two fixed fluorophores, we (i) demonstrate the accuracy and precision of our localization- and mapping-methods, using the known structure of dsDNA as benchmark; (ii) resolve 10 base pair differences in fluorophore separations; (iii) determine the unique 3D orientation of each DNA molecule.

  View details for DOI 10.1016/bs.mie.2016.08.020

  View details for PubMedID 27793279

• Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin. The Journal of experimental biology Spudich, J. A., Aksel, T., Bartholomew, S. R., Nag, S., Kawana, M., Yu, E. C., Sarkar, S. S., Sung, J., Sommese, R. F., Sutton, S., Cho, C., Adhikari, A. S., Taylor, R., Liu, C., Trivedi, D., Ruppel, K. M. 2016; 219 (Pt 2): 161-7

  Abstract

  Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

  View details for DOI 10.1242/jeb.125930

  View details for PubMedID 26792326

• Optimized measurements of separations and angles between intra-molecular fluorescent markers NATURE COMMUNICATIONS Mortensen, K. I., Sung, J., Flyvbjerg, H., Spudich, J. A. 2015; 6

  View details for DOI 10.1038/ncomms9621

  View details for PubMedID 26509412

• Contractility parameters of human ß-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function. Science advances Nag, S., Sommese, R. F., Ujfalusi, Z., Combs, A., Langer, S., Sutton, S., Leinwand, L. A., Geeves, M. A., Ruppel, K. M., Spudich, J. A. 2015; 1 (9)

  Abstract

  Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human β-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca(2+)-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human β-cardiac myosin contractility as a result of the R403Q mutation.

  View details for DOI 10.1126/sciadv.1500511

  View details for PubMedID 26601291

  View details for PubMedCentralID PMC4646805

• A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division ELIFE Manor, U., Bartholomew, S., Golani, G., Christenson, E., Kozlov, M., Higgs, H., Spudich, J., Lippincott-Schwartz, J. 2015; 4

  Abstract

  Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

  View details for DOI 10.7554/eLife.08828

  View details for Web of Science ID 000373817500001

  View details for PubMedID 26305500

  View details for PubMedCentralID PMC4574297

• Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments NATURE NANOTECHNOLOGY Hariadi, R. F., Sommese, R. F., Adhikari, A. S., Taylor, R. E., Sutton, S., Spudich, J. A., Sivaramakrishnan, S. 2015; 10 (8): 696-700

  Abstract

  The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

  View details for DOI 10.1038/NNANO.2015.132

  View details for Web of Science ID 000359754500014

  View details for PubMedID 26149240

• Harmonic force spectroscopy measures load-dependent kinetics of individual human beta-cardiac myosin molecules NATURE COMMUNICATIONS Sung, J., Nag, S., Mortensen, K. I., Vestergaard, C. L., Sutton, S., Ruppel, K., Flyvbjerg, H., Spudich, J. A. 2015; 6

  View details for DOI 10.1038/ncomms8931

  View details for Web of Science ID 000360346000013

• Ensemble Force Changes that Result from Human Cardiac Myosin Mutations and a Small-Molecule Effector CELL REPORTS Aksel, T., Yu, E. C., Sutton, S., Ruppel, K. M., Spudich, J. A. 2015; 11 (6): 910-920

  View details for DOI 10.1016/j.celrep.2015.04.006

  View details for Web of Science ID 000354406900008

  View details for PubMedID 25937279

• Mechanistic Heterogeneity in Contractile Properties of alpha-Tropomyosin (TPM1) Mutants Associated with Inherited Cardiomyopathies JOURNAL OF BIOLOGICAL CHEMISTRY Gupte, T. M., Haque, F., Gangadharan, B., Sunitha, M. S., Mukherjee, S., Anandhan, S., Rani, D. S., Mukundan, N., Jambekar, A., Thangaraj, K., Sowdhamini, R., Sommese, R. F., Nag, S., Spudich, J. A., Mercer, J. A. 2015; 290 (11): 7003-7015

  Abstract

  The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.

  View details for DOI 10.1074/jbc.M114.596676

  View details for Web of Science ID 000350991500034

  View details for PubMedID 25548289

  View details for PubMedCentralID PMC4358124

• Establishing disease causality for a novel gene variant in familial dilated cardiomyopathy using a functional in-vitro assay of regulated thin filaments and human cardiac myosin. BMC medical genetics Pan, S., Sommese, R. F., Sallam, K. I., Nag, S., Sutton, S., Miller, S. M., Spudich, J. A., Ruppel, K. M., Ashley, E. A. 2015; 16 (1): 97-?

  Abstract

  As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins.In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human β-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics.The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.

  View details for DOI 10.1186/s12881-015-0243-5

  View details for PubMedID 26498512

  View details for PubMedCentralID PMC4620603

• Observation of correlated X-ray scattering at atomic resolution. Philosophical transactions of the Royal Society of London. Series B, Biological sciences Mendez, D., Lane, T. J., Sung, J., Sellberg, J., Levard, C., Watkins, H., Cohen, A. E., Soltis, M., Sutton, S., Spudich, J., Pande, V., Ratner, D., Doniach, S. 2014; 369 (1647)

  Abstract

  Tools to study disordered systems with local structural order, such as proteins in solution, remain limited. Such understanding is essential for e.g. rational drug design. Correlated X-ray scattering (CXS) has recently attracted new interest as a way to leverage next-generation light sources to study such disordered matter. The CXS experiment measures angular correlations of the intensity caused by the scattering of X-rays from an ensemble of identical particles, with disordered orientation and position. Averaging over 15 496 snapshot images obtained by exposing a sample of silver nanoparticles in solution to a micro-focused synchrotron radiation beam, we report on experimental efforts to obtain CXS signal from an ensemble in three dimensions. A correlation function was measured at wide angles corresponding to atomic resolution that matches theoretical predictions. These preliminary results suggest that other CXS experiments on disordered ensembles-such as proteins in solution-may be feasible in the future.

  View details for DOI 10.1098/rstb.2013.0315

  View details for PubMedID 24914148

• Effects of Troponin T Cardiomyopathy Mutations on the Calcium Sensitivity of the Regulated Thin Filament and the Actomyosin Cross-Bridge Kinetics of Human beta-Cardiac Myosin PLOS ONE Sommese, R. F., Nag, S., Sutton, S., Miller, S. M., Spudich, J. A., Ruppel, K. M. 2013; 8 (12)

  Abstract

  Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca(2+)-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca(2+) sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca(2+) induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.

  View details for DOI 10.1371/journal.pone.0083403

  View details for Web of Science ID 000328740300103

  View details for PubMedID 24367593

  View details for PubMedCentralID PMC3867432

• Memories of Hugh E. Huxley (1924-2013). Molecular biology of the cell Spudich, J. 2013; 24 (18): 2769-71

  View details for DOI 10.1091/mbc.E13-08-0454

  View details for PubMedID 24030511

  View details for PubMedCentralID PMC3771940

• Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human beta-cardiac myosin motor function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sommese, R. F., Sung, J., Nag, S., Sutton, S., Deacon, J. C., Choe, E., Leinwand, L. A., Ruppel, K., Spudich, J. A. 2013; 110 (31): 12607-12612

  Abstract

  Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

  View details for DOI 10.1073/pnas.1309493110

  View details for Web of Science ID 000322441500036

  View details for PubMedID 23798412

• Single-molecule fluorescence imaging of processive myosin with enhanced background suppression using linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC) OPTICS EXPRESS Elting, M. W., Leslie, S. R., Churchman, L. S., Korlach, J., McFaul, C. M., Leith, J. S., Levene, M. J., Cohen, A. E., Spudich, J. A. 2013; 21 (1): 1189-1202

  Abstract

  Resolving single fluorescent molecules in the presence of high fluorophore concentrations remains a challenge in single-molecule biophysics that limits our understanding of weak molecular interactions. Total internal reflection fluorescence (TIRF) imaging, the workhorse of single-molecule fluorescence microscopy, enables experiments at concentrations up to about 100 nM, but many biological interactions have considerably weaker affinities, and thus require at least one species to be at micromolar or higher concentration. Current alternatives to TIRF often require three-dimensional confinement, and thus can be problematic for extended substrates, such as cytoskeletal filaments. To address this challenge, we have demonstrated and applied two new single-molecule fluorescence microscopy techniques, linear zero-mode waveguides (ZMWs) and convex lens induced confinement (CLIC), for imaging the processive motion of molecular motors myosin V and VI along actin filaments. Both technologies will allow imaging in the presence of higher fluorophore concentrations than TIRF microscopy. They will enable new biophysical measurements of a wide range of processive molecular motors that move along filamentous tracks, such as other myosins, dynein, and kinesin. A particularly salient application of these technologies will be to examine chemomechanical coupling by directly imaging fluorescent nucleotide molecules interacting with processive motors as they traverse their actin or microtubule tracks.

  View details for DOI 10.1364/OE.21.001189

  View details for Web of Science ID 000315988100142

  View details for PubMedID 23389011

  View details for PubMedCentralID PMC3632498

• Future Challenges in Single-Molecule Fluorescence and Laser Trap Approaches to Studies of Molecular Motors DEVELOPMENTAL CELL Elting, M. W., Spudich, J. A. 2012; 23 (6): 1084-1091

  Abstract

  Single-molecule analysis is a powerful modern form of biochemistry, in which individual kinetic steps of a catalytic cycle of an enzyme can be explored in exquisite detail. Both single-molecule fluorescence and single-molecule force techniques have been widely used to characterize a number of protein systems. We focus here on molecular motors as a paradigm. We describe two areas where we expect to see exciting developments in the near future: first, characterizing the coupling of force production to chemical and mechanical changes in motors, and second, understanding how multiple motors work together in the environment of the cell.

  View details for DOI 10.1016/j.devcel.2012.10.002

  View details for Web of Science ID 000312429200002

  View details for PubMedID 23237942

• Cell-Intrinsic Functional Effects of the alpha-Cardiac Myosin Arg-403-Gln Mutation in Familial Hypertrophic Cardiomyopathy BIOPHYSICAL JOURNAL Chuan, P., Sivaramakrishnan, S., Ashley, E. A., Spudich, J. A. 2012; 102 (12): 2782-2790

  Abstract

  Human familial hypertrophic cardiomyopathy is the most common Mendelian cardiovascular disease worldwide. Among the most severe presentations of the disease are those in families heterozygous for the mutation R403Q in β-cardiac myosin. Mice heterozygous for this mutation in the α-cardiac myosin isoform display typical familial hypertrophic cardiomyopathy pathology. Here, we study cardiomyocytes from heterozygous 403/+ mice. The effects of the R403Q mutation on force-generating capabilities and dynamics of cardiomyocytes were investigated using a dual carbon nanofiber technique to measure single-cell parameters. We demonstrate the Frank-Starling effect at the single cardiomyocyte level by showing that cell stretch causes an increase in amplitude of contraction. Mutant 403/+ cardiomyocytes exhibit higher end-diastolic and end-systolic stiffness than +/+ cardiomyocytes, whereas active force generation capabilities remain unchanged. Additionally, 403/+ cardiomyocytes show slowed relaxation dynamics. These phenotypes are consistent with increased end-diastolic and end-systolic chamber elastance, as well as diastolic dysfunction seen at the level of the whole heart. Our results show that these functional effects of the R403Q mutation are cell-intrinsic, a property that may be a general phenomenon in familial hypertrophic cardiomyopathy.

  View details for DOI 10.1016/j.bpj.2012.04.049

  View details for Web of Science ID 000305546500012

  View details for PubMedID 22735528

  View details for PubMedCentralID PMC3379014

• The myosin superfamily at a glance JOURNAL OF CELL SCIENCE Hartman, M. A., Spudich, J. A. 2012; 125 (7): 1627-1632

  View details for DOI 10.1242/jcs.094300

  View details for Web of Science ID 000303911300002

  View details for PubMedID 22566666

  View details for PubMedCentralID PMC3346823

• Colocalization of fluorescent probes: accurate and precise registration with nanometer resolution. Cold Spring Harbor protocols Churchman, L. S., Spudich, J. A. 2012; 2012 (2): 141-149

  Abstract

  Colocalization of fluorescent probes is commonly used in cell biology to discern the proximity of two proteins in the cell. Considering that the resolution limit of optical microscopy is on the order of 250 nm, there has not been a need for high-resolution colocalization techniques. However, with the advent of higher resolution techniques for cell biology and single-molecule biophysics, colocalization must also improve. For diffraction-limited applications, a geometric transformation (i.e., translation, scaling, and rotation) is typically applied to one color channel to align it with the other; however, to achieve high-resolution colocalization, this is not sufficient. Single-molecule high-resolution colocalization (SHREC) of single probes uses the local weighted mean transformation to achieve a colocalization resolution of at least 10 nm. This article describes the process of collecting a calibration data set of fiducials and the appropriate analysis to determine the transformation for colocalization.

  View details for DOI 10.1101/pdb.top067918

  View details for PubMedID 22301660

• Single-molecule high-resolution colocalization of single probes. Cold Spring Harbor protocols Churchman, L. S., Spudich, J. A. 2012; 2012 (2): 242-245

  Abstract

  Colocalization of fluorescent probes is commonly used in cell biology to discern the proximity of two proteins in the cell. Considering that the resolution limit of optical microscopy is on the order of 250 nm, there has not been a need for high-resolution colocalization techniques. However, with the advent of higher resolution techniques for cell biology and single-molecule biophysics, colocalization must also improve. For diffraction-limited applications, a geometric transformation (i.e., translation, scaling, and rotation) is typically applied to one color channel to align it with the other; however, to achieve high-resolution colocalization, this is not sufficient. Single-molecule high-resolution colocalization (SHREC) of single probes uses the local weighted mean transformation to achieve a colocalization resolution of at least 10 nm. This protocol describes the acquisition of registration data and the analysis required to obtain a high-resolution mapping between imaging channels. The total internal reflection fluorescence microscope (TIRFM) system described is designed to excite and image the fluorescent probes Cy3 and Cy5. Modifications may be required depending on the requirements of the individual study.

  View details for DOI 10.1101/pdb.prot067926

  View details for PubMedID 22301661

• Integrative structural modelling of the cardiac thin filament: energetics at the interface and conservation patterns reveal a spotlight on period 2 of tropomyosin. Bioinformatics and biology insights Margaret Sunitha, S., Mercer, J. A., Spudich, J. A., Sowdhamini, R. 2012; 6: 203-223

  Abstract

  Cardiomyopathies are a major health problem, with inherited cardiomyopathies, many of which are caused by mutations in genes encoding sarcomeric proteins, constituting an ever-increasing fraction of cases. To begin to study the mechanisms by which these mutations cause disease, we have employed an integrative modelling approach to study the interactions between tropomyosin and actin. Starting from the existing blocked state model, we identified a specific zone on the actin surface which is highly favourable to support tropomyosin sliding from the blocked/closed states to the open state. We then analysed the predicted actin-tropomyosin interface regions for the three states. Each quasi-repeat of tropomyosin was studied for its interaction strength and evolutionary conservation to focus on smaller surface zones. Finally, we show that the distribution of the known cardiomyopathy mutations of α-tropomyosin is consistent with our model. This analysis provides structural insights into the possible mode of interactions between tropomyosin and actin in the open state for the first time.

  View details for DOI 10.4137/BBI.S9798

  View details for PubMedID 23071391

• Structural and functional insights on the Myosin superfamily. Bioinformatics and biology insights Syamaladevi, D. P., Spudich, J. A., Sowdhamini, R. 2012; 6: 11-21

  Abstract

  The myosin superfamily is a versatile group of molecular motors involved in the transport of specific biomolecules, vesicles and organelles in eukaryotic cells. The processivity of myosins along an actin filament and transport of intracellular 'cargo' are achieved by generating physical force from chemical energy of ATP followed by appropriate conformational changes. The typical myosin has a head domain, which harbors an ATP binding site, an actin binding site, and a light-chain bound 'lever arm', followed often by a coiled coil domain and a cargo binding domain. Evolution of myosins started at the point of evolution of eukaryotes, S. cerevisiae being the simplest one known to contain these molecular motors. The coiled coil domain of the myosin classes II, V and VI in whole genomes of several model organisms display differences in the length and the strength of interactions at the coiled coil interface. Myosin II sequences have long-length coiled coil regions that are predicted to have a highly stable dimeric interface. These are interrupted, however, by regions that are predicted to be unstable, indicating possibilities of alternate conformations, associations to make thick filaments, and interactions with other molecules. Myosin V sequences retain intermittent regions of strong and weak interactions, whereas myosin VI sequences are relatively devoid of strong coiled coil motifs. Structural deviations at coiled coil regions could be important for carrying out normal biological function of these proteins.

  View details for DOI 10.4137/BBI.S8451

  View details for PubMedID 22399849

• Systematic control of protein interaction using a modular ER/K alpha-helix linker PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sivaramakrishnan, S., Spudich, J. A. 2011; 108 (51): 20467-20472

  Abstract

  Cellular functions of proteins are strongly influenced by their interactions with other proteins. The frequency of protein interactions is a function of the local concentration of two proteins and their affinity for one another. When two proteins are tethered together, the link between them influences their effective concentrations and therefore the frequency of their interaction. Currently no methods exist to systematically vary the effective concentration within this intramolecular interaction. Here we outline a modular, genetically encoded linker, namely, an ER/K [genetically encoded polypeptide motif based on alternating sequence of approximately four glutamic acid (E) followed by approximately four arginine (R) or lysine (K) residues] single α-helix that can be used to regulate the frequency of interaction between two proteins, or between a protein and a peptide, one at each end. We exploit the wide range of interaction affinities between calmodulin and its binding peptides, combined with FRET to determine the effect of the ER/K α-helix in regulating protein interactions. We find that increasing the length of the ER/K α-helix reduces the on rate of the intramolecular interaction without significantly affecting the off rate, regardless of the affinity of the bimolecular interaction. We outline a genetically encoded approach to determine the dissociation constant for both moderate (micromolar K(d)) and strong (nanomolar K(d)) protein interactions. Our studies demonstrate the use of the ER/K α-helix to systematically engineer FRET biosensors that detect changes in concentration or affinity of interacting proteins, and modulate enzyme autoinhibition. Our findings are consistent with the ER/K α-helix as a worm-like chain with rare, stochastic breaks in the helix backbone that may account for the behavior of myosin VI stepping along actin.

  View details for DOI 10.1073/pnas.1116066108

  View details for Web of Science ID 000298289400047

  View details for PubMedID 22123984

  View details for PubMedCentralID PMC3251109

• Molecular motors: forty years of interdisciplinary research MOLECULAR BIOLOGY OF THE CELL Spudich, J. A. 2011; 22 (21): 3936-3939

  Abstract

  A mere forty years ago it was unclear what motor molecules exist in cells that could be responsible for the variety of nonmuscle cell movements, including the "saltatory cytoplasmic particle movements" apparent by light microscopy. One wondered whether nonmuscle cells might have a myosin-like molecule, well known to investigators of muscle. Now we know that there are more than a hundred different molecular motors in eukaryotic cells that drive numerous biological processes and organize the cell's dynamic city plan. Furthermore, in vitro motility assays, taken to the single-molecule level using techniques of physics, have allowed detailed characterization of the processes by which motor molecules transduce the chemical energy of ATP hydrolysis into mechanical movement. Molecular motor research is now at an exciting threshold of being able to enter into the realm of clinical applications.

  View details for DOI 10.1091/mbc.E11-05-0447

  View details for Web of Science ID 000296603300008

  View details for PubMedID 22039067

  View details for PubMedCentralID PMC3204054

• Optical traps to study properties of molecular motors. Cold Spring Harbor protocols Spudich, J. A., Rice, S. E., Rock, R. S., Purcell, T. J., Warrick, H. M. 2011; 2011 (11): 1305-1318

  Abstract

  In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This article describes the use of optical traps to study processive and nonprocessive molecular motor proteins, focusing on the design of the instrument and the assays to characterize motility.

  View details for DOI 10.1101/pdb.top066662

  View details for PubMedID 22046048

• The optical trapping dumbbell assay for nonprocessive motors or motors that turn around filaments. Cold Spring Harbor protocols Spudich, J. A., Rice, S. E., Rock, R. S., Purcell, T. J., Warrick, H. M. 2011; 2011 (11): 1372-1374

  Abstract

  In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes the preparation of biotin-actin filaments and coverslips coated with polystyrene beads. These are then used in optical trapping dumbbell assays to study interactions between motors and filaments.

  View details for DOI 10.1101/pdb.prot066688

  View details for PubMedID 22046050

• Attachment of anti-GFP antibodies to microspheres for optical trapping experiments. Cold Spring Harbor protocols Spudich, J. A., Rice, S. E., Rock, R. S., Purcell, T. J., Warrick, H. M. 2011; 2011 (11): 1370-1371

  Abstract

  In vitro motility assays enabled the analysis of coupling between ATP hydrolysis and movement of myosin along actin filaments or kinesin along microtubules. Single-molecule assays using laser trapping have been used to obtain more detailed information about kinesins, myosins, and processive DNA enzymes. The combination of in vitro motility assays with laser-trap measurements has revealed detailed dynamic structural changes associated with the ATPase cycle. This protocol describes a method for attaching anti-GFP (green fluorescent protein) antibodies to microspheres. GFP-motor fusion proteins can then be adsorbed to the microspheres for use in single-molecule motility studies and optical trapping experiments.

  View details for DOI 10.1101/pdb.prot066670

  View details for PubMedID 22046049

• Proteomics approach to study the functions of Drosophila myosin VI through identification of multiple cargo-binding proteins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Finan, D., Hartman, A., Spudich, J. A. 2011; 108 (14): 5566-5571

  Abstract

  Myosin VI is a molecular motor implicated in many processes, and it likely associates with a variety of cargoes that specify its functions. Although it is critical to Drosophila development, little is known about its cellular roles. To reveal its involvement in specific pathways, we sought to identify the binding partners of Drosophila myosin VI. We used affinity chromatography and mass spectrometry to discover interacting proteins, which we tested for direct binding. Using this approach, we found that the microtubule-associated protein Cornetto bound myosin VI, and we demonstrated a role for both in secretion of the lipidated morphogen Hedgehog. We also identified a number of other binding proteins, and further characterization of their interactions with myosin VI will advance our understanding of the roles of these complexes in cellular and developmental processes. Thus, our method has provided us the means to gain valuable insight into the multifaceted roles of a motor protein in vivo.

  View details for DOI 10.1073/pnas.1101415108

  View details for Web of Science ID 000289265300020

  View details for PubMedID 21368190

  View details for PubMedCentralID PMC3078346

• Nucleotide Pocket Thermodynamics Measured by EPR Reveal How Energy Partitioning Relates Myosin Speed to Efficiency JOURNAL OF MOLECULAR BIOLOGY Purcell, T. J., Naber, N., Franks-Skiba, K., Dunn, A. R., Eldred, C. C., Berger, C. L., Malnasi-Csizmadia, A., Spudich, J. A., Swank, D. M., Pate, E., Cooke, R. 2011; 407 (1): 79-91

  Abstract

  We have used spin-labeled ADP to investigate the dynamics of the nucleotide-binding pocket in a series of myosins, which have a range of velocities. Electron paramagnetic resonance spectroscopy reveals that the pocket is in equilibrium between open and closed conformations. In the absence of actin, the closed conformation is favored. When myosin binds actin, the open conformation becomes more favored, facilitating nucleotide release. We found that faster myosins favor a more closed pocket in the actomyosin•ADP state, with smaller values of ΔH(0) and ΔS(0), even though these myosins release ADP at a faster rate. A model involving a partitioning of free energy between work-generating steps prior to rate-limiting ADP release explains both the unexpected correlation between velocity and opening of the pocket and the observation that fast myosins are less efficient than slow myosins.

  View details for DOI 10.1016/j.jmb.2010.11.053

  View details for Web of Science ID 000288725500007

  View details for PubMedID 21185304

  View details for PubMedCentralID PMC3347976

• Biochemistry. Molecular motors, beauty in complexity. Science Spudich, J. A. 2011; 331 (6021): 1143-1144

  View details for DOI 10.1126/science.1203978

  View details for PubMedID 21385703

• Detailed Tuning of Structure and Intramolecular Communication Are Dispensable for Processive Motion of Myosin VI BIOPHYSICAL JOURNAL Elting, M. W., Bryant, Z., Liao, J., Spudich, J. A. 2011; 100 (2): 430-439

  Abstract

  Dimeric myosin VI moves processively hand-over-hand along actin filaments. We have characterized the mechanism of this processive motion by measuring the impact of structural and chemical perturbations on single-molecule processivity. Processivity is maintained despite major alterations in lever arm structure, including replacement of light chain binding regions and elimination of the medial tail. We present kinetic models that can explain the ATP concentration-dependent processivities of myosin VI constructs containing either native or artificial lever arms. We conclude that detailed tuning of structure and intramolecular communication are dispensable for processive motion, and further show theoretically that one proposed type of nucleotide gating can be detrimental rather than beneficial for myosin processivity.

  View details for DOI 10.1016/j.bpj.2010.11.045

  View details for Web of Science ID 000286543600020

  View details for PubMedID 21244839

  View details for PubMedCentralID PMC3021667

• Robust Mechanosensing and Tension Generation by Myosin VI JOURNAL OF MOLECULAR BIOLOGY Chuan, P., Spudich, J. A., Dunn, A. R. 2011; 405 (1): 105-112

  Abstract

  Myosin VI is a molecular motor that is thought to function both as a transporter and as a cytoskeletal anchor in vivo. Here we use optical tweezers to examine force generation by single molecules of myosin VI under physiological nucleotide concentrations. We find that myosin VI is an efficient transporter at loads of up to ∼2 pN but acts as a cytoskeletal anchor at higher loads. Our data and the resulting model are consistent with an indirect coupling of global structural motions to nucleotide binding and release. The model provides a mechanism by which load may regulate the dual functions of myosin VI in vivo. Our results suggest that myosin VI kinetics are tuned such that the motor maintains a consistent level of mechanical tension within the cell, a property potentially shared by other mechanosensitive proteins.

  View details for DOI 10.1016/j.jmb.2010.10.010

  View details for Web of Science ID 000286700800011

  View details for PubMedID 20970430

  View details for PubMedCentralID PMC3200311

• Principles of Unconventional Myosin Function and Targeting ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 27 Hartman, M. A., Finan, D., Sivaramakrishnan, S., Spudich, J. A. 2011; 27: 133-155

  Abstract

  Unconventional myosins are a superfamily of actin-based motors implicated in diverse cellular processes. In recent years, much progress has been made in describing their biophysical properties, and headway has been made into analyzing their cellular functions. Here, we focus on the principles that guide in vivo motor function and targeting to specific cellular locations. Rather than describe each motor comprehensively, we outline the major themes that emerge from research across the superfamily and use specific examples to illustrate each. In presenting the data in this format, we seek to identify open questions in each field as well as to point out commonalities between them. To advance our understanding of myosins' roles in vivo, clearly we must identify their cellular cargoes and the protein complexes that regulate motor attachment to fully appreciate their functions on the cellular and developmental levels.

  View details for DOI 10.1146/annurev-cellbio-100809-151502

  View details for Web of Science ID 000299230700006

  View details for PubMedID 21639800

• HCM and DCM causing mutations affect the velocity and force producing capacity of human beta-cardiac myosin Annual Meeting of the American-Society-for-Cell-Biology (ASCB) Ruppel, K., Choe, E., Elting, M. W., Sung, J., Shaklee, P., Sutton, S., Leinwand, L. A., Spudich, J. A. AMER SOC CELL BIOLOGY. 2011

  View details for Web of Science ID 000305505500295

• Helicity of short E-R/K peptides PROTEIN SCIENCE Sommese, R. F., Sivaramakrishnan, S., Baldwin, R. L., Spudich, J. A. 2010; 19 (10): 2001-2005

  Abstract

  Understanding the secondary structure of peptides is important in protein folding, enzyme function, and peptide-based drug design. Previous studies of synthetic Ala-based peptides (>12 a.a.) have demonstrated the role for charged side chain interactions involving Glu/Lys or Glu/Arg spaced three (i, i + 3) or four (i, i + 4) residues apart. The secondary structure of short peptides (<9 a.a.), however, has not been investigated. In this study, the effect of repetitive Glu/Lys or Glu/Arg side chain interactions, giving rise to E-R/K helices, on the helicity of short peptides was examined using circular dichroism. Short E-R/K-based peptides show significant helix content. Peptides containing one or more E-R interactions display greater helicity than those with similar E-K interactions. Significant helicity is achieved in Arg-based E-R/K peptides eight, six, and five amino acids long. In these short peptides, each additional i + 3 and i + 4 salt bridge has substantial contribution to fractional helix content. The E-R/K peptides exhibit a strongly linear melt curve indicative of noncooperative folding. The significant helicity of these short peptides with predictable dependence on number, position, and type of side chain interactions makes them an important consideration in peptide design.

  View details for DOI 10.1002/pro.469

  View details for Web of Science ID 000282716900019

  View details for PubMedID 20669185

  View details for PubMedCentralID PMC2998735

• Determinants of Myosin II Cortical Localization during Cytokinesis CURRENT BIOLOGY Uehara, R., Goshima, G., Mabuchi, I., Vale, R. D., Spudich, J. A., Griffis, E. R. 2010; 20 (12): 1080-1085

  Abstract

  Myosin II is an essential component of the contractile ring that divides the cell during cytokinesis. Previous work showed that regulatory light chain (RLC) phosphorylation is required for localization of myosin at the cellular equator. However, the molecular mechanisms that concentrate myosin at the site of furrow formation remain unclear. By analyzing the spatiotemporal dynamics of mutant myosin subunits in Drosophila S2 cells, we show that myosin accumulates at the equator through stabilization of interactions between the cortex and myosin filaments and that the motor domain is dispensable for localization. Filament stabilization is tightly controlled by RLC phosphorylation. However, we show that regulatory mechanisms other than RLC phosphorylation contribute to myosin accumulation at three different stages: (1) turnover of thick filaments throughout the cell cycle, (2) myosin heavy chain-based control of myosin assembly at the metaphase-anaphase transition, and (3) redistribution and/or activation of myosin binding sites at the equator during anaphase. Surprisingly, the third event can occur to a degree in a Rho-independent fashion, gathering preassembled filaments to the equatorial zone via cortical flow. We conclude that multiple regulatory pathways cooperate to control myosin localization during mitosis and cytokinesis to ensure that this essential biological process is as robust as possible.

  View details for DOI 10.1016/j.cub.2010.04.058

  View details for Web of Science ID 000279304000022

  View details for PubMedID 20541410

• Optimized localization analysis for single-molecule tracking and super-resolution microscopy NATURE METHODS Mortensen, K. I., Churchman, L. S., Spudich, J. A., Flyvbjerg, H. 2010; 7 (5): 377-U59

  Abstract

  We optimally localized isolated fluorescent beads and molecules imaged as diffraction-limited spots, determined the orientation of molecules and present reliable formulas for the precision of various localization methods. Both theory and experimental data showed that unweighted least-squares fitting of a Gaussian squanders one-third of the available information, a popular formula for its precision exaggerates beyond Fisher's information limit, and weighted least-squares may do worse, whereas maximum-likelihood fitting is practically optimal.

  View details for DOI 10.1038/NMETH.1447

  View details for Web of Science ID 000277175100019

  View details for PubMedID 20364147

  View details for PubMedCentralID PMC3127582

• Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dunn, A. R., Chuan, P., Bryant, Z., Spudich, J. A. 2010; 107 (17): 7746-7750

  Abstract

  Myosin VI is proposed to act as both a molecular transporter and as an anchor in vivo. A portion of the molecule C-terminal to the canonical lever arm, termed the medial tail (MT), has been proposed to act as either a lever arm extension or as a dimerization motif. We describe constructs in which the MT is interrupted by a glycine-rich molecular swivel. Disruption of the MT results in decreased processive run lengths measured using single-molecule fluorescence microscopy and a decreased step size under applied load as measured in an optical trap. We used single-molecule gold nanoparticle tracking and optical trapping to examine the mechanism of coordination between the heads of dimeric myosin VI. We detect two rate-limiting kinetic processes at low (< 200 micromolar) ATP concentrations. Our data can be explained by a model in which intramolecular tension greatly increases the affinity of the lead head for ADP, likely by slowing ADP release from the lead head. This mechanism likely increases both the motor's processivity and its ability to act as an anchor under physiological conditions.

  View details for DOI 10.1073/pnas.1002430107

  View details for Web of Science ID 000277088700028

  View details for PubMedID 20385849

  View details for PubMedCentralID PMC2867888

• Myosin VI: an innovative motor that challenged the swinging lever arm hypothesis NATURE REVIEWS MOLECULAR CELL BIOLOGY Spudich, J. A., Sivaramakrishnan, S. 2010; 11 (2): 128-137

  Abstract

  The swinging crossbridge hypothesis states that energy from ATP hydrolysis is transduced to mechanical movement of the myosin head while bound to actin. The light chain-binding region of myosin is thought to act as a lever arm that amplifies movements near the catalytic site. This model has been challenged by findings that myosin VI takes larger steps along actin filaments than early interpretations of its structure seem to allow. We now know that myosin VI does indeed operate by an unusual approximately 180 degrees lever arm swing and achieves its large step size using special structural features in its tail domain.

  View details for DOI 10.1038/nrm2833

  View details for Web of Science ID 000273811200013

  View details for PubMedID 20094053

  View details for PubMedCentralID PMC2859320

• Functional diversity among a family of human skeletal muscle myosin motors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Resnicow, D. I., Deacon, J. C., Warrick, H. M., Spudich, J. A., Leinwand, L. A. 2010; 107 (3): 1053-1058

  Abstract

  Human skeletal muscle fibers express five highly conserved type-II myosin heavy chain (MyHC) genes in distinct spatial and temporal patterns. In addition, the human genome contains an intact sixth gene, MyHC-IIb, which is thought under most circumstances not to be expressed. The physiological and biochemical properties of individual muscle fibers correlate with the predominantly expressed MyHC isoform, but a functional analysis of homogenous skeletal muscle myosin isoforms has not been possible. This is due to the difficulties of separating the multiple isoforms usually coexpressed in muscle fibers, as well as the lack of an expression system that produces active recombinant type II skeletal muscle myosin. In this study we describe a mammalian muscle cell expression system and the functional analysis of all six recombinant human type II skeletal muscle myosin isoforms. The diverse biochemical activities and actin-filament velocities of these myosins indicate that they likely have distinct functions in muscle. Our data also show that ATPase activity and motility are generally correlated for human skeletal muscle myosins. The exception, MyHC-IIb, encodes a protein that is high in ATPase activity but slow in motility; this is the first functional analysis of the protein from this gene. In addition, the developmental isoforms, hypothesized to have low ATPase activity, were indistinguishable from adult-fast MyHC-IIa and the specialized MyHC-Extraocular isoform, that was predicted to be the fastest of all six isoforms but was functionally similar to the slower isoforms.

  View details for DOI 10.1073/pnas.0913527107

  View details for Web of Science ID 000273934100021

  View details for PubMedID 20080549

• SINGLE-MOLECULE DUAL-BEAM OPTICAL TRAP ANALYSIS OF PROTEIN STRUCTURE AND FUNCTION METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B Sung, J., Sivaramakrishnan, S., Dunn, A. R., Spudich, J. A. 2010; 475: 321-375

  Abstract

  Optical trapping is one of the most powerful single-molecule techniques. We provide a practical guide to set up and use an optical trap, applied to the molecular motor myosin as an example. We focus primarily on studies of myosin function using a dual-beam optical trap, a protocol to build such a trap, and the experimental and data analysis protocols to utilize it.

  View details for DOI 10.1016/S0076-6879(10)75014-X

  View details for Web of Science ID 000280733800014

  View details for PubMedID 20627164

• Combining Single-Molecule Optical Trapping and Small-Angle X-Ray Scattering Measurements to Compute the Persistence Length of a Protein ER/K alpha-Helix BIOPHYSICAL JOURNAL Sivaramakrishnan, S., Sung, J., Ali, M., Doniach, S., Flyvbjerg, H., Spudich, J. A. 2009; 97 (11): 2993-2999

  Abstract

  A relatively unknown protein structure motif forms stable isolated single alpha-helices, termed ER/K alpha-helices, in a wide variety of proteins and has been shown to be essential for the function of some molecular motors. The flexibility of the ER/K alpha-helix determines whether it behaves as a force transducer, rigid spacer, or flexible linker in proteins. In this study, we quantify this flexibility in terms of persistence length, namely the length scale over which it is rigid. We use single-molecule optical trapping and small-angle x-ray scattering, combined with Monte Carlo simulations to demonstrate that the Kelch ER/K alpha-helix behaves as a wormlike chain with a persistence length of approximately 15 nm or approximately 28 turns of alpha-helix. The ER/K alpha-helix length in proteins varies from 3 to 60 nm, with a median length of approximately 5 nm. Knowledge of its persistence length enables us to define its function as a rigid spacer in a translation initiation factor, as a force transducer in the mechanoenzyme myosin VI, and as a flexible spacer in the Kelch-motif-containing protein.

  View details for DOI 10.1016/j.bpj.2009.09.009

  View details for PubMedID 19948129

• Insights into Human beta-Cardiac Myosin Function from Single Molecule and Single Cell Studies JOURNAL OF CARDIOVASCULAR TRANSLATIONAL RESEARCH Sivaramakrishnan, S., Ashley, E., Leinwand, L., Spudich, J. A. 2009; 2 (4): 426-440

  Abstract

  beta-Cardiac myosin is a mechanoenzyme that converts the energy from ATP hydrolysis into a mechanical force that drives contractility in muscle. Thirty percent of the point mutations that result in hypertrophic cardiomyopathy are localized to MYH7, the gene encoding human beta-cardiac myosin heavy chain (beta-MyHC). Force generation by myosins requires a tight and highly conserved allosteric coupling between its different protein domains. Hence, the effects of single point mutations on the force generation and kinetics of beta-cardiac myosin molecules cannot be predicted directly from their location within the protein structure. Great insight would be gained from understanding the link between the functional defect in the myosin protein and the clinical phenotypes of patients expressing them. Over the last decade, several single molecule techniques have been developed to understand in detail the chemomechanical cycle of different myosins. In this review, we highlight the single molecule techniques that can be used to assess the effect of point mutations on beta-cardiac myosin function. Recent bioengineering advances have enabled the micromanipulation of single cardiomyocyte cells to characterize their force-length dynamics. Here, we briefly review single cell micromanipulation as an approach to determine the effect of beta-MyHC mutations on cardiomyocyte function. Finally, we examine the technical challenges specific to studying beta-cardiac myosin function both using single molecule and single cell approaches.

  View details for DOI 10.1007/s12265-009-9129-2

  View details for Web of Science ID 000284691000010

  View details for PubMedID 20560001

• Coupled myosin VI motors facilitate unidirectional movement on an F-actin network JOURNAL OF CELL BIOLOGY Sivaramakrishnan, S., Spudich, J. A. 2009; 187 (1): 53-60

  Abstract

  Unconventional myosins interact with the dense cortical actin network during processes such as membrane trafficking, cell migration, and mechanotransduction. Our understanding of unconventional myosin function is derived largely from assays that examine the interaction of a single myosin with a single actin filament. In this study, we have developed a model system to study the interaction between multiple tethered unconventional myosins and a model F-actin cortex, namely the lamellipodium of a migrating fish epidermal keratocyte. Using myosin VI, which moves toward the pointed end of actin filaments, we directly determine the polarity of the extracted keratocyte lamellipodium from the cell periphery to the cell nucleus. We use a combination of experimentation and simulation to demonstrate that multiple myosin VI molecules can coordinate to efficiently transport vesicle-size cargo over 10 microm of the dense interlaced actin network. Furthermore, several molecules of monomeric myosin VI, which are nonprocessive in single molecule assays, can coordinate to transport cargo with similar speeds as dimers.

  View details for DOI 10.1083/jcb.200906133

  View details for Web of Science ID 000270452800008

  View details for PubMedID 19786577

  View details for PubMedCentralID PMC2762089

• Engineered Myosin VI Motors Reveal Minimal Structural Determinants of Directionality and Processivity JOURNAL OF MOLECULAR BIOLOGY Liao, J., Elting, M. W., Delp, S. L., Spudich, J. A., Bryant, Z. 2009; 392 (4): 862-867

  Abstract

  Myosins have diverse mechanical properties reflecting a range of cellular roles. A major challenge is to understand the structural basis for generating novel functions from a common motor core. Myosin VI (M6) is specialized for processive motion toward the (-) end of actin filaments. We have used engineered M6 motors to test and refine the "redirected power stroke" model for (-) end directionality and to explore poorly understood structural requirements for processive stepping. Guided by crystal structures and molecular modeling, we fused artificial lever arms to the catalytic head of M6 at several positions, retaining varying amounts of native structure. We found that an 18-residue alpha-helical insert is sufficient to reverse the directionality of the motor, with no requirement for any calmodulin light chains. Further, we observed robust processive stepping of motors with artificial lever arms, demonstrating that processivity can arise without optimizing lever arm composition or mechanics.

  View details for DOI 10.1016/j.jmb.2009.07.046

  View details for Web of Science ID 000270601200002

  View details for PubMedID 19631216

  View details for PubMedCentralID PMC3360974

• Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells JOURNAL OF CELL BIOLOGY Vale, R. D., Spudich, J. A., Griffis, E. R. 2009; 186 (5): 727-738

  Abstract

  Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein-tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.

  View details for DOI 10.1083/jcb.200902083

  View details for Web of Science ID 000269575700012

  View details for PubMedID 19720876

• Velocity, Processivity, and Individual Steps of Single Myosin V Molecules in Live Cells BIOPHYSICAL JOURNAL Pierobon, P., Achouri, S., Courty, S., Dunn, A. R., Spudich, J. A., Dahan, M., Cappello, G. 2009; 96 (10): 4268-4275

  Abstract

  We report the tracking of single myosin V molecules in their natural environment, the cell. Myosin V molecules, labeled with quantum dots, are introduced into the cytoplasm of living HeLa cells and their motion is recorded at the single molecule level with high spatial and temporal resolution. We perform an intracellular measurement of key parameters of this molecular transporter: velocity, processivity, step size, and dwell time. Our experiments bridge the gap between in vitro single molecule assays and the indirect measurements of the motor features deduced from the tracking of organelles in live cells.

  View details for DOI 10.1016/j.bpj.2009.02.045

  View details for Web of Science ID 000266312900038

  View details for PubMedID 19450497

  View details for PubMedCentralID PMC2712235

• Dynamic Organization of Gene Loci and Transcription Compartments in the Cell Nucleus BIOPHYSICAL JOURNAL Spudich, J. A. 2008; 95 (11): 5003-5004

  View details for DOI 10.1529/biophysj.108.139196

  View details for Web of Science ID 000260999500001

  View details for PubMedID 18805930

  View details for PubMedCentralID PMC2586552

• Dynamic charge interactions create surprising rigidity in the ER/K alpha-helical protein motif PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sivaramakrishnan, S., Spink, B. J., Sim, A. Y., Doniach, S., Spudich, J. A. 2008; 105 (36): 13356-13361

  Abstract

  Protein alpha-helices are ubiquitous secondary structural elements, seldom considered to be stable without tertiary contacts. However, amino acid sequences in proteins that are based on alternating repeats of four glutamic acid (E) residues and four positively charged residues, a combination of arginine (R) and lysine (K), have been shown to form stable alpha-helices in a few proteins, in the absence of tertiary interactions. Here, we find that this ER/K motif is more prevalent than previously reported, being represented in proteins of diverse function from archaea to humans. By using molecular dynamics (MD) simulations, we characterize a dynamic pattern of side-chain interactions that extends along the backbone of ER/K alpha-helices. A simplified model predicts that side-chain interactions alone contribute substantial bending rigidity (0.5 pN/nm) to ER/K alpha-helices. Results of small-angle x-ray scattering (SAXS) and single-molecule optical-trap analyses are consistent with the high bending rigidity predicted by our model. Thus, the ER/K alpha-helix is an isolated secondary structural element that can efficiently span long distances in proteins, making it a promising tool in designing synthetic proteins. We propose that the significant rigidity of the ER/K alpha-helix can help regulate protein function, as a force transducer between protein subdomains.

  View details for DOI 10.1073/pnas.0806256105

  View details for Web of Science ID 000259251700034

  View details for PubMedID 18768817

  View details for PubMedCentralID PMC2533194

• Long single alpha-helical tail domains bridge the gap between structure and function of myosin VI NATURE STRUCTURAL & MOLECULAR BIOLOGY Spink, B. J., Sivaramakrishnan, S., Lipfert, J., Doniach, S., Spudich, J. A. 2008; 15 (6): 591-597

  Abstract

  Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take approximately 36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single alpha-helix of approximately 10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the approximately 36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

  View details for DOI 10.1038/nsmb.1429

  View details for Web of Science ID 000256388900013

  View details for PubMedID 18511944

  View details for PubMedCentralID PMC2441774

• Molecular motors: A surprising twist in myosin VI translocation CURRENT BIOLOGY Spudich, J. A. 2008; 18 (2): R68-R70

  Abstract

  A recent study has revealed an unexpected change in conformation of the myosin VI converter domain, essential for twisting the lever arm through a approximately 180 degrees rotation to achieve a large step along actin.

  View details for DOI 10.1016/j.cub.2007.11.025

  View details for Web of Science ID 000252693900016

  View details for PubMedID 18211842

• Predicting allosteric communication in myosin via a pathway of conserved residues JOURNAL OF MOLECULAR BIOLOGY Tang, S., Liao, J., Dunn, A. R., Altman, R. B., Spudich, J. A., Schmidt, J. P. 2007; 373 (5): 1361-1373

  Abstract

  We present a computational method that predicts a pathway of residues that mediate protein allosteric communication. The pathway is predicted using only a combination of distance constraints between contiguous residues and evolutionary data. We applied this analysis to find pathways of conserved residues connecting the myosin ATP binding site to the lever arm. These pathway residues may mediate the allosteric communication that couples ATP hydrolysis to the lever arm recovery stroke. Having examined pre-stroke conformations of Dictyostelium, scallop, and chicken myosin II as well as Dictyostelium myosin I, we observed a conserved pathway traversing switch II and the relay helix, which is consistent with the understood need for allosteric communication in this conformation. We also examined post-rigor and rigor conformations across several myosin species. Although initial residues of these paths are more heterogeneous, all but one of these paths traverse a consistent set of relay helix residues to reach the beginning of the lever arm. We discuss our results in the context of structural elements and reported mutational experiments, which substantiate the significance of the pre-stroke pathways. Our method provides a simple, computationally efficient means of predicting a set of residues that mediate allosteric communication. We provide a refined, downloadable application and source code (on https://simtk.org) to share this tool with the wider community (https://simtk.org/home/allopathfinder).

  View details for DOI 10.1016/j.jmb.2007.08.059

  View details for Web of Science ID 000250712600021

  View details for PubMedID 17900617

  View details for PubMedCentralID PMC2128046

• The localization of inner centromeric protein (INCENP) at the cleavage furrow is dependent on Kif12 and involves interactions of the N terminus of INCENP with the actin cytoskeleton MOLECULAR BIOLOGY OF THE CELL Chen, Q., Lakshmikanth, G. S., Spudich, J. A., De Lozanne, A. 2007; 18 (9): 3366-3374

  Abstract

  The inner centromeric protein (INCENP) and other chromosomal passenger proteins are known to localize on the cleavage furrow and to play a role in cytokinesis. However, it is not known how INCENP localizes on the furrow or whether this localization is separable from that at the midbody. Here, we show that the association of Dictyostelium INCENP (DdINCENP) with the cortex of the cleavage furrow involves interactions with the actin cytoskeleton and depends on the presence of the kinesin-6-related protein Kif12. We found that Kif12 is found on the central spindle and the cleavage furrow during cytokinesis. Kif12 is not required for the redistribution of DdINCENP from centromeres to the central spindle. However, in the absence of Kif12, DdINCENP fails to localize on the cleavage furrow. Domain analysis indicates that the N terminus of DdINCENP is necessary and sufficient for furrow localization and that it binds directly to the actin cytoskeleton. Our data suggest that INCENP moves from the central spindle to the furrow of a dividing cell by a Kif12-dependent pathway. Once INCENP reaches the equatorial cortex, it associates with the actin cytoskeleton where it then concentrates toward the end of cytokinesis.

  View details for DOI 10.1091/mbc.E06-10-0895

  View details for Web of Science ID 000249162200012

  View details for PubMedID 17567958

  View details for PubMedCentralID PMC1951774

• PHYS 589-Dissecting the reverse power stroke of myosin VI Liao, J., Bryant, Z., Delp, S. L., Spudich, J. A. AMER CHEMICAL SOC. 2007

  View details for Web of Science ID 000207593907649

• PHYS 631-Tracking single myosin molecules with high temporal resolution and low applied load Dunn, A. R., Spudich, J. A. AMER CHEMICAL SOC. 2007

  View details for Web of Science ID 000207593907608

• Precise positioning of myosin VI on Endocytic vesicles in vivo PLOS BIOLOGY Altman, D., Goswami, D., Hasson, T., Spudich, J. A., Mayor, S. 2007; 5 (8): 1712-1722

  Abstract

  Myosin VI has been studied in both a monomeric and a dimeric form in vitro. Because the functional characteristics of the motor are dramatically different for these two forms, it is important to understand whether myosin VI heavy chains are brought together on endocytic vesicles. We have used fluorescence anisotropy measurements to detect fluorescence resonance energy transfer between identical fluorophores (homoFRET) resulting from myosin VI heavy chains being brought into close proximity. We observed that, when associated with clathrin-mediated endocytic vesicles, myosin VI heavy chains are precisely positioned to bring their tail domains in close proximity. Our data show that on endocytic vesicles, myosin VI heavy chains are brought together in an orientation that previous in vitro studies have shown causes dimerization of the motor. Our results are therefore consistent with vesicle-associated myosin VI existing as a processive dimer, capable of its known trafficking function.

  View details for DOI 10.1371/journal.pbio.0050210

  View details for Web of Science ID 000249124800009

  View details for PubMedID 17683200

  View details for PubMedCentralID PMC1939883

• Identification of a minimal myosin Va binding site within an intrinsically unstructured domain of melanophilin JOURNAL OF BIOLOGICAL CHEMISTRY Geething, N. C., Spudich, J. A. 2007; 282 (29): 21518-21528

  Abstract

  Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.

  View details for DOI 10.1074/jbc.M701932200

  View details for Web of Science ID 000248047500079

  View details for PubMedID 17513864

• Dynamics of the unbound head during myosin V processive translocation NATURE STRUCTURAL & MOLECULAR BIOLOGY Dunn, A. R., Spudich, J. A. 2007; 14 (3): 246-248

  Abstract

  Myosin V moves cargoes along actin filaments by walking hand over hand. Although numerous studies support the basic hand-over-hand model, little is known about the fleeting intermediate that occurs when the rear head detaches from the filament. Here we use submillisecond dark-field imaging of gold nanoparticle-labeled myosin V to directly observe the free head as it releases from the actin filament, diffuses forward and rebinds. We find that the unbound head rotates freely about the lever-arm junction, a trait that likely facilitates travel through crowded actin meshworks.

  View details for DOI 10.1038/nsmb1206

  View details for Web of Science ID 000244715200016

  View details for PubMedID 17293871

• Extending the absorbing boundary method to fit dwell-time distributions of molecular motors with complex kinetic pathways PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liao, J., Spudich, J. A., Parker, D., Delp, S. L. 2007; 104 (9): 3171-3176

  Abstract

  Dwell-time distributions, waiting-time distributions, and distributions of pause durations are widely reported for molecular motors based on single-molecule biophysical experiments. These distributions provide important information concerning the functional mechanisms of enzymes and their underlying kinetic and mechanical processes. We have extended the absorbing boundary method to simulate dwell-time distributions of complex kinetic schemes, which include cyclic, branching, and reverse transitions typically observed in molecular motors. This extended absorbing boundary method allows global fitting of dwell-time distributions for enzymes subject to different experimental conditions. We applied the extended absorbing boundary method to experimental dwell-time distributions of single-headed myosin V, and were able to use a single kinetic scheme to fit dwell-time distributions observed under different ligand concentrations and different directions of optical trap forces. The ability to use a single kinetic scheme to fit dwell-time distributions arising from a variety of experimental conditions is important for identifying a mechanochemical model of a molecular motor. This efficient method can be used to study dwell-time distributions for a broad class of molecular motors, including kinesin, RNA polymerase, helicase, F(1) ATPase, and to examine conformational dynamics of other enzymes such as ion channels.

  View details for DOI 10.1073/pnas.0611519104

  View details for Web of Science ID 000244661400029

  View details for PubMedID 17360624

  View details for PubMedCentralID PMC1805548

• Rho Kinase's Role in Myosin Recruitment to the Equatorial Cortex of Mitotic Drosophila S2 Cells Is for Myosin Regulatory Light Chain Phosphorylation PLOS ONE Dean, S. O., Spudich, J. A. 2006; 1 (2)

  Abstract

  Myosin II recruitment to the equatorial cortex is one of the earliest events in establishment of the cytokinetic contractile ring. In Drosophila S2 cells, we previously showed that myosin II is recruited to the furrow independently of F-actin, and that Rho1 and Rok are essential for this recruitment [1]. Rok phosphorylates several cellular proteins, including the myosin regulatory light chain (RLC).Here we express phosphorylation state mimic constructs of the RLC in S2 cells to examine the role of RLC phosphorylation involving Rok in the localization of myosin. Phosphorylation of the RLC is required for myosin localization to the equatorial cortex during mitosis, and the essential role of Rok in this localization and for cytokinesis is to maintain phosphorylation of the RLC. The ability to regulate the RLC phosphorylation state spatio-temporally is not essential for the myosin localization. Furthermore, the essential role of Citron in cytokinesis is not phosphorylation of the RLC.We conclude that the Rho1 pathway leading to myosin localization to the future cytokinetic furrow is relatively straightforward, where only Rok is needed, and it is only needed to maintain phosphorylation of the myosin RLC.

  View details for DOI 10.1371/journal.pone.0000131

  View details for Web of Science ID 000207443700025

  View details for PubMedID 17205135

  View details for PubMedCentralID PMC1762308

• Molecular motors take tension in stride CELL Spudich, J. A. 2006; 126 (2): 242-244

  Abstract

  Mechanical tension controls the function of a wide variety of eukaryotic motor proteins. Single-molecule analyses have revealed how some of these proteins sense and respond to tension. The single motor studies on dynein by Reck-Peterson et al (2006) described in this issue pave the way to understand molecular mechanisms used by this unique machine.

  View details for DOI 10.1016/j.cell.2006.07.009

  View details for Web of Science ID 000239552600008

  View details for PubMedID 16873054

• A non-Gaussian distribution quantifies distances measured with fluorescence localization techniques BIOPHYSICAL JOURNAL Churchman, L. S., Flyvbjerg, H., Spudich, J. A. 2006; 90 (2): 668-671

  Abstract

  When single-molecule fluorescence localization techniques are pushed to their lower limits in attempts to measure ever-shorter distances, measurement errors become important to understand. Here we describe the non-Gaussian distribution of measured distances that is the key to proper interpretation of distance measurements. We test it on single-molecule high-resolution colocalization data for a known distance, 10 nm, and find that it gives the correct result, whereas interpretation of the same data with a Gaussian distribution gives a result that is systematically too large.

  View details for DOI 10.1529/biophysj.105.065599

  View details for Web of Science ID 000234252100027

  View details for PubMedID 16258038

  View details for PubMedCentralID PMC1367071

• A force-dependent state controls the coordination of processive myosin V PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Purcell, T. J., Sweeney, H. L., Spudich, J. A. 2005; 102 (39): 13873-13878

  Abstract

  Myosin V is an efficient processive molecular motor. Recent experiments have shown how the structure and kinetics of myosin V are specialized to produce a highly processive motor capable of taking multiple 36-nm steps on an actin filament track. Here, we examine how two identical heads coordinate their activity to produce efficient hand-over-hand stepping. We have used a modified laser-trap microscope to apply a approximately 2-pN forward or backward force on a single-headed myosin V molecule, hypothesized to simulate forces experienced by the rear or lead head, respectively. We found that pulling forward produces only a small change in the kinetics, whereas pulling backward induces a large reduction in the cycling of the head. These results support a model in which the coordination of myosin V stepping is mediated by strain-generated inhibition of the lead head.

  View details for DOI 10.1073/pnas.0506441102

  View details for Web of Science ID 000232231900031

  View details for PubMedID 16150709

  View details for PubMedCentralID PMC1236568

• Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dean, S. O., Rogers, S. L., Stuurman, N., Vale, R. D., Spudich, J. A. 2005; 102 (38): 13473-13478

  Abstract

  The correct localization of myosin II to the equatorial cortex is crucial for proper cell division. Here, we examine a collection of genes that cause defects in cytokinesis and reveal with live cell imaging two distinct phases of myosin II localization. Three genes in the rho1 signaling pathway, pebble (a Rho guanidine nucleotide exchange factor), rho1, and rho kinase, are required for the initial recruitment of myosin II to the equatorial cortex. This initial localization mechanism does not require F-actin or the two components of the centralspindlin complex, the mitotic kinesin pavarotti/MKLP1 and racGAP50c/CYK-4. However, F-actin, the centralspindlin complex, formin (diaphanous), and profilin (chickadee) are required to stably maintain myosin II at the furrow. In the absence of these latter genes, myosin II delocalizes from the equatorial cortex and undergoes highly dynamic appearances and disappearances around the entire cell cortex, sometimes associated with abnormal contractions or blebbing. Our findings support a model in which a rho kinase-dependent event, possibly myosin II regulatory light chain phosphorylation, is required for the initial recruitment to the furrow, whereas the assembly of parallel, unbranched actin filaments, generated by formin-mediated actin nucleation, is required for maintaining myosin II exclusively at the equatorial cortex.

  View details for DOI 10.1073/pnas.0506810102

  View details for Web of Science ID 000232115100023

  View details for PubMedID 16174742

  View details for PubMedCentralID PMC1200093

• A flexible domain is essential for the large step size and processivity of myosin VI MOLECULAR CELL Rock, R. S., Ramamurthy, B., Dunn, A. R., Beccafico, S., Rami, B. R., Morris, C., Spink, B. J., Franzini-Armstrong, C., Spudich, J. A., Sweeney, H. L. 2005; 17 (4): 603-609

  Abstract

  Myosin VI moves processively along actin with a larger step size than expected from the size of the motor. Here, we show that the proximal tail (the approximately 80-residue segment following the IQ domain) is not a rigid structure but, rather, a flexible domain that permits the heads to separate. With a GCN4 coiled coil inserted in the proximal tail, the heads are closer together in electron microscopy (EM) images, and the motor takes shorter processive steps. Single-headed myosin VI S1 constructs take nonprocessive 12 nm steps, suggesting that most of the processive step is covered by a diffusive search for an actin binding site. Based on these results, we present a mechanical model that describes stepping under an applied load.

  View details for DOI 10.1016/j.molcel.2005.01.015

  View details for Web of Science ID 000227143400016

  View details for PubMedID 15721263

• A mitotic kinesin-like protein required for normal karyokinesis, myosin localization to the furrow, and cytokinesis in Dictyostelium PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lakshmikanth, G. S., Warrick, H. M., Spudich, J. A. 2004; 101 (47): 16519-16524

  Abstract

  Dictyostelium mitotic kinesin Kif12 is required for cytokinesis. Myosin II localization to the cleavage furrow is severely depressed in Kif12-null (Deltakif12) cells, which accounts in part for the cytokinesis failure. Myosin II-null cells, however, undergo mitosis-coupled cytokinesis when adhering to a surface, whereas the Deltakif12 cells cannot. During mitosis, the rate of change of internuclear separation in Deltakif12 cells is reduced compared with wild-type cells, indicating multiple roles of this molecular motor during mitosis and cytokinesis. GFP-Kif12, which rescues wild-type behavior when expressed in the Deltakif12 strain, is concentrated in the nucleus in interphase cells, translocates to the cytoplasm at the onset of mitosis, appears in the centrosomes and spindle, and later is concentrated in the spindle midbody. Given these results, we hypothesize a mechanism for myosin II translocation to the furrow to set up the contractile ring.

  View details for DOI 10.1073/pnas.0407304101

  View details for Web of Science ID 000225347400027

  View details for PubMedID 15546981

  View details for PubMedCentralID PMC528903

• Two important polymers cross paths PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Spudich, J. A. 2004; 101 (45): 15825-15826

  View details for DOI 10.1073/pnas.0406932101

  View details for Web of Science ID 000225196800001

  View details for PubMedID 15522968

  View details for PubMedCentralID PMC528761

• Dictyostelium myosin bipolar thick filament formation: Importance of charge and specific domains of the myosin rod PLOS BIOLOGY Hostetter, D., Rice, S., Dean, S., Altman, D., McMahon, P. M., Sutton, S., Tripathy, A., Spudich, J. A. 2004; 2 (11): 1880-1892

  Abstract

  Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) "head" is added to the N terminus. The localization of this GFP-AD-Cterm to the cleavage furrow of dividing Dictyostelium cells depends on assembly state, similar to full-length myosin-II. This tail fragment therefore represents a good model system for the regulated formation and localization of BTFs. By reducing regulated BTF assembly to a more manageable model system, we were able to explore determinants of myosin-II self-assembly. Our data support a model in which a globular head limits the size of a BTF, and the large-scale charge character of the AD-Cterm region is important for BTF formation. Truncation analysis of AD-Cterm tail fragments shows that assembly is delicately balanced, resulting in assembled myosin-II molecules that are poised to disassemble due to the phosphorylation of only three threonines.

  View details for DOI 10.1371/journal.pbio.0020356

  View details for Web of Science ID 000225160300018

  View details for PubMedID 15492777

  View details for PubMedCentralID PMC523230

• Myosin VI walks hand-over-hand along actin NATURE STRUCTURAL & MOLECULAR BIOLOGY Okten, Z., Churchman, L. S., Rock, R. S., Spudich, J. A. 2004; 11 (9): 884-887

  Abstract

  Myosin VI is a molecular motor that can walk processively on actin filaments with a 36-nm step size. The walking mechanism of myosin VI is controversial because it takes very large steps without an apparent lever arm of required length. Therefore, myosin VI is argued to be the first exception to the widely established lever arm theory. It is therefore critical to directly demonstrate whether this motor walks hand-over-hand along actin despite its short lever arm. Here, we follow the displacement of a single myosin VI head during the stepping process. A single head is displaced 72 nm during stepping, whereas the center of mass previously has been shown to move 36 nm. The most likely explanation for this result is a hand-over-hand walking mechanism. We hypothesize the existence of a flexible element that would allow the motor to bridge the observed 72-nm distance.

  View details for DOI 10.1038/nsmb815

  View details for Web of Science ID 000223540200023

  View details for PubMedID 15286724

• Mechanics and regulation of cytokinesis CURRENT OPINION IN CELL BIOLOGY Robinson, D. N., Spudich, J. A. 2004; 16 (2): 182-188

  Abstract

  Recent advances are revealing quantitative aspects of cytokinesis. Further, genetic analyses and cell imaging are providing insights into the molecular dynamics of cleavage furrow ingression as well as further refining our knowledge of the zones of the mitotic spindle that regulate the contractile properties of the overlying cortex. Ultimately, however, cortical mechanics are the result of signals that emanate from the mitotic spindle. A genuine quantitative understanding of cytokinesis must include a thorough analysis of the mechanical properties of the cortex and how signals modify these properties to dictate a well-controlled, error-free cytokinesis.

  View details for DOI 10.1016/j.ceb.2004.02.002

  View details for Web of Science ID 000220664200011

  View details for PubMedID 15196562

• The mechanism of myosin VI translocation an its load-induced anchoring CELL Altman, D., Sweeney, H. L., Spudich, J. A. 2004; 116 (5): 737-749

  Abstract

  Myosin VI is thought to function as both a transporter and an anchor. While in vitro studies suggest possible mechanisms for processive stepping, a biochemical basis for anchoring has not been demonstrated. Using optical trapping, we observed myosin VI stepping against applied forces. Step size is not strongly affected by such loads. At saturating ATP, myosin VI kinetics shows little dependence on load until, at forces near stall, its stepping slows dramatically as load increases. At subsaturating ATP or in the presence of ADP, stepping kinetics is significantly inhibited by load. From our results, we propose a mechanism of myosin VI stepping that predicts a regulation through load of the motor's roles as transporter and anchor.

  View details for Web of Science ID 000221499700013

  View details for PubMedID 15006355

• Structure of an F-actin trimer disrupted by gelsolin and implications for the mechanism of severing JOURNAL OF BIOLOGICAL CHEMISTRY Dawson, J. F., Sablin, E. P., Spudich, J. A., Fletterick, R. J. 2003; 278 (2): 1229-1238

  Abstract

  Stable oligomers of filamentous actin were obtained by cross-linking F-actin with 1,4-N,N'-phenylenedimaleimide and depolymerization with excess segment-1 of gelsolin. Segment-1-bound and cross-linked actin oligomers containing either two or three actin subunits were purified and shown to nucleate actin assembly. Kinetic assembly data from mixtures of monomeric actin and the actin oligomers fit a nucleation model where cross-linked actin dimer or trimer reacts with an actin monomer to produce a competent nucleus for filament assembly. We report the three-dimensional structure of the segment-1-actin hexamer containing three actin subunits, each with a tightly bound ATP. Comparative analysis of this structure with twelve other actin structures provides an atomic level explanation for the preferential binding of ATP by the segment-1-complexed actin. Although the structure of segment-1-bound actin trimer is topologically similar to the helical model of F-actin (1), it has a distorted symmetry compared with that of the helical model. This distortion results from intercalation of segment-1 between actin protomers that increase the rise per subunit and rotate each of the actin subunits relative to their positions in F-actin. We also show that segment-1 of gelsolin is able to sever actin filaments, although the severing activity of segment-1 is significantly lower than full-length gelsolin.

  View details for DOI 10.1074/jbc.M209160200

  View details for Web of Science ID 000180321900070

  View details for PubMedID 12356759

• Building and using optical traps to study properties of molecular motors BIOPHOTONICS, PT B Rice, S. E., Purcell, T. J., Spudich, J. A. 2003; 361: 112-133

  View details for Web of Science ID 000181447900006

  View details for PubMedID 12624909

• Role of the lever arm in the processive stepping of myosin V PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Purcell, T. J., Morris, C., Spudich, J. A., Sweeney, H. L. 2002; 99 (22): 14159-14164

  Abstract

  Myosin V is a two-headed molecular motor that binds six light chains per heavy chain, which creates unusually long lever arms. This motor moves processively along its actin track in discrete 36-nm steps. Our model is that one head of the two-headed myosin V tightly binds to actin and swings its long lever arm through a large angle, providing a stroke. We created single-headed constructs with different-size lever arms and show that stroke size is proportional to lever arm length. In a two-headed molecule, the stroke provides the directional bias, after which the unbound head diffuses to find its binding site, 36 nm forward. Our two-headed construct with all six light chains per head reconstitutes the 36-nm processive step seen in tissue-purified myosin V. Two-headed myosin V molecules with only four light chains per head are still processive, but their step size is reduced to 24 nm. A further reduction in the length of the lever arms to one light chain per head results in a motor that is unable to walk processively. This motor produces single small approximately 6-nm strokes, and ATPase and pyrene actin quench measurements show that only one of the heads of this dimer rapidly binds to actin for a given binding event. These data show that for myosin V with its normal proximal tail domain, both heads and a long lever arm are required for large, processive steps.

  View details for Web of Science ID 000178967400037

  View details for PubMedID 12386339

• How does ATP hydrolysis control actin's associations? PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sablin, E. P., Dawson, J. F., VanLoock, M. S., Spudich, J. A., Egelman, E. H., Fletterick, R. J. 2002; 99 (17): 10945-10947

  View details for DOI 10.1073/pnas.152329899

  View details for Web of Science ID 000177606900002

  View details for PubMedID 12167670

• Differential localization in cells of myosin II heavy chain kinases cytokinesis and polarized migration BMC CELL BIOLOGY Liang, W. C., Licate, L. S., Warrick, H. M., Spudich, J. A., Egelhoff, T. T. 2002; 3

  Abstract

  Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration, and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.Biochemical analysis indicates that MHCK-C can phosphorylate MHC with concomitant disassembly of myosin II filaments. In living cells, GFP-MHCK-A displayed frequent enrichment in the anterior of polarized migrating cells, and in the polar region but not the furrow during cytokinesis. GFP-MHCK-B generally displayed a homogeneous distribution. In migrating cells GFP-MHCK-C displayed posterior enrichment similar to that of myosin II, but did not localize with myosin II to the furrow during the early stage of cytokinesis. At the late stage of cytokinesis, GFP-MHCK-C became strongly enriched in the cleavage furrow, remaining there through completion of division.MHCK-A, -B, and -C display distinct cellular localization patterns suggesting different cellular functions and regulation for each MHCK isoform. The strong localization of MHCK-C to the cleavage furrow in the late stages of cell division may reflect a mechanism by which the cell regulates the progressive removal of myosin II as furrowing progresses.

  View details for Web of Science ID 000177253700001

  View details for PubMedID 12139770

• Dynacortin is a novel actin bundling protein that localizes to dynamic actin structures JOURNAL OF BIOLOGICAL CHEMISTRY Robinson, D. N., Ocon, S. S., Rock, R. S., Spudich, J. A. 2002; 277 (11): 9088-9095

  Abstract

  Dynacortin is a novel protein that was discovered in a genetic suppressor screen of a Dictyostelium discoideum cytokinesis-deficient mutant cell line devoid of the cleavage furrow actin bundling protein, cortexillin I. While dynacortin is highly enriched in the cortex, particularly in cell-surface protrusions, it is excluded from the cleavage furrow cortex during cytokinesis. Here, we describe the biochemical characterization of this new protein. Purified dynacortin is an 80-kDa dimer with a large 5.7-nm Stokes radius. Dynacortin cross-links actin filaments into parallel arrays with a mole ratio of one dimer to 1.3 actin monomers and a 3.1 microm K(d). Using total internal reflection fluorescence microscopy, GFP-dynacortin and the actin bundling protein coronin-GFP are seen to concentrate in highly dynamic cortical structures with assembly and disassembly half-lives of about 15 s. These results indicate that cells have evolved different actin-filament cross-linking proteins with complementary cellular distributions that collaborate to orchestrate complex cell shape changes.

  View details for DOI 10.1074/jbc.M112144200

  View details for Web of Science ID 000174400600048

  View details for PubMedID 11782490

• Quantitation of the distribution and flux of myosin-II during cytokinesis BMC CELL BIOLOGY Robinson, D. N., Cavet, G., Warrick, H. M., Spudich, J. A. 2002; 3

  Abstract

  During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring.The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex.The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis.

  View details for Web of Science ID 000173744900001

  View details for PubMedID 11860600

• Myosin VI is a processive motor with a large step size PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rock, R. S., Rice, S. E., Wells, A. L., Purcell, T. J., Spudich, J. A., Sweeney, H. L. 2001; 98 (24): 13655-13659

  Abstract

  Myosin VI is a molecular motor involved in intracellular vesicle and organelle transport. To carry out its cellular functions myosin VI moves toward the pointed end of actin, backward in relation to all other characterized myosins. Myosin V, a motor that moves toward the barbed end of actin, is processive, undergoing multiple catalytic cycles and mechanical advances before it releases from actin. Here we show that myosin VI is also processive by using single molecule motility and optical trapping experiments. Remarkably, myosin VI takes much larger steps than expected, based on a simple lever-arm mechanism, for a myosin with only one light chain in the lever-arm domain. Unlike other characterized myosins, myosin VI stepping is highly irregular with a broad distribution of step sizes.

  View details for Web of Science ID 000172328100035

  View details for PubMedID 11707568

• The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle JOURNAL OF BIOLOGICAL CHEMISTRY Shih, W. M., Spudich, J. A. 2001; 276 (22): 19491-19494

  Abstract

  Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.

  View details for Web of Science ID 000169091000111

  View details for PubMedID 11278776

• The myosin swinging cross-bridge model NATURE REVIEWS MOLECULAR CELL BIOLOGY Spudich, J. A. 2001; 2 (5): 387-392

  Abstract

  No biological system has been studied by more diverse approaches than the actin-based molecular motor myosin. Biophysics, biochemistry, physiology, classical genetics and molecular genetics have all made their contributions, and myosin is now becoming one of the best-understood enzymes in biology.

  View details for Web of Science ID 000168708800019

  View details for PubMedID 11331913

• Single molecule mechanics and the myosin family of molecular motors Spudich, J. A., Shih, W., Murphy, C., Mehta, A., Rock, R., Rief, M. FEDERATION AMER SOC EXP BIOL. 2001: A165–A165

  View details for Web of Science ID 000167438100940

• A myosin II mutation uncouples ATPase activity from motility and shortens step size NATURE CELL BIOLOGY Murphy, C. T., Rock, R. S., Spudich, J. A. 2001; 3 (3): 311-315

  Abstract

  It is thought that Switch II of myosin, kinesin and G proteins has an important function in relating nucleotide state to protein conformation. Here we examine a myosin mutant containing an S456L substitution in the Switch II region. In this protein, mechanical activity is uncoupled from the chemical energy of ATP hydrolysis so that its gliding velocity on actin filaments is only one-tenth of that of the wild type. The mutant spends longer in the strongly bound state and exhibits a shorter step size, which together account for the reduction in in vitro velocity. This is the first single point mutation in myosin that has been found to affect step size.

  View details for Web of Science ID 000167365800020

  View details for PubMedID 11231583

• A myosin-II mutation uncouples ATPase activity from motility and shortens step size Murphy, C. T., Rock, R. S., Spudich, J. A. CELL PRESS. 2001: 199A–199A

  View details for Web of Science ID 000166692200910

• In vitro assays of processive myosin motors METHODS Rock, R. S., Rief, M., Mehta, A. D., Spudich, J. A. 2000; 22 (4): 373-381

  Abstract

  Myosin V is an actin-based motor thought to be involved in vesicle transport. Since the properties of such a motor may be expected to differ from those of muscle myosin II, we have examined myosin V-driven movement using a combination of gliding filament and optical trap assays to observe single molecules with high resolution. The results clearly demonstrate that brain myosin V is a highly efficient processive motor. In vitro motility assays at low myosin V densities reveal apparent single-molecule supported movement. Processive stepping was also observed in optical trapping assays of myosin V-driven motion. Here the methods that were used to demonstrate the processivity of myosin V are described. These methods include density-dependent assays that eliminate the possibility of aggregation or chance colocalization of multiple motors being responsible for apparent single-molecule motility. Such assays will be useful tools for identifying other processive classes of myosins.

  View details for Web of Science ID 000166350700010

  View details for PubMedID 11133243

• Characterization of dynacortin, a genetic link between equatorial contractility and global shape control. Robinson, D. N., Spudich, J. A. AMER SOC CELL BIOLOGY. 2000: 563A–563A

  View details for Web of Science ID 000165525902914

• TGF-beta 1 signaling inhibition by a rationally designed HIV TAT-Smad7 fusion protein that causes inhibitory Smad7 translocation Lee, F. H., Hostetter, D. R., Zhang, Y., Tan, J. L., DiChiara, M. R., Spudich, J. A., Carter, A. J., Yeung, A. C., Yock, P. G., Cooke, J. P., Topper, J. N. LIPPINCOTT WILLIAMS & WILKINS. 2000: 41–41

  View details for Web of Science ID 000090072300184

• Dynacortin, a genetic link between equatorial contractility and global shape control discovered by library complementation of a Dictyostelium discoideum cytokinesis mutant JOURNAL OF CELL BIOLOGY Robinson, D. N., Spudich, J. A. 2000; 150 (4): 823-838

  Abstract

  We have developed a system for performing interaction genetics in Dictyostelium discoideum that uses a cDNA library complementation/multicopy suppression strategy. Chemically mutagenized cells were screened for cytokinesis-deficient mutants and one mutant was subjected to library complementation. Isolates of four different genes were recovered as modifiers of this strain's cytokinesis defect. These include the cleavage furrow protein cortexillin I, a novel protein we named dynacortin, an ezrin-radixin-moesin-family protein, and coronin. The cortexillin I locus and transcript were found to be disrupted in the strain, identifying it as the affected gene. Dynacortin is localized partly to the cell cortex and becomes enriched in protrusive regions, a localization pattern that is similar to coronin and partly dependent on RacE. During cytokinesis, dynacortin is found in the cortex and is somewhat enriched at the poles. Furthermore, it appears to be reduced in the cleavage furrow. The genetic interactions and the cellular distributions of the proteins suggest a hypothesis for cytokinesis in which the contraction of the medial ring is a function of spatially restricted cortexillin I and myosin II and globally distributed dynacortin, coronin, and RacE.

  View details for Web of Science ID 000088952500012

  View details for PubMedID 10953006

• Myosin-V stepping kinetics: A molecular model for processivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rief, M., Rock, R. S., Mehta, A. D., Mooseker, M. S., Cheney, R. E., Spudich, J. A. 2000; 97 (17): 9482-9486

  Abstract

  Myosin-V is a molecular motor that moves processively along its actin track. We have used a feedback-enhanced optical trap to examine the stepping kinetics of this movement. By analyzing the distribution of time periods separating discrete approximately 36-nm mechanical steps, we characterize the number and duration of rate-limiting biochemical transitions preceding each such step. These data show that myosin-V is a tightly coupled motor whose cycle time is limited by ADP release. On the basis of these results, we propose a model for myosin-V processivity.

  View details for Web of Science ID 000088840500028

  View details for PubMedID 10944217

• Towards a molecular understanding of cytokinesis TRENDS IN CELL BIOLOGY Robinson, D. N., Spudich, J. A. 2000; 10 (6): 228-237

  Abstract

  In this review, we focus on recent discoveries regarding the molecular basis of cleavage furrow positioning and contractile ring assembly and contraction during cytokinesis. However, some of these mechanisms might have different degrees of importance in different organisms. This synthesis attempts to uncover common themes and to reveal potential relationships that might contribute to the biochemical and mechanical aspects of cytokinesis. Because the information about cytokinesis is still fairly rudimentary, our goal is not to present a definitive model but to present testable hypotheses that might lead to a better mechanistic understanding of the process.

  View details for Web of Science ID 000087147000003

  View details for PubMedID 10802538

• Variable surface loops and myosin activity: Accessories to a motor JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY Murphy, C. T., Spudich, J. A. 2000; 21 (2): 139-151

  Abstract

  The catalytic head of myosin is a globular structure that has historically been divided into three segments of 25, 50, and 20 kDa. The solvent-exposed, proteolytically-sensitive surface loops of myosin that join these three segments are highly variable in their sequences. While surface loops have not traditionally been thought to affect enzymatic activities, these loops lie near the ATP and actin-binding sites and have been implicated in the modulation of myosin's kinetic activities. In this work we review the wealth of data regarding the loops that has accumulated over the years and discuss the roles of the loops in contributing to the different activities displayed by different myosin isoforms.

  View details for Web of Science ID 000087600000004

  View details for PubMedID 10961838

• Mutational analysis of phosphorylation sites in the Dictyostelium myosin II tail: disruption of myosin function by a single charge change FEBS LETTERS Nock, S., Liang, W. C., Warrick, H. M., Spudich, J. A. 2000; 466 (2-3): 267-272

  Abstract

  The dynamic assembly/disassembly of non-muscle myosin II filaments is critical for the regulation of enzymatic activities and localization. Phosphorylation of three threonines, 1823, 1833 and 2029, in the tail of Dictyostelium discoideum myosin II has been implicated in control of myosin filament assembly. By systematically replacing the three threonines to aspartates, mimicking a phosphorylated residue, we found that position 1823 is the most critical one for the regulation of myosin filament formation and in vivo function. Surprisingly, a single charge change is able to perturb filament formation and in vivo function of myosin II.

  View details for Web of Science ID 000085122200013

  View details for PubMedID 10682841

• Myosin V is a processive actin-based motor Mehta, A. D., Rock, R. S., Rief, M., Spudich, J. A., Mooseker, M. S., Cheney, R. E. CELL PRESS. 2000: 272A–272A

  View details for Web of Science ID 000084779301586

• A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly JOURNAL OF CELL BIOLOGY Liang, W. C., Warrick, H. M., Spudich, J. A. 1999; 147 (5): 1039-1047

  Abstract

  Myosin II thick filament assembly in Dictyostelium is regulated by phosphorylation at three threonines in the tail region of the molecule. Converting these three threonines to aspartates (3 x Asp myosin II), which mimics the phosphorylated state, inhibits filament assembly in vitro, and 3 x Asp myosin II fails to rescue myosin II-null phenotypes. Here we report a suppressor screen of Dictyostelium myosin II-null cells containing 3 x Asp myosin II, which reveals a 21-kD region in the tail that is critical for the phosphorylation control. These data, combined with new structural evidence from electron microscopy and sequence analyses, provide evidence that thick filament assembly control involves the folding of myosin II into a bent monomer, which is unable to incorporate into thick filaments. The data are consistent with a structural model for the bent monomer in which two specific regions of the tail interact to form an antiparallel tetrameric coiled-coil structure.

  View details for Web of Science ID 000083995800016

  View details for PubMedID 10579723

• Single molecule mechanical studies of myosin V Mehta, A. D., Rock, R., Rief, M., Spudich, J., Mooseker, M., Cheney, R. AMER SOC CELL BIOLOGY. 1999: 163A–163A

  View details for Web of Science ID 000083673500948

• Dynacortin, a genetic link between the global cortical tension generating system of RacE and coronin and the equatorial cortical tension generating system of cortexillin I and myosin II. Robinson, D. N., Spudich, J. A. AMER SOC CELL BIOLOGY. 1999: 133A–133A

  View details for Web of Science ID 000083673500769

• Myosin-V is a processive actin-based motor NATURE Mehta, A. D., Rock, R. S., Rief, M., Spudich, J. A., Mooseker, M. S., Cheney, R. E. 1999; 400 (6744): 590-593

  Abstract

  Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.

  View details for Web of Science ID 000081854800062

  View details for PubMedID 10448864

• Biomechanics, one molecule at a time JOURNAL OF BIOLOGICAL CHEMISTRY Mehta, A. D., Rief, M., Spudich, J. A. 1999; 274 (21): 14517-14520

  View details for Web of Science ID 000081965200002

  View details for PubMedID 10329637

• The sequence of the myosin 50-20K loop affects myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity BIOCHEMISTRY Murphy, C. T., Spudich, J. A. 1999; 38 (12): 3785-3792

  Abstract

  We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.

  View details for Web of Science ID 000079510700038

  View details for PubMedID 10090768

• Specialized conservation of surface loops of myosin: Evidence that loops are involved in determining functional characteristics JOURNAL OF MOLECULAR BIOLOGY Goodson, H. V., Warrick, H. M., Spudich, J. A. 1999; 287 (1): 173-185

  Abstract

  The molecular motor myosin has been the focus of considerable structure-function analysis. Of key interest are the portions of the protein that control the rate of ATP hydrolysis, the affinity for actin, and the velocity at which myosin moves actin. Two regions that have been implicated in determining these parameters are the "loop" regions at the junctions of the 25 kDa and 50 kDa domains and the 50 kDa and 20 kDa domains of the protein. However, the sequences of these regions are poorly conserved between different myosin families, suggesting that they are not constrained evolutionarily, and thus are relatively unimportant for myosin function. In order to address this apparent incongruity, we have performed an analysis of relative rates of observed evolutionary change. We found that the sequences of these loop regions appear to be actually more constrained than the sequences of the rest of the myosin molecule, when myosins are compared that are known to be kinetically or developmentally similar. This suggests that these loop regions could play an important role in myosin function and supports the idea that they are involved in modulating the specific kinetic characteristics that functionally differentiate one myosin isoform from another. Apparently "unconserved" loops may generally play a role in determining kinetic properties of enzymes, and similar analyses of relative rates of evolution may prove useful for the study of structure-function relationships in other protein families.

  View details for Web of Science ID 000079315400014

  View details for PubMedID 10074415

• Single-molecule biomechanics with optical methods SCIENCE Mehta, A. D., Rief, M., Spudich, J. A., Smith, D. A., Simmons, R. M. 1999; 283 (5408): 1689-1695

  Abstract

  Single-molecule observation and manipulation have come of age. With the advent of optical tweezers and other methods for probing and imaging single molecules, investigators have circumvented the model-dependent extrapolation from ensemble assays that has been the hallmark of classical biochemistry and biophysics. In recent years, there have been important advances in the understanding of how motor proteins work. The range of these technologies has also started to expand into areas such as DNA transcription and protein folding. Here, recent experiments with rotary motors, linear motors, RNA polymerase, and titin are described.

  View details for Web of Science ID 000079102800040

  View details for PubMedID 10073927

• Myosin II localization during cytokinesis occurs by a mechanism that does not require its motor domain PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zang, J. H., Spudich, J. A. 1998; 95 (23): 13652-13657

  Abstract

  Myosin II generates force for the division of eukaryotic cells. The molecular basis of the spatial and temporal localization of myosin II to the cleavage furrow is unknown, although models often imply that interaction between myosin II and actin filaments is essential. We examined the localization of a chimeric protein that consists of the green fluorescent protein fused to the N terminus of truncated myosin II heavy chain in Dictyostelium cells. This chimera is missing the myosin II motor domain, and it does not bind actin filaments. Surprisingly, it still localizes to the cleavage furrow region during cytokinesis. These results indicate that myosin II localization during cytokinesis occurs through a mechanism that does not require it to be the force-generating element or to interact with actin filaments directly.

  View details for Web of Science ID 000076997000054

  View details for PubMedID 9811855

• Genetic analysis of Dictyostelium cytokinesis. Robinson, D. N., Spudich, J. A. AMER SOC CELL BIOLOGY. 1998: 400A–400A

  View details for Web of Science ID 000076906702324

• Kinetic analysis of "uncoupled" mutant myosins Murphy, C. T., Spudich, J. A. AMER SOC CELL BIOLOGY. 1998: 144A–144A

  View details for Web of Science ID 000076906700832

• Nucleotide-dependent conformational change near the fulcrum region in Dictyostelium myosin II PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liang, W. C., Spudich, J. A. 1998; 95 (22): 12844-12847

  Abstract

  In skeletal muscle myosin, the reactive thiols (SH1 and SH2) are close to a proposed fulcrum region that is thought to undergo a large conformational change. The reactive thiol region is thought to transmit the conformational changes induced by the actin-myosin-ATP interactions to the lever arm, which amplifies the power stroke. In skeletal muscle myosin, SH1 and SH2 can be chemically cross-linked in the presence of nucleotide, trapping the nucleotide in its pocket. Although the flexibility of the reactive thiol region has been well studied in skeletal muscle myosin, crystal structures of truncated nonmuscle myosin II from Dictyostelium in the presence of various ATP analogs do not show changes at the reactive thiol region that would be consistent with the SH1-SH2 cross-linking observed for muscle myosin. To examine the dynamics of the reactive thiol region in Dictyostelium myosin II, we have examined a modified myosin II that has cysteines at the muscle myosin SH1 and SH2 positions. This myosin is specifically cross-linked at SH1-SH2 by a chemical cross-linker in the presence of ADP, but not in its absence. Furthermore, the cross-linked species traps the nucleotide, as in the case of muscle myosin. Thus, the Dictyostelium myosin II shares the same dynamic behavior in the fulcrum region of the molecule as the skeletal muscle myosin. This result emphasizes the importance of nucleotide-dependent changes in this part of the molecule.

  View details for Web of Science ID 000076757300024

  View details for PubMedID 9789002

• Direct regulation of myosin by nitric oxide Tan, J. L., Heidecker, W., Cohen, J. D., Fowler, M. B., Spudich, J. A. LIPPINCOTT WILLIAMS & WILKINS. 1998: 682–82

  View details for Web of Science ID 000076594403602

• MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Silveira, L. A., Smith, J. L., Tan, J. L., Spudich, J. A. 1998; 95 (22): 13000-13005

  Abstract

  Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.

  View details for Web of Science ID 000076757300052

  View details for PubMedID 9789030

• Kinetic characterization of myosin head fragments with long-lived myosin center dot ATP states BIOCHEMISTRY Friedman, A. L., Geeves, M. A., Manstein, D. J., Spudich, J. A. 1998; 37 (27): 9679-9687

  Abstract

  We have separately expressed the Dictyosteliumdiscoideum myosin II nonhydrolyzer point mutations E459V and E476K [Ruppel, K. M., and Spudich, J. A. (1996) Mol. Biol. Cell 7, 1123-1136] in the soluble myosin head fragment M761-1R [Anson et al. (1996) EMBO J. 15, 6069-6074] and performed transient kinetic analyses to characterize the ATPase cycles of the mutant proteins. While the mutations cause some changes in mantATP [2'(3')-O-(N-methylanthraniloyl)-ATP] and mantADP binding, the most dramatic effect is on the hydrolysis step of the ATPase cycle, which is reduced by 4 (E476K) and 6 (E459V) orders of magnitude. Thus, both mutant myosin constructs do in fact catalyze ATP hydrolysis but have very long-lived myosin.ATP states. The E459V mutation allowed for a direct measurement of the ATP off rate constant from myosin, which was found to be 2 x 10(-)5 s-1. Actin accelerated ATP release from this E459V construct by at least 100-fold. Additionally, we found that the affinity of the E476K construct for actin is significantly weaker than for the wild-type construct, while the E459V mutant interacts with actin normally. Their functional properties and the fact that they can be produced and purified in large amounts make the E476K and E459V constructs ideal tools to elucidate key structural features of the myosin ATPase cycle. These constructs should allow us to address important questions, including how binding of ATP to myosin heads results in a >3 order of magnitude reduction in actin affinity.

  View details for Web of Science ID 000074758600009

  View details for PubMedID 9657680

• Single molecule biochemistry using optical tweezers FEBS LETTERS Mehta, A. D., Pullen, K. A., Spudich, J. A. 1998; 430 (1-2): 23-27

  Abstract

  The use of optical trapping to create extremely compliant mechanical probes has ushered in a new field of biological inquiry, the mechanical and kinetic study of proteins at the single molecule level. This review focuses on three examples of such study and includes methods of extracting parameters of interest from the raw data such experiments generate.

  View details for Web of Science ID 000074771700005

  View details for PubMedID 9678588

• Conditional loss-of-myosin-II-function mutants reveal a position in the tail that is critical for filament nucleation MOLECULAR CELL Moores, S. L., Spudich, J. A. 1998; 1 (7): 1043-1050

  Abstract

  Myosin-II must be assembled into filaments to perform its cellular functions. Two conditional loss-of-myosin-II-function mutants were recovered from a previous genetic screen with defects that were mapped to the coiled-coil tail region of Dictyostelium myosin-II. Strikingly, both tail mutations affected the same arginine residue at position 1880. A single amino acid substitution, R1880P, disrupted both the dimerization and tetramerization steps of filament nucleation. Even a single charge reversal at this position, R1880D, was sufficient to inhibit filament assembly, while other single charge reversals in the region of antiparallel contract suppressed these filament assembly mutants. The considerable impact of small electrostatic forces on nucleation suggests that these steps are delicately balanced and easily reversible.

  View details for Web of Science ID 000074389200011

  View details for PubMedID 9651587

• Dictyostelium myosin 25-50K loop substitutions specifically affect ADP release rates BIOCHEMISTRY Murphy, C. T., Spudich, J. A. 1998; 37 (19): 6738-6744

  Abstract

  While most of the sequence of myosin's motor domain is highly conserved among various organisms and tissue types, the junctions between the 25 and 50 kDa domains and the 50 and 20 kDa domains are strikingly divergent. The 50-20K loop is positioned to interact with actin, while the 25-50K loop is situated nearer the ATP binding site [Rayment, I., et al. (1993) Science 261, 50-58]. Chimeric studies of the 50-20K loop [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-569; Rovner, A. S., et al. (1995) J. Biol. Chem. 270 (51), 30260-30263] have shown that this loop affects actin activation of ATPase activity. Given the function of myosin as a molecular motor, it was proposed that the 25-50K loop might specifically alter ADP release [Spudich, J. A. (1994) Nature 374, 515-518]. Here we study the role of this loop by engineering chimeras containing the Dictyostelium myosin heavy chain with loops from two enzymatically diverse myosins, rabbit skeletal and Acanthamoeba. The chimeric myosins complement the myosin null phenotype in vivo, bind nucleotide normally, interact normally with actin, and display wild-type levels of actin-activated ATPase activity. However, the rate of ADP release from the myosins, normally the slowest step involved in motility, was changed in a manner that reflects the activity of the donor myosin. In summary, studies of Dictyostelium myosin heavy chain chimeras have shown that the 50-20K sequence specifically affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994)] while the 25-50K sequence helps determine the rate of ADP release.

  View details for Web of Science ID 000073797300013

  View details for PubMedID 9578557

• Cysteine engineering studies on Dictyostelium myosin II Shih, W. M., Spudich, J. A. CELL PRESS. 1998: A130–A130

  View details for Web of Science ID 000073445400747

• New strategies in site-specific immobilization of proteins on micro- and nanostructured surfaces Wagner, P., Nock, S., Heidecker, M., Shih, W., Ulman, N., Spudich, J. A. CELL PRESS. 1998: A295–A295

  View details for Web of Science ID 000073445401705

• Single molecule myosin mechanics examined using optical tweezers Mehta, A. D., Spudich, J. A. CELL PRESS. 1998: A225–A225

  View details for Web of Science ID 000073445401301

• Reflections of a lucid dreamer: Optical trap design considerations METHODS IN CELL BIOLOGY, VOL 55 Mehta, A. D., Finer, J. T., Spudich, J. A. 1998; 55: 47-69

  View details for Web of Science ID 000073062000004

  View details for PubMedID 9352511

• Single myosin molecule mechanics Workshop on Molecular Biophysics of the Cytoskeleton - Microtubule Formation, Structure, Function, and Interactions Mehta, A. D., Spudich, J. A. JAI PRESS INC. 1998: 229–270

  View details for Web of Science ID 000078851000011

• Use of optical traps in single-molecule study of nonprocessive biological motors MOLECULAR MOTORS AND THE CYTOSKELETON, PT B Mehta, A. D., Finer, J. T., Spudich, J. A. 1998; 298: 436-459

  View details for Web of Science ID 000076246300037

  View details for PubMedID 9751902

• On the role of myosin-II in cytokinesis: Division of Dictyostelium cells under adhesive and nonadhesive conditions MOLECULAR BIOLOGY OF THE CELL Zang, J. H., Cavet, G., Sabry, J. H., Wagner, P., Moores, S. L., Spudich, J. A. 1997; 8 (12): 2617-2629

  Abstract

  We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild-type and myosin-null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild-type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms that we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain-binding sites were deleted (DeltaBLCBS-myosin). Although this myosin is a slower motor than wild-type myosin and has constitutively high activity due to the abolition of regulation by light-chain phosphorylation, cells expressing DeltaBLCBS-myosin were previously shown to divide in suspension (Uyeda et al., 1996). However, we suspected their behavior during cytokinesis to be different from wild-type cells given the large alteration in their myosin. Surprisingly, DeltaBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a DeltaBLCBS-myosin is similar to that in wild-type cells.

  View details for Web of Science ID A1997YK93000020

  View details for PubMedID 9398680

• Myosin heavy chain phosphorylation sites regulate myosin localization during cytokinesis in live cells MOLECULAR BIOLOGY OF THE CELL Sabry, J. H., Moores, S. L., Ryan, S., Zang, J. H., Spudich, J. A. 1997; 8 (12): 2605-2615

  Abstract

  Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.

  View details for Web of Science ID A1997YK93000019

  View details for PubMedID 9398679

• The role of the 50-20K loop in myosin function Murphy, C. T., Spudich, J. A. AMER SOC CELL BIOLOGY. 1997: 916–916

  View details for Web of Science ID A1997YF09600915

• Cold-sensitive mutants G680V and G691C of Dictyostelium myosin II confer dramatically different biochemical defects JOURNAL OF BIOLOGICAL CHEMISTRY Patterson, B., Ruppel, K. M., Wu, Y., Spudich, J. A. 1997; 272 (44): 27612-27617

  Abstract

  Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

  View details for Web of Science ID A1997YD47300024

  View details for PubMedID 9346898

• Reversible, site-specific immobilization of polyarginine-tagged fusion proteins on mica surfaces FEBS LETTERS Nock, S., Spudich, J. A., Wagner, P. 1997; 414 (2): 233-238

  Abstract

  A large variety of genes is expressed as fusion proteins for the purpose of characterization and purification in molecular biology. We have used this strategy to append polyarginine peptides in order to achieve specific binding of the Arg-tag to atomically flat, negatively charged mica surfaces. We show that the model protein, hexaarginine-tagged green fluorescent protein (GFP), binds to mica via its Arg-tag based on ion exchange of naturally occurring potassium cations. Only non-specific binding was observed with the control protein that is free of the Arg-tag. This novel technology will be widely applicable to orient functional proteins on flat surfaces.

  View details for Web of Science ID A1997XW75900012

  View details for PubMedID 9315692

• Single myosin molecule mechanics: Nanometer steps, piconewton forces, millisecond kinetics, and stiffness of elastic elements measured with a dual-beam laser trap. Spudich, J. A., Mehta, A. FEDERATION AMER SOC EXP BIOL. 1997: A855–A855

  View details for Web of Science ID 000073305600480

• Detection of single-molecule interactions using correlated thermal diffusion Colloquium on Genetics and the Origin of Species Mehta, A. D., Finer, J. T., Spudich, J. A. NATL ACAD SCIENCES. 1997: 7927–31

  Abstract

  Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.

  View details for Web of Science ID A1997XM42800040

  View details for PubMedID 9223289

• Phenotypically selected mutations in myosin's actin binding domain demonstrate intermolecular contacts important for motor functions BIOCHEMISTRY Giese, K. C., Spudich, J. A. 1997; 36 (28): 8465-8473

  Abstract

  Here, we biochemically characterize Dictyostelium myosin II mutants that were previously phenotypically selected following random mutagenesis and shown to lie in the actin binding domain [Patterson, B., & Spudich, J. A. (1996) Genetics 143, 801-810]. We show that the conditional loss of myosin-dependent activity in vivo, which results from the mutations E531Q, P536R, and R562L, is likely due to the loss of important contacts with actin. Purified wild-type and mutant myosin subfragments 1 (S1), expressed in Dictyostelium, are alike in binding to actin and releasing it in an ATP-dependent manner. Furthermore, the rates of ATP hydrolysis without actin are similar for the mutant and wild-type S1s. Thus, the mutations in the actin binding site have little effect on ATP binding or product release in the absence of actin. All three mutants, however, have impaired actin-activated ATPase activity, with apparent second-order rate constants for actin interactions that are 4-25-fold smaller than that of wild-type S1 at 30 degrees C. The mutations also cause defects in the ability to move actin, as measured by in vitro motility assays of full-length myosins. On the basis of motility of a mixture of wild-type and mutant myosins, there appears to be at least two classes of mutations, with the primary defect in either a weak or a strong actin binding state. In summary, the activities in vitro of myosins with mutations in the actin binding site suggest losses of important contacts with actin.

  View details for Web of Science ID A1997XK85600005

  View details for PubMedID 9214290

• Probing conformational changes in myosin during the ATPase cycle. Liang, W. C., Shih, W., Spudich, J. A. CELL PRESS. 1997: MPMA7–MPMA7

  View details for Web of Science ID A1997WE74700087

• Correlated motion of actin filament segments on either side of a single attached crossbridge. Mehta, A. D., Spudich, J. A. CELL PRESS. 1997: WAMJ1–WAMJ1

  View details for Web of Science ID A1997WE74701336

• Site-specific immobilization of biomolecules on micro- and nanofabricated gold and silicon surfaces. Wagner, P., Spudich, A., Ulman, N., Chidsey, C. E., Spudich, J. A. CELL PRESS. 1997: MP452–MP452

  View details for Web of Science ID A1997WE74700585

• Bioreactive self-assembled monolayers on hydrogen-passivated Si(111) as a new class of atomically flat substrates for biological scanning probe microscopy JOURNAL OF STRUCTURAL BIOLOGY Wagner, P., Nock, S., Spudich, J. A., VOLKMUTH, W. D., Chu, S., Cicero, R. L., Wade, C. P., Linford, M. R., Chidsey, C. E. 1997; 119 (2): 189-201

  Abstract

  This is the first report of bioreactive self-assembled monolayers, covalently bound to atomically flat silicon surfaces and capable of binding biomolecules for investigation by scanning probe microscopy and other surface-related assays and sensing devices. These monolayers are stable under a wide range of conditions and allow tailor-made functionalization for many purposes. We describe the substrate preparation and present an STM and SFM characterization, partly performed with multiwalled carbon nanotubes as tapping-mode supertips. Furthermore, we present two strategies of introducing in situ reactive headgroup functionalities. One method entails a free radical chlorosulfonation process with subsequent sulfonamide formation. A second method employs singlet carbenemediated hydrogen-carbon insertion of a heterobifunctional, amino-reactive trifluoromethyl-diazirinyl crosslinker. We believe that this new substrate is advantageous to others, because it (i) is atomically flat over large areas and can be prepared in a few hours with standard equipment, (ii) is stable under most conditions, (iii) can be modified to adjust a certain degree of reactivity and hydrophobicity, which allows physical adsorption or covalent crosslinking of the biological specimen, (iv) builds the bridge between semiconductor microfabrication and organic/biological molecular systems, and (v) is accessible to nanopatterning and applications requiring conductive substrates.

  View details for Web of Science ID A1997XN38200014

  View details for PubMedID 9245759

• Single molecule myosin mechanics measured using optical trapping NATO Advanced Study Institute on Structure and Function of Interacting Protein Domains in Signal and Energy Transduction Mehta, A. D., Spudich, J. A. SPRINGER-VERLAG BERLIN. 1997: 247–259

  View details for Web of Science ID 000075441300038

• Cysteine engineering studies on Dictyostelium myosin II Shih, W. M., Liang, W. C., Spudich, J. A. AMER SOC CELL BIOLOGY. 1996: 1139–1139

  View details for Web of Science ID A1996WB01801138

• The role of nonconserved surface loops in myosin function Murphy, C. T., Uyeda, T. Q., Spudich, J. A. AMER SOC CELL BIOLOGY. 1996: 1141–1141

  View details for Web of Science ID A1996WB01801142

• Activation of Dictyostelium myosin light chain kinase a by phosphorylation of Thr166 EMBO JOURNAL Smith, J. L., Silveira, L. A., Spudich, J. A. 1996; 15 (22): 6075-6083

  Abstract

  Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.

  View details for Web of Science ID A1996VW41800008

  View details for PubMedID 8947030

• Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: A role for MLCK-A in cytokinesis and evidence for multiple MLCKs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Smith, J. L., Silveira, L. A., Spudich, J. A. 1996; 93 (22): 12321-12326

  Abstract

  We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.

  View details for Web of Science ID A1996VP93700050

  View details for PubMedID 8901579

• The Dictyostelium dual-specificity kinase splA is essential for spore differentiation DEVELOPMENT Nuckolls, G. H., Osherov, N., Loomis, W. F., Spudich, J. A. 1996; 122 (10): 3295-3305

  Abstract

  We have studied the structure and function of the Dictyostelium kinase splA. A truncated form of the splA protein exhibited primarily tyrosine kinase activity in vitro; however, it also autophosphorylated on serine and threonine residues. The kinase domain of splA exhibits approximately 38% identity to the CTR1 kinase of Arabidopsis, which is a member of the Raf family. Outside its kinase domain, splA shares homology with the byr2 kinase of S. pombe. By aligning the sequences of splA, byr2 and STE11, a homologue of byr2 in S. cerevisiae, we have identified a conserved motif that is also found in members of the Eph family of growth factor receptor tyrosine kinases. SplA is expressed throughout development with a peak during the mound stage of morphogenesis. Strains in which the splA gene had been disrupted completed fruiting body formation; however, spore cells spontaneously lysed before completing their differentiation. Northern analysis revealed the expression of the prespore marker cotB and the prestalk markers ecmA and ecmB in the mutant strain during development. The spore differentiation marker spiA was detected in the mutant spores both by northern and immunoblotting, but these cells failed to assemble spore coats. Immunoblot analysis of the developmental pattern of tyrosine phosphorylation revealed a protein that was phosphorylated in mutants but was not phosphorylated in the wild-type cells. SplA is a novel dual specificity kinase that regulates the differentiation of spore cells.

  View details for Web of Science ID A1996VP65300032

  View details for PubMedID 8898241

• Structure-function studies of the myosin motor domain: Importance of the 50-kDa cleft MOLECULAR BIOLOGY OF THE CELL Ruppel, K. M., Spudich, J. A. 1996; 7 (7): 1123-1136

  Abstract

  We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.

  View details for Web of Science ID A1996UX91800010

  View details for PubMedID 8862525

• Synthetic lethality screen identifies a novel yeast myosin I gene (MYO5): Myosin I proteins are required for polarization of the actin cytoskeleton JOURNAL OF CELL BIOLOGY Goodson, H. V., Anderson, B. L., Warrick, H. M., Pon, L. A., Spudich, J. A. 1996; 133 (6): 1277-1291

  Abstract

  The organization of the actin cytoskeleton plays a critical role in cell physiology in motile and nonmotile organisms. Nonetheless, the function of the actin based motor molecules, members of the myosin superfamily, is not well understood. Deletion of MYO3, a yeast gene encoding a "classic" myosin I, has no detectable phenotype. We used a synthetic lethality screen to uncover genes whose functions might overlap with those of MYO3 and identified a second yeast myosin 1 gene, MYO5. MYO5 shows 86 and 62% identity to MYO3 across the motor and non-motor regions. Both genes contain an amino terminal motor domain, a neck region containing two IQ motifs, and a tail domain consisting of a positively charged region, a proline-rich region containing sequences implicated in ATP-insensitive actin binding, and an SH3 domain. Although myo5 deletion mutants have no detectable phenotype, yeast strains deleted for both MYO3 and MYO5 have severe defects in growth and actin cytoskeletal organization. Double deletion mutants also display phenotypes associated with actin disorganization including accumulation of intracellular membranes and vesicles, cell rounding, random bud site selection, sensitivity to high osmotic strength, and low pH as well as defects in chitin and cell wall deposition, invertase secretion, and fluid phase endocytosis. Indirect immunofluorescence studies using epitope-tagged Myo5p indicate that Myo5p is localized at actin patches. These results indicate that MYO3 and MYO5 encode classical myosin I proteins with overlapping functions and suggest a role for Myo3p and Myo5p in organization of the actin cytoskeleton of Saccharomyces cerevisiae.

  View details for Web of Science ID A1996UT40700011

  View details for PubMedID 8682864

• Cold-sensitive mutations of Dictyostelium myosin heavy chain highlight functional domains of the myosin motor GENETICS Patterson, B., Spudich, J. A. 1996; 143 (2): 801-810

  Abstract

  Dictyostelium provides a powerful environment for characterization of myosin II function. It provides well-established biochemical methods for in vitro analysis of myosin's properties as well as an array of molecular genetic tools. The absence of myosin function results in an array of phenotypes that can be used to genetically manipulate myosin function. We have previously reported methods for the isolation and identification of rapid-effect cold-sensitive myosin II mutations in Dictyostelium. Here, we report the development and utilization of a rapid method for localizing these point mutations. We have also sequenced 19 mutants. The mutations show distinct clustering with respect to three-dimensional location and biochemically characterized functional domains of the protein. We conclude that these mutants represent powerful tools for understanding the mechanisms driving this protein motor.

  View details for Web of Science ID A1996UN68400016

  View details for PubMedID 8725228

• Quantitative measurements of force and displacement using an optical trap BIOPHYSICAL JOURNAL Simmons, R. M., Finer, J. T., Chu, S., Spudich, J. A. 1996; 70 (4): 1813-1822

  Abstract

  We combined a single-beam gradient optical trap with a high-resolution photodiode position detector to show that an optical trap can be used to make quantitative measurements of nanometer displacements and piconewton forces with millisecond resolution. When an external force is applied to a micron-sized bead held by an optical trap, the bead is displaced from the center of the trap by an amount proportional to the applied force. When the applied force is changed rapidly, the rise time of the displacement is on the millisecond time scale, and thus a trapped bead can be used as a force transducer. The performance can be enhanced by a feedback circuit so that the position of the trap moves by means of acousto-optic modulators to exert a force equal and opposite to the external force applied to the bead. In this case the position of the trap can be used to measure the applied force. We consider parameters of the trapped bead such as stiffness and response time as a function of bead diameter and laser beam power and compare the results with recent ray-optic calculations.

  View details for Web of Science ID A1996UB81800027

  View details for PubMedID 8785341

  View details for PubMedCentralID PMC1225151

• Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Coppin, C. M., Finer, J. T., Spudich, J. A., Vale, R. D. 1996; 93 (5): 1913-1917

  Abstract

  Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.

  View details for Web of Science ID A1996TY96400033

  View details for PubMedID 8700858

• Myosin dynamics iri live Dictyostelium cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Moores, S. L., Sabry, J. H., Spudich, J. A. 1996; 93 (1): 443-446

  Abstract

  Conventional myosin plays a key role in the cytoskeletal reorganization necessary for cytokinesis, migration, and morphological changes associated with development in nonmuscle cells. We have made a fusion between the green fluorescent protein (GFP) and the Dictyostelium discoideum myosin heavy chain (GFP-myosin). The unique Dictyostelium system allows us to test the GFP-tagged myosin for activity both in vivo and in vitro. Expression of GFP-myosin rescues all myosin null cell defects. Additionally, GFP-myosin purified from these cells exhibits the same ATPase activities and in vitro motility as wild-type myosin. GFP-myosin is concentrated in the cleavage furrow during cytokinesis and in the posterior cortex of migrating cells. Surprisingly, GFP-myosin concentration increases transiently in the tips of retracting pseudopods. Contrary to previous thinking, this suggests that conventional myosin may play an important role in the dynamics of pseudopods as well as filopodia, lamellipodia, and other cellular protrusions.

  View details for Web of Science ID A1996TP36700088

  View details for PubMedID 8552657

• Structure-function analysis of the motor domain of myosin ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Ruppel, K. M., Spudich, J. A. 1996; 12: 543-573

  Abstract

  Motor proteins perform a wide variety of functions in all eukaryotic cells. Recent advances in the structural and mutagenic analysis of the myosin motor has led to insights into how these motors transduce chemical energy into mechanical work. This review focuses on the analysis of the effects of myosin mutations from a variety of organisms on the in vivo and in vitro properties of this ubiquitous motor and illustrates the positions of these mutations on the high-resolution three-dimensional structure of the myosin motor domain.

  View details for Web of Science ID A1996VY42800019

  View details for PubMedID 8970737

• A NOVEL POSITIVE SELECTION FOR IDENTIFYING COLD-SENSITIVE MYOSIN-II MUTANTS IN DICTYOSTELIUM GENETICS Patterson, B., Spudich, J. A. 1995; 140 (2): 505-515

  Abstract

  We developed a positive selection for myosin heavy chain mutants in Dictyostelium. This selection is based on the fact that brief exposure to azide causes wild-type cells to release from the substrate, whereas myosin null cells remain adherent. This procedure assays myosin function on a time scale of minutes and has therefore allowed us to select rapid-onset cold-sensitive mutants after random chemical mutagenesis of Dictyostelium cells. We developed a rapid technique for determining which mutations lie in sequences of the myosin gene that encode the head (motor) domain and localized 27 of 34 mutants to this domain. We recovered the appropriate sequences from five of the mutants and demonstrated that they retain their cold-sensitive properties when expressed from extrachromosomal plasmids.

  View details for Web of Science ID A1995RA36600009

  View details for PubMedID 7498732

• Characterization of single actin-myosin interactions. Biophysical journal Finer, J. T., Mehta, A. D., Spudich, J. A. 1995; 68 (4): 291S-296S

  Abstract

  The feedback-enhanced laser trap assay (Finer et al., 1994) allows the measurement of force and displacement produced by single myosin molecules interacting with an actin filament suspended in solution by two laser traps. The average displacement of 11 nm at low load and the average force of 4 pN near isometric conditions are consistent with the conventional swinging cross-bridge model of muscle contraction (Huxley, 1969). The durations of single actin-myosin interactions at low load, 3-7 ms, suggest a relatively small duty ratio. Event durations can be increased either by reducing the ATP concentration until ATP binding is rate-limiting or by lowering the temperature. For sufficiently long interactions near isometric conditions, low frequency force fluctuations were observed within the time frame of a single event. Single myosin events can be measured at ionic strengths that disrupt weak binding actomyosin interactions, supporting the postulate of distinct weak and strong binding states. Myosin-generated force and displacement were measured simultaneously against several different loads to generate a force-displacement curve. The linear appearance of this curve suggests that the myosin powerstroke is driven by the release of a strained linear elastic element with a stiffness of approximately 0.4 pN nm-1.

  View details for PubMedID 7787094

• Measurement of the isometric force exerted by a single kinesin molecule. Biophysical journal Coppin, C. M., Finer, J. T., Spudich, J. A., Vale, R. D. 1995; 68 (4): 242S-244S

  View details for PubMedID 7787084

• MEASUREMENT OF THE ISOMETRIC FORCE EXERTED BY A SINGLE KINESIN MOLECULE 7th Biophysical Discussions on Molecular Motors - Structure, Mechanics and Energy Transduction Coppin, C. M., Finer, J. T., Spudich, J. A., Vale, R. D. BIOPHYSICAL SOCIETY. 1995: S242–S244

  View details for Web of Science ID A1995RK09000055

• CHARACTERIZATION OF SINGLE ACTIN-MYOSIN INTERACTIONS 7th Biophysical Discussions on Molecular Motors - Structure, Mechanics and Energy Transduction Finer, J. T., Mehta, A. D., Spudich, J. A. BIOPHYSICAL SOCIETY. 1995: S291–S297

  View details for Web of Science ID A1995RK09000064

• MYOSIN STRUCTURE-FUNCTION - A COMBINED MUTAGENESIS-CRYSTALLOGRAPHY APPROACH CURRENT OPINION IN STRUCTURAL BIOLOGY Ruppel, K. M., Lorenz, M., Spudich, J. A. 1995; 5 (2): 181-186

  Abstract

  In the past year, the structure of the regulatory domain of scallop myosin has joined that of the chicken skeletal muscle myosin subfragment 1 and provided insights into the regulation of myosin function. Mutagenesis studies in a variety of systems have used the information provided by these structures to create mutant myosins to test models of chemomechanical transduction and its regulation.

  View details for Web of Science ID A1995RA49400005

  View details for PubMedID 7648319

• MYOSIN MOTOR FUNCTION - STRUCTURAL AND MUTAGENIC APPROACHES CURRENT OPINION IN CELL BIOLOGY Ruppel, K. M., Spudich, J. A. 1995; 7 (1): 89-93

  Abstract

  Recent advances in three areas of myosin research--structural biology, in vitro motility assays, and mutagenesis--are leading to a new understanding of the molecular mechanism of chemomechanical transduction by this motor protein. Highlights include rational design of mutants using the crystal structure of subfragment 1, combined in vivo and in vitro mutant analyses using Dictyostelium, and the emergence of baculovirus as an in vitro system for expression of mutated mammalian myosins.

  View details for Web of Science ID A1995QE87300013

  View details for PubMedID 7755994

• IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF A YEAST MYOSIN-I CELL MOTILITY AND THE CYTOSKELETON Goodson, H. V., Spudich, J. A. 1995; 30 (1): 73-84

  Abstract

  The family of myosin motors is comprised of numerous classes distributed among a diverse set of organisms and cell types. We have identified an unconventional myosin gene (MYO3) in the yeast Saccharomyces cerevisiae and show that it is member of a subclass of unconventional myosin proteins originally found only in the amoeboid organisms Dictyostelium and Acanthamoeba. Identification of this protein in these genetically and morphologically divergent organisms suggests that it will be ubiquitous in eukaryotes and that it has a role in the basic functions of the eukaryotic cell. We have constructed a strain of yeast missing 99% of the MYO3 coding sequence. This mutation has no observable phenotypic effect, placing MYO3 into a growing class of yeast genes which are dispensable under laboratory conditions, perhaps due to genetic redundancy. Alignment of MYO3 with other unconventional myosins shows that it shares with a subset of them a previously unrecognized region of homology in the tail; this region falls within a domain identified as important for mediating nonspecific electrostatic interactions with membranes. The existence of this region suggests that it may be involved in mediating specific protein-protein interactions, possibly helping to localize this myosin to specific membranes or membrane regions. In addition, we show that "classic" myosin I proteins share a region of hyper-proline-richness 10 amino acids before the SH3 domain. Proline-rich regions have recently been implicated as SH3 binding sites, which suggests that this region might be involved with regulating or in other ways interacting with SH3 domains.

  View details for Web of Science ID A1995QJ72100008

  View details for PubMedID 7728870

• Myosin structure and function Cold Spring Harbor Symposia on Quantitative Biology - Protein Kinesis: The Dynamics of Protein Trafficking and Stability Spudich, J. A., Finer, J., Simmons, B., Ruppel, K., Patterson, B., Uyeda, T. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1995: 783–791

  View details for Web of Science ID A1995VA12500083

  View details for PubMedID 8824453

• MOW MOLECULAR MOTORS WORK NATURE Spudich, J. A. 1994; 372 (6506): 515-518

  Abstract

  What is the molecular basis of cell movement and changes in cell shape? The integration of three approaches is revealing how the molecular motors that drive these processes move and produce force.

  View details for Web of Science ID A1994PW08200042

  View details for PubMedID 7990922

• MOLECULAR-GENETIC TRUNCATION ANALYSIS OF FILAMENT ASSEMBLY AND PHOSPHORYLATION DOMAINS OF DICTYOSTELIUM MYOSIN HEAVY-CHAIN JOURNAL OF CELL SCIENCE Lee, R. J., Egelhoff, T. T., Spudich, J. A. 1994; 107: 2875-2886

  Abstract

  Conventional myosin ('myosin II') is a major component of the cytoskeleton in a wide variety of eukaryotic cells, ranging from lower amoebae to mammalian fibroblasts and neutrophils. Gene targeting technologies available in the Dictyostelium discoideum system have provided the first genetic proof that this molecular motor protein is essential for normal cytokinesis, capping of cell surface receptors, normal chemotactic cell locomotion and morphogenetic shape changes during development. Although the roles of myosin in a variety of cell functions are becoming clear, the mechanisms that regulate myosin assembly into functional bipolar filaments within cells are poorly understood. Dictyostelium is currently the only system where mutant forms of myosin can be engineered in vitro, then expressed in their native context in cells that are devoid of the wild-type isoform. We have utilized this technology in combination with nested truncation and deletion analysis to map domains of the myosin tail necessary for in vivo and in vitro filament assembly, and for normal myosin heavy chain (MHC) phosphorylation. This analysis defines a region of 35 amino acids within the tail that is critical for filament formation both for purified myosin molecules and for myosin within the in vivo setting. Phosphorylation analysis of these mutants in intact cytoskeletons demonstrates that the carboxy-terminal tip of the myosin heavy chain is required for complete phosphorylation of the myosin tail.

  View details for Web of Science ID A1994PQ38000020

  View details for PubMedID 7876354

• STAGE-SPECIFIC REQUIREMENT FOR MYOSIN-II DURING DICTYOSTELIUM DEVELOPMENT DEVELOPMENT Springer, M. L., Patterson, B., Spudich, J. A. 1994; 120 (9): 2651-2660

  Abstract

  Dictyostelium cells that lack a functional myosin II heavy chain are motile and are capable of aggregation, but fail to undergo further multicellular development. We have used a Dictyostelium mutant expressing a cold-sensitive myosin heavy chain to examine the requirement for myosin throughout the course of development. The loss of myosin function upon cooling is rapid and reversible. Temperature-shift experiments reveal that myosin is essential during two different stages of development. During aggregation, myosin function appears to be necessary for cells to sort correctly in a way that allows further development to occur. During the final stage of development, it is required for the formation of a complete stalk and the raising of the spore head. Development between those stages, however, proceeds normally in the absence of myosin function. Aggregates at non-permissive temperature undergo an aberrant form of development resulting in a ball of cells. Calcofluor staining and reporter gene fusions reveal that these structures contain defective spores and a miniature stalk.

  View details for Web of Science ID A1994PF17600027

  View details for PubMedID 7956839

• ROLE OF HIGHLY CONSERVED LYSINE-130 OF MYOSIN MOTOR DOMAIN - IN-VIVO AND IN-VITRO CHARACTERIZATION OF SITE-SPECIFICALLY MUTATED MYOSIN JOURNAL OF BIOLOGICAL CHEMISTRY Ruppel, K. M., Uyeda, T. Q., Spudich, J. A. 1994; 269 (29): 18773-18780

  Abstract

  We have created a mutant Dictyostelium myosin II heavy chain gene in which a highly conserved lysine residue (Lys-130) is changed to leucine. Lys-130 is a residue that is known to be trimethylated in skeletal muscle myosin and had been thought to play an integral role in the interaction of myosin with ATP during the actomyosin chemomechanical cycle. We report here the first in vivo and in vitro characterization of an engineered missense mutation in the motor domain of myosin. Expression of the K130L myosin in a Dictyostelium strain that lacks the myosin II heavy chain gene is sufficient to restore the ability of that cell line to undergo cytokinesis and multicellular development, processes that require functional myosin. The K130L myosin purified from these cells displays maximal actin-activated ATPase activities and promotes maximal sliding velocities of actin filaments in an in vitro motility assay that are comparable with those of wild-type myosin. These results demonstrate that this lysine residue is not required for the enzymatic or motile activities of myosin. However, the mutant protein exhibits a 4-fold increase in Km for ATP over wild-type myosin, indicating that this residue participates in the interaction of myosin with its nucleotide substrate.

  View details for Web of Science ID A1994NX32700016

  View details for PubMedID 8034630

• ENZYMATIC-ACTIVITIES CORRELATE WITH CHIMERIC SUBSTITUTIONS AT THE ACTIN-BINDING FACE OF MYOSIN NATURE Uyeda, T. Q., Ruppel, K. M., Spudich, J. A. 1994; 368 (6471): 567-569

  Abstract

  Myosins are a functionally divergent group of mechanochemical enzymes involved in various motile activities in cells. Despite a high degree of conservation in the amino-acid sequence of the 130K motor domain (head region) of the molecule, there are large differences in the enzymatic and motile activities (Tables 1 and 2) of myosins from diverse species and cell types. However, the degree of conservation is not uniform throughout the head sequence; therefore, one reasonable hypothesis is that the functional differences between myosins derive from the poorly conserved areas. The most prominent divergent region occurs at the 50K/20K junction, a region of the molecule sensitive to proteolytic digestion and a binding site for actin. We have now constructed chimaeras of this region of myosin by substituting the 9-amino-acid Dictyostelium junction region with those from myosins from other species and find that the actin-activated ATPase correlates well with the activity of the myosin from which the junction region was derived. Our results suggest that this region, likely to be part of the myosin head that interacts directly with actin, is important in determining the enzymatic activity of myosin.

  View details for Web of Science ID A1994NE33500061

  View details for PubMedID 8139694

• FUNCTIONAL-ANALYSIS OF A CARDIAC MYOSIN ROD IN DICTYOSTELIUM-DISCOIDEUM CELL MOTILITY AND THE CYTOSKELETON LEBLANCSTRACESKI, J. M., Fukui, Y., Sohn, R. L., Spudich, J. A., Leinwand, L. A. 1994; 27 (4): 313-326

  Abstract

  Manipulation of the single conventional myosin heavy chain (mhc) gene in Dictyostelium discoideum (Dd) has delineated an essential role for the filament-forming, or light meromyosin (LMM) domain of the myosin molecule in cytokinesis, development, and in the capping of cell surface receptors (see Spudich: Cell Regulation 1:1-11, 1989; Egelhoff et al.: Journal of Cell Biology, 112:677-688, 1991a). In order to assess the functional relationship between sarcomeric and cytoplasmic myosins, a chimeric gene encoding the Dd myosin head and subfragment 2 fused to rat beta cardiac LMM was transfected into both wild-type and Dd mhc null cells. Chimeric myosin was organized into dense cortical patches in the cytoplasm of both wild-type and Dd mhc null cells. Although null cells expressing chimeric mhc at approximately 10% of Dd mhc levels were unable to grow in shaking suspension or to complete development, chimeric myosin was able to rescue capping of cell surface receptors, to associate with filamentous actin, and to localize to the correct subcellular position during aggregation. Deletion of 29 amino acids in the rod corresponding to a previously defined filament assembly competent region eliminated the cortical patches and the posterior localization during chemotaxis. Taken together, these observations suggest that sarcomeric and cytoplasmic myosin rods are functionally interchangeable in several aspects of nonmuscle motility.

  View details for Web of Science ID A1994NB82600003

  View details for PubMedID 8069939

• A FUNCTIONAL RECOMBINANT MYOSIN-II LACKING A REGULATORY LIGHT-CHAIN BINDING-SITE SCIENCE Uyeda, T. Q., Spudich, J. A. 1993; 262 (5141): 1867-1870

  Abstract

  Myosin II, which converts the energy of adenosine triphosphate hydrolysis into the movement of actin filaments, is a hexamer of two heavy chains, two essential light chains, and two regulatory light chains (RLCs). Dictyostelium myosin II is known to be regulated in vitro by phosphorylation of the RLC. Cells in which the wild-type myosin II heavy chain was replaced with a recombinant form that lacks the binding site for RLC carried out cytokinesis and almost normal development, processes known to be dependent on functional myosin II. Characterization of the purified recombinant protein suggests that a complex of RLC and the RLC binding site of the heavy chain plays an inhibitory role for adenosine triphosphatase activity and a structural role for the movement of myosin along actin.

  View details for Web of Science ID A1993MM51100034

  View details for PubMedID 8266074

• DICTYOSTELIUM MYOSIN HEAVY-CHAIN PHOSPHORYLATION SITES REGULATE MYOSIN FILAMENT ASSEMBLY AND LOCALIZATION IN-VIVO CELL Egelhoff, T. T., Lee, R. J., Spudich, J. A. 1993; 75 (2): 363-371

  Abstract

  Three threonine residues in the tail region of Dictyostelium myosin II heavy chain have been implicated previously in control of myosin filament formation. Here we report the in vitro and in vivo consequences of converting these sites to alanine residues, which eliminates phosphorylation at these positions, or to aspartate residues, which mimics the negative charge state of the phosphorylated molecule. Alanine substitution allows in vitro assembly and in vivo contractile activity, although this myosin shows substantial over-assembly in vivo. Aspartate substitution eliminates filament assembly in vitro and renders the myosin unable to drive any tested contractile event in vivo. These results demonstrate that heavy chain phosphorylation plays a key modulatory role in controlling myosin function in vivo.

  View details for Web of Science ID A1993MD88500017

  View details for PubMedID 7691416

• THE UNCONVENTIONAL MYOSIN ENCODED BY THE MYOA GENE PLAYS A ROLE IN DICTYOSTELIUM MOTILITY MOLECULAR BIOLOGY OF THE CELL Titus, M. A., Wessels, D., Spudich, J. A., Soll, D. 1993; 4 (2): 233-246

  Abstract

  The myoA gene of Dictyostelium is a member of a gene family of unconventional myosins. The myosin Is share homologous head and basic domains, but the myoA gene product lacks the glycine-, proline-, alanine-rich and src homology 3 domains typical of several of the other myosin Is. A mutant strain of Dictyostelium lacking a functional myoA gene was produced by gene targeting, and the motility of this strain in buffer and a spatial gradient of the chemoattractant cyclic AMP was analyzed by computer-assisted methods. The myoA- cells have a normal elongate morphology in buffer but exhibit a decrease in the instantaneous velocity of cellular translocation, an increase in the frequency of lateral pseudopod formation, and an increase in turning. In a spatial gradient, in which the frequency of pseudopod formation is depressed, myoA- cells exhibit positive chemotaxis but still turn several times more frequently than control cells. These results demonstrate that the other members of the unconventional myosin family do not fully compensate for the loss of functional myoA gene product. Surprisingly, the phenotype of the myoA- strain closely resembles that of the myoB- strain, suggesting that both play a role in the frequency of pseudopod formation and turning during cellular translocation.

  View details for Web of Science ID A1993KQ09200009

  View details for PubMedID 8382977

• MOLECULAR EVOLUTION OF THE MYOSIN FAMILY - RELATIONSHIPS DERIVED FROM COMPARISONS OF AMINO-ACID-SEQUENCES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goodson, H. V., Spudich, J. A. 1993; 90 (2): 659-663

  Abstract

  To examine the evolutionary relationships between members of the myosin family, we have used two different phylogenetic methods, distance matrix and maximum parsimony, to analyze all available myosin head sequences. We find that there are at least three equally divergent classes of myosin, demonstrating that the current classification of myosin into only two classes needs to be reexamined. In the myosin II class, smooth muscle myosin is more closely related to nonmuscle myosin than to striated muscle myosin, implying that smooth muscle and skeletal muscle myosins were independently derived from nonmuscle myosin and suggesting that similarities between these types of muscle are the result of convergent evolution. The grouping of head sequences produced by phylogenetic analysis is consistent with classifications based on enzymology and structural localization and is generally consistent with grouping based on common tail structure elements. This result demonstrates that specific head sequences are tightly coupled to specific tail sequences throughout evolution and challenges the idea that myosin heads are freely interchangeable units whose unique function is determined only by the tail structure to which it is attached.

  View details for Web of Science ID A1993KH51600062

  View details for PubMedID 8421702

• IN-VITRO METHODS FOR MEASURING FORCE AND VELOCITY OF THE ACTIN-MYOSIN INTERACTION USING PURIFIED PROTEINS METHODS IN CELL BIOLOGY, VOL 39 Warrick, H. M., Simmons, R. M., Finer, J. T., Uyeda, T. Q., Chu, S., Spudich, J. A. 1993; 39: 1-21

  View details for Web of Science ID A1993BZ58U00001

  View details for PubMedID 8246790

• A DICTYOSTELIUM MYOSIN-II LACKING A PROXIMAL 58-KDA PORTION OF THE TAIL IS FUNCTIONAL INVITRO AND INVIVO MOLECULAR BIOLOGY OF THE CELL KUBALEK, E. W., Uyeda, T. Q., Spudich, J. A. 1992; 3 (12): 1455-1462

  Abstract

  We used molecular genetic approaches to delete 521 amino acid residues from the proximal portion of the Dictyostelium myosin II tail. The deletion encompasses approximately 40% of the tail, including the S2-LMM junction, a region that in muscle myosin II has been proposed to be important for contraction. The functions of the mutant myosin II are indistinguishable from the wild-type myosin II in our in vitro assays. It binds to actin in a typical rigor configuration in the absence of ATP and it forms filaments in a normal salt-dependent manner. In an in vitro motility assay, both monomeric and filamentous forms of the mutant myosin II translocate actin filaments at 2.4 microns/s at 30 degrees C, similar to that of wild-type myosin II. The mutant myosin II is also functional in vivo. Cells expressing the mutant myosin II in place of the native myosin II perform myosin II-dependent activities such as cytokinesis and formation of fruiting bodies, albeit inefficiently. Growth of the mutant cells in suspension gives rise to many large multinucleated cells, demonstrating that cytokinesis often fails. The majority of the fruiting bodies are also morphologically abnormal. These results demonstrate that this region of the myosin II tail is not required for motile activities but its presence is necessary for optimum function in vivo.

  View details for Web of Science ID A1992KF45200013

  View details for PubMedID 1493338

• MEMBRANE-BOUND DICTYOSTELIUM MYOSIN HEAVY-CHAIN KINASE - A DEVELOPMENTALLY REGULATED SUBSTRATE-SPECIFIC MEMBER OF THE PROTEIN-KINASE-C FAMILY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ravid, S., Spudich, J. A. 1992; 89 (13): 5877-5881

  Abstract

  A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the Dictyostelium MHCK is a distinct member of the protein kinase C family and imply that this kinase family, which may include members with very specific cellular functions, may be even more heterogeneous than previously thought.

  View details for Web of Science ID A1992JC86800037

  View details for PubMedID 1321427

• YEAST ACTIN-FILAMENTS DISPLAY ATP-DEPENDENT SLIDING MOVEMENT OVER SURFACES COATED WITH RABBIT MUSCLE MYOSIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kron, S. J., Drubin, D. G., Botstein, D., Spudich, J. A. 1992; 89 (10): 4466-4470

  Abstract

  The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because it is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act1 mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been significant conservation of actin function, specifically the ability to be moved by interaction with myosin.

  View details for Web of Science ID A1992HU97700051

  View details for PubMedID 1533933

• CONTROL OF NONMUSCLE MYOSINS BY PHOSPHORYLATION ANNUAL REVIEW OF BIOCHEMISTRY Tan, J. L., Ravid, S., Spudich, J. A. 1992; 61: 721-759

  View details for Web of Science ID A1992JE31500025

  View details for PubMedID 1497323

• Molecular genetic approaches to the cytoskeleton in Dictyostelium. Current opinion in genetics & development Patterson, B., Ruppel, K. M., Spudich, J. A. 1991; 1 (3): 378-382

  Abstract

  Recent advances in molecular genetic techniques are being applied in Dictyostelium to test and expand prevailing views on the functioning of the actin-based cytoskeleton. Current research involves the disruption, by homologous recombination, of genes encoding cytoskeletal elements. We suggest combining classical and molecular genetic approaches to supplement these investigations.

  View details for PubMedID 1840895

• CHARACTERIZATION AND BACTERIAL EXPRESSION OF THE DICTYOSTELIUM MYOSIN LIGHT CHAIN KINASE CDNA - IDENTIFICATION OF AN AUTOINHIBITORY DOMAIN JOURNAL OF BIOLOGICAL CHEMISTRY Tan, J. L., Spudich, J. A. 1991; 266 (24): 16044-16049

  Abstract

  A full-length cDNA corresponding to the Dictyostelium myosin light chain kinase gene has been isolated and characterized. Sequence analysis of the cDNA confirms conserved protein kinase subdomains and reveals that the Dictyostelium sequence is highly homologous to those of calcium/calmodulin-dependent protein kinases, including myosin light chain kinases from higher eukaryotes. Despite the high homologies to calcium/calmodulin-dependent protein kinases, there is no recognizable calmodulin-binding domain within the Dictyostelium sequence. However, the Dictyostelium myosin light chain kinase possesses a putative auto-inhibitory domain near its carboxyl terminus. To further characterize this domain, the full-length enzyme as well as a truncated form lacking this domain were expressed in bacterial cells and purified. The full-length enzyme expressed in bacteria exhibits essentially the same biochemical characteristics as the enzyme isolated from Dictyostelium. The truncated form however exhibits a Vmax that is approximately ten times greater than that of the native enzyme. In addition, unlike the native kinase and the full-length kinase expressed in bacteria, the truncated enzyme does not undergo autophosphorylation. These results suggest that the Dictyostelium enzyme, like myosin light chain kinases from higher eukaryotes, is regulated by an autoinhibitory domain but that the specific molecular signals necessary for activation of the Dictyostelium enzyme are entirely distinct.

  View details for Web of Science ID A1991GB97700078

  View details for PubMedID 1651931

• QUANTIZED VELOCITIES AT LOW MYOSIN DENSITIES IN AN INVITRO MOTILITY ASSAY NATURE Uyeda, T. Q., Warrick, H. M., Kron, S. J., Spudich, J. A. 1991; 352 (6333): 307-311

  Abstract

  An in vitro motility assay has been developed in which single actin filaments move on one or a few heavy meromyosin (HMM) molecules. This movement is slower than when many HMM molecules are involved, in contrast to analogous experiments with microtubules and kinesin. Frequency analysis shows that sliding speeds distribute around integral multiples of a unitary velocity. This discreteness may be due to differences in the numbers of HMM molecules interacting with each actin filament, where the unitary velocity reflects the activity of one HMM molecule. The value of the unitary velocity predicts a step size of 5-20 nm per ATP, which is consistent with the conventional swinging crossbridge model for myosin function.

  View details for Web of Science ID A1991FY28900060

  View details for PubMedID 1852205

• MOLECULAR-GENETICS OF CELL-MIGRATION - DICTYOSTELIUM AS A MODEL SYSTEM TRENDS IN GENETICS Egelhoff, T. T., Spudich, J. A. 1991; 7 (5): 161-166

  Abstract

  A central unresolved issue in modern cell biology concerns how eukaryotic cell migration is achieved. Although the underlying mechanics of cell locomotion appear similar in cells ranging from amoebae to leukocytes, the organisms that have been historically studied have not been amenable to the techniques of modern molecular genetics. The recent development of high-efficiency gene targeting technology for Dictyostelium discoideum, coupled with the classic cell migration behavior of this organism, offers an opportunity to resolve many of the controversial issues concerning cell locomotion.

  View details for Web of Science ID A1991FJ47600007

  View details for PubMedID 2068788

• SPATIAL AND TEMPORAL CONTROL OF NONMUSCLE MYOSIN LOCALIZATION - IDENTIFICATION OF A DOMAIN THAT IS NECESSARY FOR MYOSIN FILAMENT DISASSEMBLY INVIVO JOURNAL OF CELL BIOLOGY Egelhoff, T. T., Brown, S. S., Spudich, J. A. 1991; 112 (4): 677-688

  Abstract

  Myosin null mutants of Dictyostelium are defective for cytokinesis, multicellular development, and capping of surface proteins. We have used these cells as transformation recipients for an altered myosin heavy chain gene that encodes a protein bearing a carboxy-terminal 34-kD truncation. This truncation eliminates threonine phosphorylation sites previously shown to control filament assembly in vitro. Despite restoration of growth in suspension, development, and ability to cap cell surface proteins, these delta C34-truncated myosin transformants display severe cytoskeletal abnormalities, including excessive localization of the truncated myosin to the cortical cytoskeleton, impaired cell shaped dynamics, and a temporal defect in myosin dissociation from beneath capped surface proteins. These data demonstrate that the carboxy-terminal domain of myosin plays a critical role in regulating the disassembly of the protein from contractile structures in vivo.

  View details for Web of Science ID A1991EX92000014

  View details for PubMedID 1899668

• MANIPULATION AND EXPRESSION OF MOLECULAR MOTORS IN DICTYOSTELIUM-DISCOIDEUM WORKSHOP ON MOTOR PROTEINS Manstein, D. J., Ruppel, K. M., KUBALEK, L., Spudich, J. A. COMPANY OF BIOLOGISTS LTD. 1991: 63–65

  Abstract

  The eukaryote Dictyostelium discoideum is an attractive model organism for the study of cytoskeletal proteins and cell motility. The appearance and behavior of this cell closely resembles that of mammalian cells, but unlike mammalian cells, Dictyostelium offers the opportunity specifically to alter the cell physiology by molecular genetic approaches.

  View details for Web of Science ID A1991FK90200014

  View details for PubMedID 1885661

• ASSAYS FOR ACTIN SLIDING MOVEMENT OVER MYOSIN-COATED SURFACES METHODS IN ENZYMOLOGY Kron, S. J., Toyoshima, Y. Y., Uyeda, T. Q., Spudich, J. A. 1991; 196: 399-416

  View details for Web of Science ID A1991FN84100034

  View details for PubMedID 2034132

• MOLECULAR GENETIC TOOLS FOR STUDY OF THE CYTOSKELETON IN DICTYOSTELIUM METHODS IN ENZYMOLOGY Egelhoff, T. T., Titus, M. A., Manstein, D. J., Ruppel, K. M., Spudich, J. A. 1991; 196: 319-334

  View details for Web of Science ID A1991FN84100028

  View details for PubMedID 2034127

• AN APPROACH TO RECONSTITUTING MOTILITY OF SINGLE MYOSIN MOLECULES WORKSHOP ON MOTOR PROTEINS Kron, S. J., Uyeda, T. Q., Warrick, H. M., Spudich, J. A. COMPANY OF BIOLOGISTS LTD. 1991: 129–133

  Abstract

  Over the last five years, the value of in vitro motility assays as probes of the mechanical properties of the actin-myosin interaction has been amply demonstrated. Motility assays in which single fluorescent actin filaments are observed moving over surfaces coated with myosin or its soluble fragments are now used in many laboratories. They have been applied to a wide range of problems including the study of structure-function relationships in the myosin molecule and measurement of fundamental properties of the myosin head. However, one limitation of these assays has been uncertainty over the number of myosin heads interacting with each sliding filament, that frustrates attempts to determine properties of individual heads. In order to address this limitation, we have modified the conditions of the actin sliding filament assay to reduce the number of heads interacting with each filament. Our goal is to establish an assay in which the motor function of a single myosin head can be characterized from the movement of a single actin filament.

  View details for Web of Science ID A1991FK90200027

  View details for PubMedID 1885651

• GENETICALLY ENGINEERED TRUNCATED MYOSIN IN DICTYOSTELIUM - THE CARBOXYL-TERMINAL REGULATORY DOMAIN IS NOT REQUIRED FOR THE DEVELOPMENTAL CYCLE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA OHALLORAN, T. J., Spudich, J. A. 1990; 87 (20): 8110-8114

  Abstract

  The study of engineered Dictyostelium mutants with altered or missing myosin has revealed the molecule to be essential both for cytokinesis and for completion of the complex Dictyostelium developmental cycle. To explore the biological role of the carboxyl-terminal portion of the myosin tail, we have created a Dictyostelium cell line bearing a mutation designated my delta C34 in the myosin (mhcA) locus. This cell line produces a truncated myosin protein lacking the 34-kDa carboxyl terminus of the wild-type tail. Southern blots of the mutant cells show that the myosin gene was disrupted by homologous recombination of the transforming plasmid into the myosin locus. Based on in vitro studies of myosin functional domains, the 200-kDa truncated myosin was designed to include a domain important for assembly but to eliminate a domain important for threonine phosphorylation. The mutant cells are defective in cytokinesis, similar to those mutants that are either devoid of myosin (null cells) or contain a truncated 140-kDa myosin (hmm cells). However, unlike previous mutants, the cells carrying the my delta C34 mutation are able to complete the Dictyostelium developmental cycle to form fruiting bodies. Thus a truncated 200-kDa myosin can substitute for native myosin to function in developing cells. These results demonstrate that the 34-kDa carboxyl terminus of myosin, which contributes regulated phosphorylation sites and 20% of the total length of the rod, is not required for the developmental cycle of Dictyostelium.

  View details for Web of Science ID A1990ED61200066

  View details for PubMedID 2236024

• THE MYOSIN STEP SIZE - MEASUREMENT OF THE UNIT DISPLACEMENT PER ATP HYDROLYZED IN AN INVITRO ASSAY PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Toyoshima, Y. Y., Kron, S. J., Spudich, J. A. 1990; 87 (18): 7130-7134

  Abstract

  Chemomechanical coupling in muscle contraction may be due to "swinging crossbridges," such that a change in the angle at which the myosin head binds to the actin filament is tightly coupled to release of products of ATP hydrolysis. This model would limit the step size, the unit displacement of actin produced by a single ATP hydrolysis, to less than twice the chord length of the myosin head. Recent measurements have found the step size to be significantly larger than this geometric limit, bringing into question any direct correspondence between the crossbridge and ATP-hydrolysis cycles. We have measured the rate of ATP hydrolysis due to actin sliding movement in an in vitro motility assay consisting of purified actin and purified myosin. We have calculated an apparent myosin step size well within the geometric limit set by the size of the myosin head. These data are consistent with tight coupling between myosin crossbridge movement and ATP hydrolysis.

  View details for Web of Science ID A1990DZ45300043

  View details for PubMedID 2144900

• DICTYOSTELIUM MYOSIN LIGHT CHAIN KINASE - PURIFICATION AND CHARACTERIZATION JOURNAL OF BIOLOGICAL CHEMISTRY Tan, J. L., Spudich, J. A. 1990; 265 (23): 13818-13824

  Abstract

  A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.

  View details for Web of Science ID A1990DU27500064

  View details for PubMedID 2380188

• MYOSIN STEP SIZE - ESTIMATION FROM SLOW SLIDING MOVEMENT OF ACTIN OVER LOW-DENSITIES OF HEAVY-MEROMYOSIN JOURNAL OF MOLECULAR BIOLOGY Uyeda, T. Q., Kron, S. J., Spudich, J. A. 1990; 214 (3): 699-710

  Abstract

  We have estimated the step size of the myosin cross-bridge (d, displacement of an actin filament per one ATP hydrolysis) in an in vitro motility assay system by measuring the velocity of slowly moving actin filaments over low densities of heavy meromyosin on a nitrocellulose surface. In previous studies, only filaments greater than a minimum length were observed to undergo continuous sliding movement. These filaments moved at the maximum speed (Vo), while shorter filaments dissociated from the surface. We have now modified the assay system by including 0.8% methylcellulose in the ATP solution. Under these conditions, filaments shorter than the previous minimum length move, but significantly slower than Vo, as they are propelled by a limited number of myosin heads. These data are consistent with a model that predicts that the sliding velocity (v) of slowly moving filaments is determined by the product of vo and the fraction of time when at least one myosin head is propelling the filament, that is, v = vo [1-(1-ts/tc)N], where ts is the time the head is strongly bound to actin, tc is the cycle time of ATP hydrolysis, and N is the average number of myosin heads that can interact with the filament. Using this equation, the optimum value of ts/tc to fit the measured relationship between v and N was calculated to be 0.050. Assuming d = vots, the step size was then calculated to be between 10nm and 28 nm per ATP hydrolyzed, the latter value representing the upper limit. This range is within that of geometric constraint for conformational change imposed by the size of the myosin head, and therefore is not inconsistent with the swinging cross-bridge model tightly coupled with ATP hydrolysis.

  View details for Web of Science ID A1990DU27400013

  View details for PubMedID 2143785

• SUBTILISIN CLEAVAGE OF ACTIN INHIBITS INVITRO SLIDING MOVEMENT OF ACTIN-FILAMENTS OVER MYOSIN JOURNAL OF CELL BIOLOGY SCHWYTER, D. H., Kron, S. J., Toyoshima, Y. Y., Spudich, J. A., Reisler, E. 1990; 111 (2): 465-470

  Abstract

  Subtilisin cleaved actin was shown to retain several properties of intact actin including the binding of heavy meromyosin (HMM), the dissociation from HMM by ATP, and the activation of HMM ATPase activity. Similar Vmax but different Km values were obtained for acto-HMM ATPase with the cleaved and intact actins. The ATPase activity of HMM stimulated by copolymers of intact and cleaved actin showed a linear dependence on the fraction of intact actin in the copolymer. The most important difference between the intact and cleaved actin was observed in an in vitro motility assay for actin sliding movement over an HMM coated surface. Only 30% of the cleaved actin filaments appeared mobile in this assay and moreover, the velocity of the mobile filaments was approximately 30% that of intact actin filaments. These results suggest that the motility of actin filaments can be uncoupled from the activation of myosin ATPase activity and is dependent on the structural integrity of actin and perhaps, dynamic changes in the actin molecule.

  View details for Web of Science ID A1990DT67800016

  View details for PubMedID 2143196

• DEVELOPMENTALLY REGULATED PROTEIN-TYROSINE KINASE GENES IN DICTYOSTELIUM-DISCOIDEUM MOLECULAR AND CELLULAR BIOLOGY Tan, J. L., Spudich, J. A. 1990; 10 (7): 3578-3583

  Abstract

  Dictyostelium discoideum, an organism that undergoes development and that is amenable to biochemical and molecular genetic approaches, is an attractive model organism with which to study the role of tyrosine phosphorylation in cell-cell communication. We report the presence of protein-tyrosine kinase genes in D. discoideum. Screening of a Dictyostelium cDNA expression library with an anti-phosphotyrosine antibody identifies fusion proteins that exhibit protein-tyrosine kinase activity. Two distinct cDNAs were identified and isolated. Though highly homologous to protein kinases in general, these kinases do not exhibit many of the hallmarks of protein-tyrosine kinases of higher eucaryotes. In addition, these genes are developmentally regulated, which suggests a role for tyrosine phosphorylation in controlling Dictyostelium development.

  View details for Web of Science ID A1990DP60000030

  View details for PubMedID 1972546

• PURIFICATION OF A FUNCTIONAL RECOMBINANT MYOSIN FRAGMENT FROM DICTYOSTELIUM-DISCOIDEUM ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Ruppel, K. M., Egelhoff, T. T., Spudich, J. A. 1990; 582: 147-155

  View details for Web of Science ID A1990DF83200014

  View details for PubMedID 2141452

• COMPLEMENTATION OF MYOSIN NULL MUTANTS IN DICTYOSTELIUM-DISCOIDEUM BY DIRECT FUNCTIONAL SELECTION DEVELOPMENTAL BIOLOGY Egelhoff, T. T., Manstein, D. J., Spudich, J. A. 1990; 137 (2): 359-367

  Abstract

  The eukaryotic slime mold Dictyostelium discoideum contains a single conventional myosin heavy chain gene (mhcA). Cell lines in which this gene was deleted via homologous recombination have been previously reported. These myosin null cells were shown to be defective for cytokinesis and for sporogenesis. We demonstrate here that the cloned mhcA gene can be reintroduced into these cells by the use of a direct functional selection. This selection was imposed by demanding that cells be capable of growth in suspension. The resulting transformants appear normal for cytokinesis, and also are fully competent for sporogenesis, confirming that reintroduction of the myosin gene is sufficient to restore these properties. These results demonstrate a method for rescuing mutants in Dictyostelium which may be generally applicable for genetically created mutations as well as for mutations which have been engineered.

  View details for Web of Science ID A1990CM89300012

  View details for PubMedID 2406175

• Molecular genetics: a key to the cytoskeleton's closet CURRENT OPINION IN CELL BIOLOGY Titus, M. A., Warrick, H. M., Spudich, J. A. 1990; 2 (1): 116-120

  View details for Web of Science ID 000209395300020

• STRUCTURE AND FUNCTION OF THE CYTOSKELETON OF A DICTYOSTELIUM MYOSIN-DEFECTIVE MUTANT JOURNAL OF CELL BIOLOGY Fukui, Y., DeLozanne, A., Spudich, J. A. 1990; 110 (2): 367-378

  Abstract

  To study the role of conventional myosin in nonmuscle cells, we determined the cytoskeletal organization and physiological responses of a Dictyostelium myosin-defective mutant. Dictyostelium hmm cells were created by insertional mutagenesis of the myosin heavy chain gene (De Lozanne, A., and J. A. Spudich. 1987. Science (Wash. DC). 236: 1086-1091). Western blot analysis using different mAbs confirms that hmm cells express a truncated myosin fragment of 140 kD (HMM-140 protein) instead of the normal 243-kD myosin heavy chain (MHC). Spontaneous revertants appear at a frequency less than 4 x 10(-5), which synthesize normal myosin and are capable of forming thick filaments. In hmm cells, the HMM-140 protein is diffusely distributed in the cytoplasm, indicating that it cannot assemble into thick filaments. The actin distribution in these mutant cells appears similar to that of wild-type cells. However, there is a significant abnormality in the organization of cytoplasmic microtubules, which penetrate into lamellipodial regions. The microtubule networks consist of approximately 13 microtubules on average and their pattern is abnormal. Although hmm cells can form mitotic spindles, mitosis is not coordinated with normal furrow formation. The hmm cells are clearly defective in the contractile events that lead to normal cytokinesis. The retraction of different regions of the cell can result in the occasional pinching off of part of the cell. This process is not coupled with formation of mitotic spindles. There is no specific accumulation of HMM-140 in such constrictions, whereas 73% of such cells show actin concentrated in these regions. The mutant hmm cells are also deficient in capping of Con-A-bound surface receptors, but instead internalize this complex into the cytoplasm. The hmm cells display active phagocytosis of bacteria. Whereas actin is concentrated in the phagocytic cups, HMM-140 protein is not localized in these regions. cAMP, a chemoattractant that induces drastic rounding up and formation of surface blebs in wild type cells, does not induce rounding up in the hmm cells. A Triton-permeabilized cell model of the wild-type amebae contracts on reactivation with Mg-ATP, whereas a model of the hmm cell shows no detectable contraction. Our data demonstrate that the conventional myosin participates in the significant cortical motile activities of Dictyostelium cells, which include rounding up, constriction of cleavage furrows, capping surface receptors, and establishing cell polarity.

  View details for Web of Science ID A1990CM83300013

  View details for PubMedID 2404992

• Molecular genetics: a key to the cytoskeleton's closet. Current opinion in cell biology Titus, M. A., Warrick, H. M., Spudich, J. A. 1990; 2 (1): 116-120

  View details for PubMedID 2183835

• EXPRESSION OF DICTYOSTELIUM MYOSIN TAIL SEGMENTS IN ESCHERICHIA-COLI - DOMAINS REQUIRED FOR ASSEMBLY AND PHOSPHORYLATION JOURNAL OF CELL BIOLOGY OHALLORAN, T. J., Ravid, S., Spudich, J. A. 1990; 110 (1): 63-70

  Abstract

  The assembly of myosins into filaments is a property common to all conventional myosins. The ability of myosins to form filaments is conferred by the tail of the large asymmetric molecule. We are studying cloned portions of the Dictyostelium myosin gene expressed in Escherichia coli to investigate functional properties of defined segments of the myosin tail. We have focused on five segments derived from the 68-kD carboxyl-terminus of the myosin tail. These have been expressed and purified to homogeneity from E. coli, and thus the boundaries of each segment within the myosin gene and protein sequence are known. We identified an internal 34-kD segment of the tail, N-LMM-34, which is required and sufficient for assembly. This 287-amino acid domain represents the smallest tail segment purified from any myosin that is capable of forming highly ordered paracrystals characteristic of myosin. Because the assembly of Dictyostelium myosin can be regulated by phosphorylation of the heavy chain, we have studied the in vitro phosphorylation of the expressed tail segments. We have determined which segments are phosphorylated to a high level by a Dictyostelium myosin heavy chain kinase purified from developed cells. While LMM-68, the 68-kD carboxyl terminus of Dictyostelium myosin, or LMM-58, which lacks the 10-kD carboxyl terminus of LMM-68, are phosphorylated to the same extent as purified myosin, subdomains of these segments do not serve as efficient substrates for the kinase. Thus LMM-58 is one minimal substrate for efficient phosphorylation by the myosin heavy chain kinase purified from developed cells. Taken together these results identify two functional domains in Dictyostelium myosin: a 34-kD assembly domain bounded by amino acids 1533-1819 within the myosin sequence and a larger 58-kD phosphorylation domain bounded by amino acids 1533-2034 within the myosin sequence.

  View details for Web of Science ID A1990CJ68900007

  View details for PubMedID 2404023

• PURIFICATION OF A FUNCTIONAL RECOMBINANT MYOSIN FRAGMENT FROM DICTYOSTELIUM-DISCOIDEUM WORKSHOP ON CYTOKINESIS : MECHANISMS OF FURROW FORMATION DURING CELL DIVISION Ruppel, K. M., Egelhoff, T. T., Spudich, J. A. NEW YORK ACAD SCIENCES. 1990: 147–155

  View details for Web of Science ID A1990BQ86N00014

• EXPRESSION AND CHARACTERIZATION OF A FUNCTIONAL MYOSIN HEAD FRAGMENT IN DICTYOSTELIUM-DISCOIDEUM SCIENCE Manstein, D. J., Ruppel, K. M., Spudich, J. A. 1989; 246 (4930): 656-658

  Abstract

  The isolated head fragment of myosin is a motor protein that is able to use energy liberated from the hydrolysis of adenosine triphosphate to cause sliding movement of actin filaments. Expression of a myosin fragment nearly equivalent to the amino-terminal globular head domain, generally referred to as subfragment 1, has been achieved by transforming the eukaryotic organism Dictyostelium discoideum with a plasmid that carries a 2.6-kilobase fragment of the cloned Dictyostelium myosin heavy chain gene under the control of the Dictyostelium actin-15 promoter. The recombinant fragment of the myosin heavy chain was purified 2400-fold from one of the resulting cell lines and was found to be functional by the following criteria: the myosin head fragment copurified with the essential and regulatory myosin light chains, decorated actin filaments, and displayed actin-activated adenosine triphosphatase activity. In addition, motility assays in vitro showed that the recombinant myosin fragment is capable of supporting sliding movement of actin filaments.

  View details for Web of Science ID A1989AX71200039

  View details for PubMedID 2530629

• MULTIPLE ACTIN-BASED MOTOR GENES IN DICTYOSTELIUM CELL REGULATION Titus, M. A., Warrick, H. M., Spudich, J. A. 1989; 1 (1): 55-63

  Abstract

  Dictyostelium cells, devoid of conventional myosin, display a variety of motile activities, consistent with the presence of other molecular motors. The Dictyostelium genome was probed at low stringency with a gene fragment containing the conserved conventional myosin head domain sequences to identify other actin-based motors that may play a role in the observed motility of these mutant cells. One gene (abmA) has been characterized and encodes a polypeptide of approximately 135 kDa with a head region homologous to other myosin head sequences and a tail region that is not predicted to form either an alpha-helical structure of coiled-coil interactions. Comparisons of the amino acid sequences of the tail regions of abmA, Dictyostelium myosin I, and Acanthamoeba myosins IB and IL reveal an area of sequence similarity in the amino terminal half of the tail that may be a membrane-binding domain. The abmA gene, however, does not contain an unusual Gly, Pro, Ala stretch typical of many of the previously described myosin Is. Two additional genes (abmB and abmC) were identified using this approach and also found to contain sequences that encode proteins with typical conserved myosin head sequences. The abm genes may be part of a large family of actin-based motors that play various roles in diverse aspects of cellular motility.

  View details for Web of Science ID A1989DM09500006

  View details for PubMedID 2519618

• IN PURSUIT OF MYOSIN FUNCTION CELL REGULATION Spudich, J. A. 1989; 1 (1): 1-11

  View details for Web of Science ID A1989DM09500001

  View details for PubMedID 2519609

• CAPPING OF SURFACE-RECEPTORS AND CONCOMITANT CORTICAL TENSION ARE GENERATED BY CONVENTIONAL MYOSIN NATURE Pasternak, C., Spudich, J. A., Elson, E. L. 1989; 341 (6242): 549-551

  Abstract

  We have investigated the role of cytoskeletal contraction in the capping of surface proteins crosslinked by concanavalin A on mutant Dictyostelium cells lacking conventional myosin. Measurements of cellular deformability to indicate the development of cortical tension show that cells of the wild-type parental strain, AX4, stiffen early during capping and relax back towards the softer resting state as the process is completed. Mutant cells lacking myosin (mhcA-) have a lower resting-state stiffness, and fail to stiffen and to cap crosslinked proteins on binding concanavalin A. Hence conventional myosin is essential both for capping and for the concomitant increase in cell stiffness. Furthermore, depletion of cellular ATP by azide causes a 'rigor' contraction in AX4 cells which makes them stiffen and become spherical. By contrast, the mhcA- cells fail to respond in these ways. These measurements of cortical tension in non-muscle cells can thus be directly correlated with the presence of conventional myosin, demonstrating that contractile tension generated by myosin can drive both a change of cell shape and the capping of crosslinked surface receptors.

  View details for Web of Science ID A1989AU72100061

  View details for PubMedID 2797182

• BIDIRECTIONAL MOVEMENT OF ACTIN-FILAMENTS ALONG TRACKS OF MYOSIN HEADS NATURE Toyoshima, Y. Y., Toyoshima, C., Spudich, J. A. 1989; 341 (6238): 154-156

  Abstract

  It is well established that muscle contraction results from the relative sliding of actin and myosin filaments. Both filaments have definite polarities and well-ordered structures. Thick filaments, however, are not vital for supporting movement in vitro. Previously we have demonstrated that actin filaments can move continuously on myosin fragments (subfragment-1 or heavy meromyosin (HMM] that are bound to a nitrocellulose surface. Here we report that actin filaments can move in opposite directions on tracks of myosin heads formed when actin filaments decorated with HMM are placed on a nitrocellulose surface. The actin filaments always move forward, frequently changing the direction of the movement, but never move backward reversing the polarity of the movement. The direction of movement is therefore determined by the polarity of the actin filament. These results indicate that myosin heads have considerable flexibility.

  View details for Web of Science ID A1989AP72600065

  View details for PubMedID 2674720

• MYOSIN HEAVY-CHAIN KINASE FROM DEVELOPED DICTYOSTELIUM CELLS - PURIFICATION AND CHARACTERIZATION JOURNAL OF BIOLOGICAL CHEMISTRY Ravid, S., Spudich, J. A. 1989; 264 (25): 15144-15150

  Abstract

  We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.

  View details for Web of Science ID A1989AN59700092

  View details for PubMedID 2549052

• INTERMOLECULAR VERSUS INTRAMOLECULAR INTERACTIONS OF DICTYOSTELIUM MYOSIN - POSSIBLE REGULATION BY HEAVY-CHAIN PHOSPHORYLATION JOURNAL OF CELL BIOLOGY Pasternak, C., Flicker, P. F., Ravid, S., Spudich, J. A. 1989; 109 (1): 203-210

  Abstract

  Dictyostelium myosin has been examined under conditions that reveal intramolecular and intermolecular interactions that may be important in the process of assembly and its regulation. Rotary shadowed myosin molecules exhibit primarily two configurations under these conditions: straight parallel dimers and folded monomers. All of the monomers bend in a specific region of the 1860-A-long tail that is 1200 A from the head-tail junction. Molecules in parallel dimers are staggered by 140 A, which is a periodicity in the packing of myosin molecules originally observed in native thick filaments of muscle. The most common region for interaction in the dimers is a segment of the tail about 200-A-long, extending from 900 to 1100 A from the head-tail junction. Parallel dimers form tetramers by way of antiparallel interactions in their tail regions with overlaps in multiples of 140 A. The folded configuration of the myosin molecules is promoted by phosphorylation of the heavy chain by Dictyostelium myosin heavy chain kinase. It appears that the bent monomers are excluded from filaments formed upon addition of salt while the dimeric molecules assemble. These results may provide the structural basis for primary steps in myosin filament assembly and its regulation by heavy chain phosphorylation.

  View details for Web of Science ID A1989AF18400018

  View details for PubMedID 2745547

• HYGROMYCIN RESISTANCE AS A SELECTABLE MARKER IN DICTYOSTELIUM-DISCOIDEUM MOLECULAR AND CELLULAR BIOLOGY Egelhoff, T. T., Brown, S. S., Manstein, D. J., Spudich, J. A. 1989; 9 (5): 1965-1968

  Abstract

  We have constructed an expression cartridge which has the bacterial hygromycin resistance gene (hph) fused to the Dictyostelium discoideum actin 15 promoter, with a segment of 3'-flanking DNA from the actin 15 locus placed downstream of the hph gene to serve as a transcription terminator. The plasmid pDE109, which contained this cartridge and a Dictyostelium origin of replication, transformed D. discoideum with high efficiency under hygromycin selection. The availability of this selectable marker circumvents the previous limitation of having G418 resistance as the only selectable marker for this organism; secondary transformation can now be used to introduce DNA into previously transformed cell lines.

  View details for Web of Science ID A1989U275100018

  View details for PubMedID 2546056

• LINKAGE ANALYSIS OF THE MYOSIN HEAVY-CHAIN GENE IN DICTYOSTELIUM-DISCOIDEUM USING A MUTATION GENERATED BY HOMOLOGOUS RECOMBINATION MOLECULAR AND GENERAL GENETICS Welker, D. L., DeLozanne, A., Spudich, J. A. 1989; 216 (2-3): 498-502

  Abstract

  A mutation (mhcA1 in strain HMM) created by insertional gene inactivation was used to map the Dictyostelium discoideum myosin heavy chain gene (mhcA) to linkage group IV. Three phenotypic traits associated with this mutation (slow colony growth, inability of the mutant to develop past aggregation, and the presence of five to ten integrated vector copies) cosegregated as expected for the consequences of a single insertional event. This linkage was confirmed using a restriction fragment length polymorphism. The mhcA1 mutation was recessive to wild type and was nonallelic with mutations at the following loci on linkage group IV: aggJ, aggL. couH, minA, phgB and tsgB. This work demonstrates the ability to apply standard techniques developed for D. discoideum parasexual genetic analyses to mutants generated by transformation, which is of particular relevance to analysis of genes for which no classical mutations or restriction fragment length polymorphisms are available.

  View details for Web of Science ID A1989U365500043

  View details for PubMedID 2568578

• GENE REPLACEMENT IN DICTYOSTELIUM - GENERATION OF MYOSIN NULL MUTANTS EMBO JOURNAL Manstein, D. J., Titus, M. A., DeLozanne, A., Spudich, J. A. 1989; 8 (3): 923-932

  Abstract

  The eukaryotic slime mold Dictyostelium discoideum has a single conventional myosin heavy chain gene (mhcA). The elimination of the mhcA gene was achieved by homologous recombination. Two gene replacement plasmids were constructed, each carrying the G418 resistance gene as a selective marker and flanked by either 0.7 kb of 5' coding sequence and 0.9 kb of 3' coding sequence or 1.5 kb of 5' flanking sequence and 1.1 kb of 3' flanking sequence. Myosin null mutants (mhcA- cells) were obtained after transformation with either of these plasmids. The mhcA- cells are genetically stable and are capable of a variety of motile processes. Our results provide genetic proof that in Dictyostelium the conventional myosin gene is required for growth in suspension, normal cell division and sporogenesis, and illustrate how gene targeting can be used as a tool in Dictyostelium.

  View details for Web of Science ID A1989T691000033

  View details for PubMedID 2721503

• CHEMOATTRACTANT-ELICITED TRANSLOCATION OF MYOSIN IN MOTILE DICTYOSTELIUM CELL MOTILITY AND THE CYTOSKELETON Nachmias, V. T., Fukui, Y., Spudich, J. A. 1989; 13 (3): 158-169

  Abstract

  The distribution of myosin was studied in amebae of the Ax-3 and NC-4 strains of Dictyostelium migrating at room temperature, using indirect immunofluorescence of aggregation-competent amebae and the agar-overlay technique. Amebae were fixed in methanol-formaldehyde or absolute acetone at -15 degrees C before or after stimulation with micromolar cyclic AMP at room temperature (20-25 degrees C). Myosin was detected by monoclonal antibodies to Dictyostelium myosin heavy chain followed by a fluorescent secondary antibody that had been preabsorbed to remove nonspecific staining. In both strains there was a striking increase in intensity of anti-myosin immunofluorescence in the cortex where it appeared as a continuous ring 30 seconds after addition of cyclic AMP. This correlated with a rounding up of the cell body. Sixty seconds after stimulation there was a clear reduction of cytoplasmic myosin rods in conjunction with the increased cortical localization. At this time extensions of largely hyaline cytoplasm were observed that extended beyond the cortical shell of myosin. Two minutes after the stimulus the immunofluorescence remained as a distinct line at the cortex, but the cells began to resume in elongated shape. By 3 minutes (NC-4 strain) or 5 minutes (Ax-3 strain) the amebae had largely returned to the control shape, and myosin had returned to its control distribution. Counts of the treated cells at different time points substantiated the observations of individual cells. The time course of translocation of myosin in the Ax-3 strain parallels the time course of myosin phosphorylation reported in previous studies. The results are interpreted in terms of a working hypothesis for the mechanism of translocation.

  View details for Web of Science ID A1989AF11500003

  View details for PubMedID 2550149

• COEXPRESSION AND ASSEMBLY OF MYOSIN HEAVY-CHAIN AND MYOSIN LIGHT CHAIN IN ESCHERICHIA-COLI PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McNally, E. M., Goodwin, E. B., Spudich, J. A., Leinwand, L. A. 1988; 85 (19): 7270-7273

  Abstract

  A fragment of the Dictyostelium discoideum myosin heavy chain gene representing heavy meromyosin was coexpressed in Escherichia coli with the entire essential myosin light chain from the scallop. The expressed myosin heavy chain and essential myosin light chain copurify through ammonium sulfate fractionation, anion exchange, and gel filtration chromatography. The purified complex consists of about 1 mol of light chain per mol of heavy chain. This stoichiometry, which is that of native myosin, suggests that no special eukaryotic machinery is required for coassembly of these two proteins. By coexpressing different myosin heavy chain and myosin light chain combinations, it should be possible to study various isoforms of these two proteins, which are both products of multigene families in mammals. E. coli is thus an ideal system in which to study expression and multimeric assembly of individual components of the eukaryotic contractile apparatus.

  View details for Web of Science ID A1988Q358500049

  View details for PubMedID 3050991

• Codon preference in Dictyostelium discoideum. Nucleic acids research Warrick, H. M., Spudich, J. A. 1988; 16 (14A): 6617-6635

  Abstract

  Dictyostelium discoideum is of increasing interest as a model eukaryotic cell because its many attributes have recently been expanded to include improved genetic and biochemical manipulability. The ability to transform Dictyostelium using drug resistance as a selectable marker (1) and to gene target by high frequency homologous integration (2) makes this organism particularly useful for molecular genetic approaches to cell structure and function. Given this background, it becomes important to analyze the codon preference used in this organism. Dictyostelium displays a strong and unique overall codon preference. This preference varies between different coding regions and even varies between coding regions from the same gene family. The degree of codon preference may be correlated with expression levels but not with the developmental time of expression of the gene product. The strong codon preference can be applied to identify coding regions in Dictyostelium DNA and aid in the design of oligonucleotide probes for cloning Dictyostelium genes.

  View details for PubMedID 3399410

• CODON PREFERENCE IN DICTYOSTELIUM-DISCOIDEUM NUCLEIC ACIDS RESEARCH Warrick, H. M., Spudich, J. A. 1988; 16 (14): 6617-6635

  View details for Web of Science ID A1988P435100026

• CELL MOTILITY AND CHEMOTAXIS IN DICTYOSTELIUM AMEBAE LACKING MYOSIN HEAVY-CHAIN DEVELOPMENTAL BIOLOGY Wessels, D., Soll, D. R., Knecht, D., Loomis, W. F., DeLozanne, A., Spudich, J. 1988; 128 (1): 164-177

  Abstract

  Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.

  View details for Web of Science ID A1988P134900020

  View details for PubMedID 2838348

• SIGNAL TRANSDUCTION, CHEMOTAXIS, AND CELL-AGGREGATION IN DICTYOSTELIUM-DISCOIDEUM CELLS WITHOUT MYOSIN HEAVY-CHAIN DEVELOPMENTAL BIOLOGY Peters, D. J., Knecht, D. A., Loomis, W. F., DeLozanne, A., Spudich, J., VANHAASTERT, P. J. 1988; 128 (1): 158-163

  Abstract

  Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.

  View details for Web of Science ID A1988P134900019

  View details for PubMedID 2838347

• EXPRESSION IN ESCHERICHIA-COLI OF A FUNCTIONAL DICTYOSTELIUM MYOSIN TAIL FRAGMENT JOURNAL OF CELL BIOLOGY DeLozanne, A., Berlot, C. H., Leinwand, L. A., Spudich, J. A. 1987; 105 (6): 2999-3005

  Abstract

  The amino acid sequence of the myosin tail determines the specific manner in which myosin molecules are packed into the myosin filament, but the details of the molecular interactions are not known. Expression of genetically engineered myosin tail fragments would enable a study of the sequences important for myosin filament formation and its regulation. We report here the expression in Escherichia coli of a 1.5-kb fragment of the Dictyostelium myosin heavy chain gene coding for a 58-kD fragment of the myosin tail. The expressed protein (DdLMM-58) was purified to homogeneity from the soluble fraction of E. coli extracts. The expressed protein was found to be functional by the following criteria: (a) it appears in the electron microscope as a 74-nm-long rod, the predicted length for an alpha-helical coiled coil of 500 amino acids; (b) it assembles into filamentous structures that show the typical axial periodicity of 14 nm found in muscle myosin native filaments; (c) its assembly into filaments shows the same ionic strength dependence as Dictyostelium myosin; (d) it serves as a substrate for the Dictyostelium myosin heavy chain kinase which phosphorylates myosin in response to chemotactic signaling; (e) in its phosphorylated form it has the same phosphoamino acids and similar phosphopeptide maps to those of phosphorylated Dictyostelium myosin heavy chain; (f) it competes with myosin for the heavy chain kinase. Thus, all the information required for filament formation and phosphorylation is contained within this expressed protein.

  View details for Web of Science ID A1987L620600013

  View details for PubMedID 3320060

• MOVEMENT OF MYOSIN FRAGMENTS INVITRO - DOMAINS INVOLVED IN FORCE PRODUCTION CELL Hynes, T. R., Block, S. M., White, B. T., Spudich, J. A. 1987; 48 (6): 953-963

  Abstract

  We have used the Nitella-based movement assay to localize the site of force production in myosin. Methods were developed to use nonfilamentous myosin or proteolytic fragments of myosin in place of the thick filaments used in the original assay. In the experiments described here, the tail of myosin or its subfragments is anchored via antibodies to the surface of small particles. Nonfilamentous myosin or its subfragments move along Nitella actin cables at speeds similar to those obtained with filamentous myosin. We generated short HMM, a myosin fragment containing the heads and only 400 A of the tail. Although short HMM lacks the "hinge" region proposed by Harrington to be the site of force generation, and is incapable of forming thick filaments, it moves along actin at speeds above 1 micron/sec. Therefore, neither a thick filament nor the carboxy-terminal 1100 A of the tail is required for movement along actin. The results indicate that force production occurs in or near the myosin heads.

  View details for Web of Science ID A1987G694600006

  View details for PubMedID 3548997

• CHEMOATTRACTANT-ELICITED INCREASES IN DICTYOSTELIUM MYOSIN PHOSPHORYLATION ARE DUE TO CHANGES IN MYOSIN LOCALIZATION AND INCREASES IN KINASE-ACTIVITY JOURNAL OF BIOLOGICAL CHEMISTRY Berlot, C. H., Devreotes, P. N., Spudich, J. A. 1987; 262 (8): 3918-3926

  Abstract

  We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.

  View details for Web of Science ID A1987G477800078

  View details for PubMedID 3029131

• MYOSIN STRUCTURE AND FUNCTION IN CELL MOTILITY ANNUAL REVIEW OF CELL BIOLOGY Warrick, H. M., Spudich, J. A. 1987; 3: 379-421

  View details for Web of Science ID A1987K913700013

  View details for PubMedID 3318880

• INTRODUCTORY-REMARKS AND SOME BIOCHEMICAL CONSIDERATIONS METHODS IN CELL BIOLOGY Spudich, J. A. 1987; 28: 3-8

  View details for Web of Science ID A1987J148200001

  View details for PubMedID 3600410

• CONSERVED PROTEIN DOMAINS IN A MYOSIN HEAVY-CHAIN GENE FROM DICTYOSTELIUM-DISCOIDEUM PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Warrick, H. M., DeLozanne, A., Leinwand, L. A., Spudich, J. A. 1986; 83 (24): 9433-9437

  Abstract

  The 2116-amino acid myosin heavy chain sequence from Dictyostelium discoideum was determined from DNA sequence analysis of the cloned gene. The gene product can be divided into two distinct regions, a globular head region and a long alpha-helical, rod-like tail. In comparisons with nematode and mammalian muscle myosins, specific areas of the head region are highly conserved. These areas presumably reflect conserved functional and structural domains. Certain features that are present in the head region of nematode and mammalian muscle myosins, and that have been assumed to be important for myosin function, are missing in the Dictyostelium myosin sequence. The protein sequence of the Dictyostelium tail region is very poorly conserved with respect to the other myosins but displays the periodicities similar to those of muscle myosins. These periodicities are believed to play a role in filament formation. The 196-residue repeating unit that determines the 14.3-nm repeat seen in muscle thick filaments, the 28-residue charge repeating unit, and the 1,4 hydrophobic repeat previously described for the nematode myosin are all present in the Dictyostelium myosin rod sequence, suggesting that the filament structures of muscle and Dictyostelium myosins must be similar.

  View details for Web of Science ID A1986F500600033

  View details for PubMedID 3540939

• MYOSIN MOVEMENT INVITRO - A QUANTITATIVE ASSAY USING ORIENTED ACTIN CABLES FROM NITELLA METHODS IN ENZYMOLOGY Sheetz, M. P., Block, S. M., Spudich, J. A. 1986; 134: 531-544

  View details for Web of Science ID A1986H889700050

  View details for PubMedID 3821577

• SITE-SPECIFIC INHIBITION OF MYOSIN-MEDIATED MOTILITY INVITRO BY MONOCLONAL-ANTIBODIES JOURNAL OF CELL BIOLOGY Flicker, P. F., Peltz, G., Sheetz, M. P., Parham, P., Spudich, J. A. 1985; 100 (4): 1024-1030

  Abstract

  Monoclonal antibodies directed against seven different sites on Dictyostelium myosin (Peltz, G., J. A. Spudich, and P. Parham, 1985, J. Cell Biol., 100: 1016-1023) were tested for their ability to inhibit movement of myosin in vitro, using the Nitella-based myosin-mediated bead movement assay (Sheetz, M. P., R. Chasan, and J. A. Spudich, 1984, J. Cell Biol., 99: 1867-1871). To complement this functional assay, we located the binding sites of these antibodies by electron microscopy, using the rotary shadowing technique. One antibody bound to the 18,000-dalton light chain and inhibited movement completely. All of the remaining antibodies bound to various positions along the rod portion of the myosin molecule, which is approximately 1,800 A long. Antibodies that bound to the rod about 470, 680, and 1400 A from the head-tail junction did not alter myosin movement. One antibody appeared to bind very close to the head-tail junction and to inhibit movement 50%. Surprisingly, three antibodies that bound about 1,200 A from the head-tail junction inhibited movement completely. This inhibition did not depend on using intact IgG, since Fab' fragments had the same effect.

  View details for Web of Science ID A1985AEQ9400004

  View details for PubMedID 3980577

• MONOCLONAL-ANTIBODIES AGAINST 7 SITES ON THE HEAD AND TAIL OF DICTYOSTELIUM MYOSIN JOURNAL OF CELL BIOLOGY Peltz, G., Spudich, J. A., Parham, P. 1985; 100 (4): 1016-1023

  Abstract

  Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

  View details for Web of Science ID A1985AEQ9400003

  View details for PubMedID 2579955

• LIGHT CHAIN PHOSPHORYLATION REGULATES THE MOVEMENT OF SMOOTH-MUSCLE MYOSIN ON ACTIN-FILAMENTS JOURNAL OF CELL BIOLOGY Sellers, J. R., Spudich, J. A., Sheetz, M. P. 1985; 101 (5): 1897-1902

  Abstract

  In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.

  View details for Web of Science ID A1985ATG7400029

  View details for PubMedID 3840488

• CLONING AND CHARACTERIZATION OF A NONMUSCLE MYOSIN HEAVY-CHAIN CDNA PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA DeLozanne, A., Lewis, M., Spudich, J. A., LEINWAD, L. A. 1985; 82 (20): 6807-6810

  Abstract

  Despite many biochemical and structural similarities between muscle and nonmuscle myosins, their genes appear to have completely diverged, since muscle myosin molecular clones will not hybridize to RNA from nonmuscle sources. Here we report the isolation and characterization of a partial myosin heavy chain (MHC) cDNA clone from the slime mold Dictyostelium discoideum. We have isolated this clone from a lambda gt11 expression cDNA library by antibody screening. In contrast to the highly conserved sarcomeric muscle MHC multigene families in other organisms, there appears to be only one gene encoding MHC in the Dictyostelium genome. The cloned portion of this gene does not hybridize to the genomic DNAs of other eukaryotic organisms. Analysis of the predicted amino acid sequence of the partial Dictyostelium MHC clone shows that while there is no sequence homology to known striated muscle MHCs, the structure- and coiled-coil-forming capacities have been conserved.

  View details for Web of Science ID A1985ATC6000019

  View details for PubMedID 3901008

• CHEMOATTRACTANT-ELICITED INCREASES IN MYOSIN PHOSPHORYLATION IN DICTYOSTELIUM CELL Berlot, C. H., Spudich, J. A., Devreotes, P. N. 1985; 43 (1): 307-314

  Abstract

  Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [32P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a gamma-[32P]ATP-containing reaction mixture.

  View details for Web of Science ID A1985AVE3900033

  View details for PubMedID 3000604

• MONOCLONAL-ANTIBODIES PREPARED AGAINST DICTYOSTELIUM ACTIN - CHARACTERIZATION AND INTERACTIONS WITH ACTIN JOURNAL OF CELL BIOLOGY Simpson, P. A., Spudich, J. A., Parham, P. 1984; 99 (1): 287-295

  Abstract

  Three mouse monoclonal antibodies, Act I, Act II, and Act IV, against actin from the cellular slime mold Dictyostelium discoideum, have been made and characterized. All three antibodies are IgG1 and share the following properties: They form stable complexes with monomeric Dictyostelium actin, which prevents polymerization of the actin into filaments. On addition to preformed actin filaments, they cause a reduction in filament size and in the viscosity of the actin solution. They cross-react strongly with actins from the lower eucaryotes Physarum and Acanthamoeba, but not with alpha-actins from rabbit and human muscle or beta- and gamma-actins from human erythrocytes and a human B lymphoid cell line. Act II and Act IV recognize a similar antigenic determinant that is topographically distinct from that identified by Act I. In protein immunoblotting, only Act I bound strongly to Dictyostelium actin. Analysis of actin fragments with this technique showed that amino acids 13 to about 50 are required for Act I binding to actin. A comparison of the amino acid sequences of actins from lower eucaryotes and higher vertebrates implicates threonine 41 as a critical residue in the Act I antigenic site. The properties of Act II and Act IV suggest that they recognize antigenic sites involving the NH2-terminal six residues.

  View details for Web of Science ID A1984SY60700034

  View details for PubMedID 6203918

• CA-2+-DEPENDENT BINDING OF SEVERIN TO ACTIN - A ONE-TO-ONE COMPLEX IS FORMED JOURNAL OF CELL BIOLOGY Giffard, R. G., Weeds, A. G., Spudich, J. A. 1984; 98 (5): 1796-1803

  Abstract

  Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.

  View details for Web of Science ID A1984SR27800021

  View details for PubMedID 6427234

• MOVEMENT OF MYOSIN-COATED FLUORESCENT BEADS ON ACTIN CABLES INVITRO NATURE Sheetz, M. P., Spudich, J. A. 1983; 303 (5912): 31-35

  Abstract

  Myosin-coated fluorescent beads are observed to move unidirectionally along organized actin filament arrays in the alga, Nitella, with an average velocity similar to in vivo rates of movement in muscle and other cells. The myosin produces the motive force, as the movement is ATP dependent and is blocked by inactivation of the myosin heads. The movement, which occurs in the absence of bipolar thick filaments, provides a quantitative assay for myosin motility.

  View details for Web of Science ID A1983QN77000036

  View details for PubMedID 6682486

• MOVEMENT OF MYOSIN-COATED STRUCTURES ON ACTIN CABLES CELL MOTILITY AND THE CYTOSKELETON Sheetz, M. P., Spudich, J. A. 1983; 3 (5-6): 485-489

  Abstract

  Myosin-coated spheres from 0.6 to 120 microns in diameter move in vitro on a substratum of polar arrays of actin cables derived from the alga Nitella. The force for this movement is provided by skeletal muscle myosin since it is ATP-dependent, and N-ethylmaleimide (NEM) inactivation of the myosin blocks movement. These observations demonstrate that attachment of myosin in a random orientation to structures will enable those structures to move along polar arrays of actin filaments.

  View details for Web of Science ID A1983RW49400014

  View details for PubMedID 6661767

• CA-2+-SENSITIVE ISOLATION OF A CORTICAL ACTIN MATRIX FROM DICTYOSTELIUM AMEBAS JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY Giffard, R. G., Spudich, J. A., Spudich, A. 1983; 4 (1): 115-131

  Abstract

  A cortical actin matrix has been isolated from amoebae of Dictyostelium discoideum grown in liquid culture. The existence of this actin matrix in whole cells is indicated in electron micrographs as an area free of cytoplasmic organelles. The actin beneath the membrane is more clearly visible in sections of cells that are lysed gently with 0.5% Triton X-100 and fixed with 1% glutaraldehyde. Such Triton-lysed cells have fragments of plasma membrane associated with the cortical actin matrix. Isolation of the actin matrix, which sediments at 400 g, is inhibited by Ca2+. As much as 50% of the actin of the cell and about 12% of the total protein is found in the matrix isolated in lysis buffer containing no added Ca2+ and 2.5 mM EGTA, whereas less than 15% of the actin of the cell is recovered in a 400 g pellet when cells are lysed in buffer containing 2.5 mM Ca2+ and 2.5 mM EGTA. A 40 000 molecular weight protein that fragments F-actin in a Ca2+-dependent manner is not found in the isolated cortical actin matrix.

  View details for Web of Science ID A1983QD97100007

  View details for PubMedID 6404931

• A 40,000-DALTON PROTEIN FROM DICTYOSTELIUM-DISCOIDEUM AFFECTS ASSEMBLY PROPERTIES OF ACTIN IN A CA-2+-DEPENDENT MANNER JOURNAL OF CELL BIOLOGY Brown, S. S., Yamamoto, K., Spudich, J. A. 1982; 93 (1): 205-210

  Abstract

  A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000-dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.

  View details for Web of Science ID A1982NH70900024

  View details for PubMedID 7068756

• DICTYOSTELIUM-DISCOIDEUM - METHODS AND PERSPECTIVES FOR STUDY OF CELL MOTILITY METHODS IN CELL BIOLOGY Spudich, J. A. 1982; 25: 359-364

  View details for Web of Science ID A1982NT58900018

  View details for PubMedID 7109964

• MECHANISM OF K+-INDUCED ACTIN ASSEMBLY JOURNAL OF CELL BIOLOGY Pardee, J. D., Spudich, J. A. 1982; 93 (3): 648-654

  Abstract

  The assembly of highly purified actin from Dictyostelium discoideum amoebae and rabbit skeletal muscle by physiological concentrations of KCI proceeds through successive stages of (a) rapid formation of a distinct monomeric species referred to as KCI-monomer, (b) incorporation of KCI-monomers into an ATP-containing filament, and (c) ATP hydrolysis that occurs significantly after the incorporation event. KCI-monomer has a conformation which is distinct from that of either conventional G- or F-actin, as judged by UV spectroscopy at 210-220 nm and by changes in ATP affinity. ATP is not hydrolyzed during conversion of G-actin to KCI-monomer. KCI-monomer formation precedes filament formation and may be necessary for the assembly event. Although incorporation of KCI-monomers into filaments demonstrates lagphase kinetics by viscometry, both continuous absorbance monitoring at 232 nm and rapid sedimentation of filaments demonstrate hyperbolic assembly curves. ATP hydrolysis significantly lags the formation of actin filaments. When half of the actin has assembled, only 0.1 to 0.2 mole of ATP are hydrolyzed per mole of actin present as filaments.

  View details for Web of Science ID A1982NW96400014

  View details for PubMedID 6889598

• PURIFICATION OF MUSCLE ACTIN METHODS IN CELL BIOLOGY Pardee, J. D., Spudich, J. A. 1982; 24: 271-289

  View details for Web of Science ID A1982NS69900019

  View details for PubMedID 7098993

• PURIFICATION OF MUSCLE ACTIN METHODS IN ENZYMOLOGY Pardee, J. D., Spudich, J. A. 1982; 85: 164-181

  View details for Web of Science ID A1982PB65600018

  View details for PubMedID 7121269

• Control of assembly of Dictyostelium myosin and actin filaments. Cold Spring Harbor symposia on quantitative biology Spudich, J. A., Kuczmarski, E. R., Pardee, J. D., Simpson, P. A., Yamamoto, K., Stryer, L. 1982; 46: 553-561

  View details for PubMedID 6955100

• MECHANISM OF INTERACTION OF DICTYOSTELIUM SEVERIN WITH ACTIN-FILAMENTS JOURNAL OF CELL BIOLOGY Yamamoto, K., Pardee, J. D., REIDLER, J., Stryer, L., Spudich, J. A. 1982; 95 (3): 711-719

  Abstract

  Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.

  View details for Web of Science ID A1982PT01600004

  View details for PubMedID 6897549

• ACTIN-FILAMENTS UNDERGO LIMITED SUBUNIT EXCHANGE IN PHYSIOLOGICAL SALT CONDITIONS JOURNAL OF CELL BIOLOGY Pardee, J. D., Simpson, P. A., Stryer, L., Spudich, J. A. 1982; 94 (2): 316-324

  Abstract

  The exchange of actin filament subunits for unpolymerized actin or for subunits in other filaments has been quantitated by three experimental techniques: fluorescence energy transfer, incorporation of 35S-labeled actin monomers into unlabeled actin filaments, and exchange of [14C]ATP with filament-bound ADP. In the fluorescence energy transfer experiments, actin labeled with 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid (IAENS) served as the fluorescent energy donor, and actin labeled with either fluorescein-5-isothiocyanate (FITC) or fluorescein-5-maleimide (FM) served as the energy acceptor. Fluorescent-labeled actins from Dictyostelium amoebae and rabbit skeletal muscle were very similar to their unlabeled counterparts with respect to critical actin concentration for filament assembly, assembly rate, ATP hydrolysis upon assembly, and steady-state ATPase. As evidenced by two different types of fluorescence energy transfer experiments, less than 5% of the actin filament subunits exchanged under a variety of buffer conditions at actin concentrations greater than 0.5 mg/ml. At all actin concentrations limited exchange to a plateau level occurred with a half-time of about 20 min. Nearly identical results were obtained when exchange was quantitated by incorporation of 35S-labeled Dictyostelium actin monomers into unlabeled muscle actin or Dictyostelium actin filaments. Furthermore, the proportion of filament-bound ADP which exchanged with [14C]-ATP was nearly the same as actin subunit exchange measured by fluorescence energy transfer and 35S-labeled actin incorporation. These experiments demonstrate that under approximately physiologic ionic conditions only a small percentage of subunits in highly purified skeletal muscle or Dictyostelium F-actin participate in exchange.

  View details for Web of Science ID A1982NZ60200009

  View details for PubMedID 7202009

• ACTIN AND MYOSIN - CONTROL OF FILAMENT ASSEMBLY PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON SERIES B-BIOLOGICAL SCIENCES Spudich, J. A., Pardee, J. D., Simpson, P. A., Yamamoto, K., Kuczmarski, E. R., Stryer, L. 1982; 299 (1095): 247-261

  Abstract

  Actin filaments, assembled from highly purified actin from either skeletal muscle or Dictyostelium amoebae, are very stable under physiological ionic conditions. A small and limited amount of exchange of actin filament subunits for unpolymerized actin or subunits in other filaments has been measured by three techniques: fluorescence energy transfer, incorporation of 35S-labelled actin monomers into unlabelled actin filaments, and exchange of [14C]ATP with filament-bound ADP. A 40 kDa protein purified from amoebae destabilizes these otherwise stable filaments in a Ca2+-dependent manner. Myosin purified from Dictyostelium amoebae is phosphorylated both in the tail region of the heavy chain and in one of the light chains. Phosphorylation appears to regulate myosin thick-filament formation.

  View details for Web of Science ID A1982PP66100010

  View details for PubMedID 6129660

• MOLECULAR ASPECTS OF CORTICAL ACTIN FILAMENT FORMATION UPON FERTILIZATION CELL DIFFERENTIATION Spudich, A., Giffard, R. G., Spudich, J. A. 1982; 11 (5-6): 281-284

  View details for Web of Science ID A1982PL51900010

• MECHANISM OF ACTION OF CYTOCHALASIN - EVIDENCE THAT IT BINDS TO ACTIN FILAMENT ENDS JOURNAL OF CELL BIOLOGY Brown, S. S., Spudich, J. A. 1981; 88 (3): 487-491

  Abstract

  To test the idea that cytochalasin retards actin assembly by binding to filament ends, we have designed a new assay for cytochalasin binding in which the number of filament ends can be varied independently of the total actin concentration. Actin is reacted with polylysine-coated polystyrene beads to make filament ends (Brown and Spudich, 1979, J. Cell Biol. 80:499-504) and then reacted with [3H]cytochalasin B. We have found that cytochalasin B binds to beads in the presence of actin, and that the number of cytochalasin B binding sites can be varied as a function of the number of filament ends independent of the total actin concentration by varying the bead concentration.

  View details for Web of Science ID A1981LE20200002

  View details for PubMedID 6894300

• DICTYOSTELIUM MYOSIN - CHARACTERIZATION OF CHYMOTRYPTIC FRAGMENTS AND LOCALIZATION OF THE HEAVY-CHAIN PHOSPHORYLATION SITE JOURNAL OF CELL BIOLOGY Peltz, G., Kuczmarski, E. R., Spudich, J. A. 1981; 89 (1): 104-108

  Abstract

  Chymotrypsin cleaves Dictyostelium myosin in half, splitting the heavy chain (210,000 daltons) into two fragments of 105,000 daltons each. One of the two major fragments is soluble at low ionic strength and has a native molecular weight of 130,000. As judged by SDS polyacrylamide gel electrophoresis, this soluble fragment consists of the two intact myosin light chains of 18,000 and 16,000 daltons and a 105,000-dalton polypeptide derived from the myosin heavy chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin-activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin, although its apparent Km for actin is 12-fold greater than that of myosin. In addition to the major soluble 105,000-dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin also generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments that are devoid of myosin heads. A less prevalent insoluble fragment has a molecular weight of 83,000 and is probably a subfragment of the insoluble 105,000-dalton fragment. The heavy chain of myosin is phosphorylated in vivo and the phosphorylation site has been localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.

  View details for Web of Science ID A1981LH41600013

  View details for PubMedID 7228895

• CONTROL OF ASSEMBLY OF DICTYOSTELIUM MYOSIN AND ACTIN-FILAMENTS COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Spudich, J. A., Kuczmarski, E. R., Pardee, J. D., Simpson, P. A., Yamamoto, K., Stryer, L. 1981; 46: 553-561

  View details for Web of Science ID A1981NV38800006

• PURIFICATION OF A CA++ SENSITIVE K-38 DALTON PROTEIN FROM DICTYOSTELIUM-DISCOIDEUM WHICH AFFECTS THE ASSEMBLY STATE OF ACTIN Brown, S. S., Spudich, J. A. ROCKEFELLER UNIV PRESS. 1980: A224–A224

  View details for Web of Science ID A1980KN77600852

• ATP-DRIVEN STEADY-STATE EXCHANGE OF MONOMERIC AND FILAMENTOUS ACTIN FROM DICTYOSTELIUM-DISCOIDEUM PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Simpson, P. A., Spudich, J. A. 1980; 77 (8): 4610-4613

  Abstract

  When [35S]actin monomer from the slime mold Dictyostelium discoideum is added in trace amounts to a population of unlabeled Dictyostelium actin molecules assembled to steady state, it rapidly exchanges with the pool of actin filaments. This exchange between monomeric and filamentous actin is dependent on the presence of ATP. In addition, the exchange appears to occur via filament ends, because cytochalasin D, a drug that interacts specifically with actin filament ends to inhibit filament assenbly, inhibits the exchange reaction.

  View details for Web of Science ID A1980KG46700043

  View details for PubMedID 6933508

• REGULATION OF MYOSIN SELF-ASSEMBLY - PHOSPHORYLATION OF DICTYOSTELIUM HEAVY-CHAIN INHIBITS FORMATION OF THICK FILAMENTS PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Kuczmarski, E. R., Spudich, J. A. 1980; 77 (12): 7292-7296

  Abstract

  Dictyostelium myosin is composed of two heavy chains and two pairs of light chains in a 1:1:1 stoichiometry. Myosin purified from amoebae grown in medium containing [32P]phosphate had two of the subunits labeled (0.2-0.3 mol of phosphate per mol of 210,000-dalton heavy chains and approximately 0.1 mol of phosphate per mol of 18,000-dalton light chain). Kinase activities specific for the 210,000-dalton and for the 18,000-dalton subunits have been identified in extracts of Dictyostelium amoebae, and the heavy chain kinase has been purified 50-fold. This kinase phosphorylated Dictyostelium myosin to a maximum of 0.5-1.0 mol of phosphate per mol of heavy chain. Heavy chain phosphate, but not light chain phosphate, can be removed with bacterial alkaline phosphatase. Actin-activated myosin ATPase increased 80% when phosphorylated myosin was dephosphorylated to a level of approximately 0.06 mol of phosphate per mol of heavy chain. This effect could be reversed by rephosphorylating the myosin. The ability of myosin to self-assemble into thick filaments was inhibited by heavy chain phosphorylation. For example, in 80-100 mM KCl, only 10-20% of the myosin was assembled into thick filaments when the heavy chains were fully phosphorylated. Removal of the heavy chain phosphate resulted in 70-90% thick filament formation. This effect on self-assembly could be reversed by rephosphorylating the dephosphorylated myosin. These findings suggest that heavy chain phosphorylation may regulate cell contractile events by altering the state of myosin assembly.

  View details for Web of Science ID A1980LC49400063

  View details for PubMedID 6452632

• PURIFICATION AND PROPERTIES OF SOLUBLE ACTIN FROM SEA-URCHIN EGGS JOURNAL OF BIOCHEMISTRY Mabuchi, I., Spudich, J. A. 1980; 87 (3): 785-802

  Abstract

  Unfertilized eggs of the sea urchin, Strongylocentrotus purpuratus, were homogenized in a buffer containing 0.1 M KCl and 2 mM MgCl2 at pH 6.85. About 50% of the actin was recovered in the high-speed supernate of the homogenate. More than 80% of the actin in this supernate was found to be monomeric upon gel filtration chromatography through a Sephadex G-150 column or by a DNase I inhibition assay. The critical concentration for polymerization of this actin prior to further purification was 0.3-0.9 mg/ml under various conditions. Actin was purified to near homogeneity from the Sephadex G-150 pool with high yield. The purified actin had a critical concentration for polymerization of 0.02-0.03 mg/ml. The isoelectric point of the crude actin and the purified actin was the same. Indeed, we found that there is only one isoelectric focusing species of actin in the sea urchin egg, and it has an isoelectric point more basic than rabbit skeletal muscle actin. The discrepancy between the polymerizability of the crude and purified actin may be due to the presence of factors in the crude fraction which inhibit the polymerization of actin.

  View details for Web of Science ID A1980JH15000013

  View details for PubMedID 6893042

• NUCLEATION OF POLAR ACTIN FILAMENT ASSEMBLY BY A POSITIVELY CHARGED SURFACE JOURNAL OF CELL BIOLOGY Brown, S. S., Spudich, J. A. 1979; 80 (2): 499-504

  Abstract

  Polylysine-coated polystyrene beads can nucleate polar assembly of monomeric actin into filamentous form. This nucleation has been demonstrated by a combination of biochemical and structural experiments. The polylysine-coated beads accelerate the rate of actin assembly as detected by two different biochemical assays. Subsequent examination of the beads by electron microscopy reveals numerous actin filaments of similar length radiating from the beads. ATP promotes this bead-induced acceleration of assembly. Decoration of the filaments with the myosin fragment S1 shows that these filaments all have the same polarity, with the arrowhead pattern pointing toward the bead. The relevance of the system to in vitro mechanisms and its usefulness in other studies are discussed.

  View details for Web of Science ID A1979GG61700023

  View details for PubMedID 572366

• STRUCTURE OF ACTIN FILAMENT BUNDLES FROM MICROVILLI OF SEA-URCHIN EGGS JOURNAL OF MOLECULAR BIOLOGY Spudich, J. A., Amos, L. A. 1979; 129 (2): 319-331

  View details for Web of Science ID A1979GQ36600009

  View details for PubMedID 573333

• ACTIN IN TRITON-TREATED CORTICAL PREPARATIONS OF UNFERTILIZED AND FERTILIZED SEA-URCHIN EGGS JOURNAL OF CELL BIOLOGY Spudich, A., Spudich, J. A. 1979; 82 (1): 212-226

  Abstract

  Triton-treated cortical fragments of unfertilized and fertilized sea urchin eggs prepared in the presence of greater than or equal to 5 mM EGTA contain 15-30% of the total egg actin. However, actin filaments are not readily apparent by electron microscopy on the cortical fragments of unfertilized eggs but are numerous on those of fertilized eggs. The majority of the actin associated with cortical fragments of unfertilized eggs is solubilized by dialysis against a low ionic strength buffer at pH 7.5. This soluble actin preparation (less than 50% pure actin) does not form proper filaments in 0.1 M KCl and 3 mM MgCl2, whereas actin purified from this preparation does, as judged by electron microscopy. Optical diffraction analysis reveals that these purified actin filaments have helical parameters very similar to those of muscle actin. Furthermore, the properties of the purified actin with regard to activation of myosin ATPase are similar to those of actin from other cell types. The possibility that actin is maintained in a nonfilamentous form on the inner surface of the unfertilized egg plasma membrane and is induced to assemble upon fertilization is discussed.

  View details for Web of Science ID A1979HB11800017

  View details for PubMedID 573270

• CYTOCHALASIN INHIBITS THE RATE OF ELONGATION OF ACTIN FILAMENT FRAGMENTS JOURNAL OF CELL BIOLOGY Brown, S. S., Spudich, J. A. 1979; 83 (3): 657-662

  Abstract

  Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.

  View details for Web of Science ID A1979HX33600014

  View details for PubMedID 574873

• BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF ACTIN FROM DICTYOSTELIUM-DISCOIDEUM JOURNAL OF BIOLOGICAL CHEMISTRY UYEMURA, D. G., Brown, S. S., Spudich, J. A. 1978; 253 (24): 9088-9096

  Abstract

  Actin has been purified from amoebae of Dictyostelium discoideum by a procedure which is notable in that proteolysis has been diminished to undetectable levels and "selective" purification steps have been avoided. The overall yield of this procedure is 5- to 10- fold greater than that of a previous report (Spudich, J. A. (1974) J. Biol. Chem. 249, 6013-6020). The detailed biochemical and structural properties of this new preparation (preparation B) have been compared to those of Dictyostelium actin prepared by the previous procedure (preparation A) as well as to rabbit skeletal muscle actin. Preparation B actin is similar to muscle actin in its molecular weight, ability to activate myosin, filament structure, and polymerization properties. Preparation B actin has the same molecular weight and isoelectric point as preparation A actin, which is more acidic than that of skeletal muscle actin. However, preparation B actin and muscle actin form longer filaments than preparation A actin, as judged by viscometry and electron microscopy.

  View details for Web of Science ID A1978GC40800066

  View details for PubMedID 152763

• SUPRAMOLECULAR FORMS OF ACTIN FROM AMEBAS OF DICTYOSTELIUM-DISCOIDEUM JOURNAL OF BIOLOGICAL CHEMISTRY Spudich, J. A., Cooke, R. 1975; 250 (18): 7485-7491

  Abstract

  Actin purified from amoebae of Dictyostelium discoideum polymerizes into filaments at 24 degrees upon addition of KCl, as judged by a change in optical density at 232 nm and by electron microscopy. The rate and extent of formation of this supramolecular assembly and the optimal KCl concentrations (0.1 M) for assembly are similar to those of striated muscle actin. The apparent equilibrium constant for the monomer-polymer transition is 1.3 muM for both Dictyostelium and muscle actin. Although assembly of highly purified Dictyostelium actin monomers into individual actin filaments resembles that of muscle actin, Dictyostelium actin but not muscle actin was observed to assemble into two-dimensional nets in 10 mM CaCl2. The Dictyostelium actin also forms filament bundles which are 0.1 mum in diameter and which assemble in the presence of 5 mM MgCl2. These bundles formed from partially purified Dictyostelium actin preparations but not from highly purified preparations, suggesting that their formation may depend on the presence of another component. These actin bundles reconstituted in vitro resemble the actin-containing bundles found in situ by microscopy in many non-muscle cells.

  View details for Web of Science ID A1975AR59200059

  View details for PubMedID 1172503