Mrs. George A. Winzer Professor in Cell Biology, Stanford University School of Medicine (2009 - Present)
Chair, Department of Chemical & Systems Biology, Stanford University School of Medicine (2011 - Present)
Honors & Awards
Packard Fellow for Science and Engineering, David and Lucile Packard Foundation (1992-97)
Mrs. George A Winzer Professor in Cell Biology, Stanford University School of Medicine (2011)
Boards, Advisory Committees, Professional Organizations
Fellowship Award Committee Member, Damon Runyon Cancer Research Foundation (2013 - Present)
Editorial Board, Journal of Biological Chemistry (1996 - 1998)
Vordiplom, University of Basel, Basel, Switzerland, Mathematics and Biology (1980)
Masters, University of Basel/CERN, Geneva, Switzerland, Nuclear Physics (1983)
PhD, Biocenter of the University of Basel, Switzerland, Biophysics (1986)
Current Research and Scholarly Interests
UNDERSTANDING SIGNAL TRANSDUCTION NETWORKS The fundamental problem in signal transduction research is to understand how different receptor-stimuli and other cellular inputs control particular cell functions with a high degree of specificity. Similar to the regulation of organ function by complex hormonal feedback mechanisms, it is believed that interactions between several hundred intracellular signaling proteins and many types of second messengers guarantee the specificity of particular cellular responses. The goal of the research is our laboratory is to identify the fundamental properties of such intracellular Signal Transduction Networks and to determine how perturbations in networks can cause disease. In particular, we are currently using three mammalian model systems to understand and model the signaling systems that control neuronal plasticity, chemotaxis and regulated secretion. Many signaling events for these and other cell functions occur as oscillations or all-or-none responses and the precise timing and localization between parallel signaling events is essential for the induction of downstream functions. Therefore, single cell measurements of signaling responses are essential to develop models of signal transduction networks. Over the last years, we have developed several new strategies to monitor signaling events in single cells using different fluorescent microscopy techniques and fluorescent probes and we are now in a unique position to be able to monitor in the same experiment signaling responses in each of many thousands of cells. By combining such imaging techniques with quantitative single cell perturbation strategies, we are pursuing a strategy for a genome-wide exploration on how signaling networks are wired in health and disease.
- Cell Signaling
CSB 210 (Win)
- Chemical and Systems Biology Bootcamp
CSB 201 (Aut)
- Quantitative single cell analysis of live cell images
BIOS 238 (Aut)
Independent Studies (10)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Cancer Biology
CBIO 299 (Aut, Win, Spr, Sum)
- Directed Reading in Chemical and Systems Biology
CSB 299 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Graduate Research
CSB 399 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Win, Spr, Sum)
- Medical Scholars Research
CSB 370 (Aut, Win, Spr, Sum)
- Undergraduate Research
CSB 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
- Prior Year Courses
p53 suppresses muscle differentiation at the myogenin step in response to genotoxic stress
CELL DEATH AND DIFFERENTIATION
2015; 22 (4): 560-573
Acute muscle injury and physiological stress from chronic muscle diseases and aging lead to impairment of skeletal muscle function. This raises the question of whether p53, a cellular stress sensor, regulates muscle tissue repair under stress conditions. By investigating muscle differentiation in the presence of genotoxic stress, we discovered that p53 binds directly to the myogenin promoter and represses transcription of myogenin, a member of the MyoD family of transcription factors that plays a critical role in driving terminal muscle differentiation. This reduction of myogenin protein is observed in G1-arrested cells and leads to decreased expression of late but not early differentiation markers. In response to acute genotoxic stress, p53-mediated repression of myogenin reduces post-mitotic nuclear abnormalities in terminally differentiated cells. This study reveals a mechanistic link previously unknown between p53 and muscle differentiation, and suggests new avenues for managing p53-mediated stress responses in chronic muscle diseases or during muscle aging.
View details for DOI 10.1038/cdd.2014.189
View details for Web of Science ID 000350857200007
View details for PubMedID 25501595
- Systematic Discovery of Human Gene Function and Principles of Modular Organization through Phylogenetic Profiling CELL REPORTS 2015; 10 (6): 993-1006
Using light to shape chemical gradients for parallel and automated analysis of chemotaxis.
Molecular systems biology
2015; 11 (4): 804-?
Numerous molecular components have been identified that regulate the directed migration of eukaryotic cells toward sources of chemoattractant. However, how the components of this system are wired together to coordinate multiple aspects of the response, such as directionality, speed, and sensitivity to stimulus, remains poorly understood. Here we developed a method to shape chemoattractant gradients optically and analyze cellular chemotaxis responses of hundreds of living cells per well in 96-well format by measuring speed changes and directional accuracy. We then systematically characterized migration and chemotaxis phenotypes for 285 siRNA perturbations. A key finding was that the G-protein Giα subunit selectively controls the direction of migration while the receptor and Gβ subunit proportionally control both speed and direction. Furthermore, we demonstrate that neutrophils chemotax persistently in response to gradients of fMLF but only transiently in response to gradients of ATP. The method we introduce is applicable for diverse chemical cues and systematic perturbations, can be used to measure multiple cell migration and signaling parameters, and is compatible with low- and high-resolution fluorescence microscopy.
View details for DOI 10.15252/msb.20156027
View details for PubMedID 25908733
Dynamic recruitment of the curvature-sensitive protein ArhGAP44 to nanoscale membrane deformations limits exploratory filopodia initiation in neurons
In the vertebrate central nervous system, exploratory filopodia transiently form on dendritic branches to sample the neuronal environment and initiate new trans-neuronal contacts. While much is known about the molecules that control filopodia extension and subsequent maturation into functional synapses, the mechanisms that regulate initiation of these dynamic, actin-rich structures have remained elusive. Here, we find that filopodia initiation is suppressed by recruitment of ArhGAP44 to actin-patches that seed filopodia. Recruitment is mediated by binding of a membrane curvature-sensing ArhGAP44 N-BAR domain to plasma membrane sections that were deformed inward by acto-myosin mediated contractile forces. A GAP domain in ArhGAP44 triggers local Rac-GTP hydrolysis, thus reducing actin polymerization required for filopodia formation. Additionally, ArhGAP44 expression increases during neuronal development, concurrent with a decrease in the rate of filopodia formation. Together, our data reveals a local auto-regulatory mechanism that limits initiation of filopodia via protein recruitment to nanoscale membrane deformations.
View details for DOI 10.7554/eLife.03116
View details for Web of Science ID 000346448900001
View details for PubMedID 25498153
Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (41): E4386-E4393
Phenotypic heterogeneity within a population of genetically identical cells is emerging as a common theme in multiple biological systems, including human cell biology and cancer. Using live-cell imaging, flow cytometry, and kinetic modeling, we showed that two states--quiescence and cell cycling--can coexist within an isogenic population of human cells and resulted from low basal expression levels of p21, a Cyclin-dependent kinase (CDK) inhibitor (CKI). We attribute the p21-dependent heterogeneity in cell cycle activity to double-negative feedback regulation involving CDK2, p21, and E3 ubiquitin ligases. In support of this mechanism, analysis of cells at a point before cell cycle entry (i.e., before the G1/S transition) revealed a p21-CDK2 axis that determines quiescent and cycling cell states. Our findings suggest a mechanistic role for p21 in generating heterogeneity in both normal tissues and tumors.
View details for DOI 10.1073/pnas.1409797111
View details for Web of Science ID 000342922000017
View details for PubMedID 25267623
A polarized Ca2+, diacylglycerol and STIM1 signalling system regulates directed cell migration
NATURE CELL BIOLOGY
2014; 16 (2): 133-144
Ca(2+) signals control cell migration by regulating forward movement and cell adhesion. However, it is not well understood how Ca(2+)-regulatory proteins and second messengers are spatially organized in migrating cells. Here we show that receptor tyrosine kinase and phospholipase C signalling are restricted to the front of migrating endothelial leader cells, triggering local Ca(2+) pulses, local depletion of Ca(2+) in the endoplasmic reticulum and local activation of STIM1, supporting pulsatile front retraction and adhesion. At the same time, the mediator of store-operated Ca(2+) influx, STIM1, is transported by microtubule plus ends to the front. Furthermore, higher Ca(2+) pump rates in the front relative to the back of the plasma membrane enable effective local Ca(2+) signalling by locally decreasing basal Ca(2+). Finally, polarized phospholipase C signalling generates a diacylglycerol gradient towards the front that promotes persistent forward migration. Thus, cells employ an integrated Ca(2+) control system with polarized Ca(2+) signalling proteins and second messengers to synergistically promote directed cell migration.
View details for DOI 10.1038/ncb2906
View details for Web of Science ID 000331161400003
View details for PubMedID 24463606
Parallel measurement of dynamic changes in translation rates in single cells
2014; 11 (1): 86-?
Protein concentrations are often regulated by dynamic changes in translation rates. Nevertheless, it has been challenging to directly monitor changes in translation in living cells. We have developed a reporter system to measure real-time changes of translation rates in human or mouse individual cells by conjugating translation regulatory motifs to sequences encoding a nuclear targeted fluorescent protein and a controllable destabilization domain. Application of the method showed that individual cells undergo marked fluctuations in the translation rate of mRNAs whose 5' terminal oligopyrimidine (5' TOP) motif regulates the synthesis of ribosomal proteins. Furthermore, we show that small reductions in amino acid levels signal through different mTOR-dependent pathways to control TOP mRNA translation, whereas larger reductions in amino acid levels control translation through eIF2A. Our study demonstrates that dynamic measurements of single-cell activities of translation regulatory motifs can be used to identify and investigate fundamental principles of translation.
View details for DOI 10.1038/NMETH.2729
View details for Web of Science ID 000329178200032
View details for PubMedID 24213167
Formin-mediated actin polymerization promotes Salmonella invasion
2013; 15 (12): 2051-2063
Salmonella invade host cells using Type 3 secreted effectors, which modulate host cellular targets to promote actin rearrangements at the cell surface that drive bacterial uptake. The Arp2/3 complex contributes to Salmonella invasion but is not essential, indicating other actin regulatory factors are involved. Here, we show a novel role for FHOD1, a formin family member, in Salmonella invasion. FHOD1 and Arp2/3 occupy distinct microdomains at the invasion site and control distinct aspects of membrane protrusion formation. FHOD1 is phosphorylated during infection and this modification is required for promoting bacterial uptake by host cells. ROCK II, but not ROCK I, is recruited to the invasion site and is required for FHOD1 phosphorylation and for Salmonella invasion. Together, our studies revealan important phospho-dependent FHOD1 actin polymerization pathway in Salmonella invasion.
View details for DOI 10.1111/cmi.12173
View details for Web of Science ID 000326934000009
View details for PubMedID 23869992
- Neuropilin-2 contributes to tumorigenicity in a mouse model of Hedgehog pathway medulloblastoma JOURNAL OF NEURO-ONCOLOGY 2013; 115 (2): 161-168
The Proliferation-Quiescence Decision Is Controlled by a Bifurcation in CDK2 Activity at Mitotic Exit
2013; 155 (2): 369-383
Tissue homeostasis in metazoans is regulated by transitions of cells between quiescence and proliferation. The hallmark of proliferating populations is progression through the cell cycle, which is driven by cyclin-dependent kinase (CDK) activity. Here, we introduce a live-cell sensor for CDK2 activity and unexpectedly found that proliferating cells bifurcate into two populations as they exit mitosis. Many cells immediately commit to the next cell cycle by building up CDK2 activity from an intermediate level, while other cells lack CDK2 activity and enter a transient state of quiescence. This bifurcation is directly controlled by the CDK inhibitor p21 and is regulated by mitogens during a restriction window at the end of the previous cell cycle. Thus, cells decide at the end of mitosis to either start the next cell cycle by immediately building up CDK2 activity or to enter a transient G0-like state by suppressing CDK2 activity.
View details for DOI 10.1016/j.cell.2013.08.062
View details for Web of Science ID 000325719800014
Dosage of Dyrk1a Shifts Cells within a p21-Cyclin D1 Signaling Map to Control the Decision to Enter the Cell Cycle
2013; 52 (1): 87-100
Mammalian cells have a remarkable capacity to compensate for heterozygous gene loss or extra gene copies. One exception is Down syndrome (DS), where a third copy of chromosome 21 mediates neurogenesis defects and lowers the frequency of solid tumors. Here we combine live-cell imaging and single-cell analysis to show that increased dosage of chromosome 21-localized Dyrk1a steeply increases G1 cell cycle duration through direct phosphorylation and degradation of cyclin D1 (CycD1). DS-derived fibroblasts showed analogous cell cycle changes that were reversed by Dyrk1a inhibition. Furthermore, reducing Dyrk1a activity increased CycD1 expression to force a bifurcation, with one subpopulation of cells accelerating proliferation and the other arresting proliferation by costabilizing CycD1 and the CDK inhibitor p21. Thus, dosage of Dyrk1a repositions cells within a p21-CycD1 signaling map, directing each cell to either proliferate or to follow two distinct cell cycle exit pathways characterized by high or low CycD1 and p21 levels.
View details for DOI 10.1016/j.molcel.2013.09.009
View details for Web of Science ID 000326195800009
View details for PubMedID 24119401
- Regulators of Calcium Homeostasis Identified by Inference of Kinetic Model Parameters from Live Single Cells Perturbed by siRNA SCIENCE SIGNALING 2013; 6 (283)
Inside-Out Connections: The ER Meets the Plasma Membrane.
2013; 153 (7): 1423-1424
Junctions that connect the endoplasmic reticulum (ER) and the plasma membrane (PM) are unique yet ubiquitous subcellular compartments. Giordano et al. now report that extended synaptotagmins (E-Syts) promote their formation, providing fundamental insight into the molecular machinery controlling ER and plasma membrane crosstalk.
View details for DOI 10.1016/j.cell.2013.05.054
View details for PubMedID 23791170
A Localized Wnt Signal Orients Asymmetric Stem Cell Division in Vitro
2013; 339 (6126): 1445-1448
Developmental signals such as Wnts are often presented to cells in an oriented manner. To examine the consequences of local Wnt signaling, we immobilized Wnt proteins on beads and introduced them to embryonic stem cells in culture. At the single-cell level, the Wnt-bead induced asymmetric distribution of Wnt-?-catenin signaling components, oriented the plane of mitotic division, and directed asymmetric inheritance of centrosomes. Before cytokinesis was completed, the Wnt-proximal daughter cell expressed high levels of nuclear ?-catenin and pluripotency genes, whereas the distal daughter cell acquired hallmarks of differentiation. We suggest that a spatially restricted Wnt signal induces an oriented cell division that generates distinct cell fates at predictable positions relative to the Wnt source.
View details for DOI 10.1126/science.1231077
View details for Web of Science ID 000316740700046
View details for PubMedID 23520113
Brg1 governs distinct pathways to direct multiple aspects of mammalian neural crest cell development
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (5): 1738-1743
Development of the cerebral vessels, pharyngeal arch arteries (PAAs). and cardiac outflow tract (OFT) requires multipotent neural crest cells (NCCs) that migrate from the neural tube to target tissue destinations. Little is known about how mammalian NCC development is orchestrated by gene programming at the chromatin level, however. Here we show that Brahma-related gene 1 (Brg1), an ATPase subunit of the Brg1/Brahma-associated factor (BAF) chromatin-remodeling complex, is required in NCCs to direct cardiovascular development. Mouse embryos lacking Brg1 in NCCs display immature cerebral vessels, aberrant PAA patterning, and shortened OFT. Brg1 suppresses an apoptosis factor, Apoptosis signal-regulating kinase 1 (Ask1), and a cell cycle inhibitor, p21(cip1), to inhibit apoptosis and promote proliferation of NCCs, thereby maintaining a multipotent cell reservoir at the neural crest. Brg1 also supports Myosin heavy chain 11 (Myh11) expression to allow NCCs to develop into mature vascular smooth muscle cells of cerebral vessels. Within NCCs, Brg1 partners with chromatin remodeler Chromodomain-helicase-DNA-binding protein 7 (Chd7) on the PlexinA2 promoter to activate PlexinA2, which encodes a receptor for semaphorin to guide NCCs into the OFT. Our findings reveal an important role for Brg1 and its downstream pathways in the survival, differentiation, and migration of the multipotent NCCs critical for mammalian cardiovascular development.
View details for DOI 10.1073/pnas.1218072110
View details for Web of Science ID 000314558100038
View details for PubMedID 23319608
Regulators of Calcium Homeostasis Identified by Inference of Kinetic Model Parameters from Live Single Cells Perturbed by siRNA.
2013; 6 (283): ra56
Assigning molecular functions and revealing dynamic connections between large numbers of partially characterized proteins in regulatory networks are challenges in systems biology. We showed that functions of signaling proteins can be discovered with a differential equations model of the underlying signaling process to extract specific molecular parameter values from single-cell, time-course measurements. By analyzing the effects of 250 small interfering RNAs on Ca(2+) signals in single cells over time, we identified parameters that were specifically altered in the Ca(2+) regulatory system. Analysis of the screen confirmed known functions of the Ca(2+) sensors STIM1 (stromal interaction molecule 1) and calmodulin and of Ca(2+) channels and pumps localized in the endoplasmic reticulum (ER) or plasma membrane. Furthermore, we showed that the Alzheimer's disease-linked protein presenilin-2 and the channel protein ORAI2 prevented overload of ER Ca(2+) and that feedback from Ca(2+) to phosphatidylinositol 4-kinase and PLC? (phospholipase C?) may regulate the abundance of the plasma membrane lipid PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) to control Ca(2+) extrusion. Thus, functions of signaling proteins and dynamic regulatory connections can be identified by extracting molecular parameter values from single-cell, time-course data.
View details for PubMedID 23838183
Coordinated oscillations in cortical actin and Ca2+ correlate with cycles of vesicle secretion
NATURE CELL BIOLOGY
2012; 14 (12): 1261-?
The actin cortex both facilitates and hinders the exocytosis of secretory granules. How cells consolidate these two opposing roles was not well understood. Here we show that antigen activation of mast cells induces oscillations in Ca(2+) and PtdIns(4,5)P(2) lipid levels that in turn drive cyclic recruitment of N-WASP and cortical actin level oscillations. Experimental and computational analysis argues that vesicle fusion correlates with the observed actin and Ca(2+) level oscillations. A vesicle secretion cycle starts with the capture of vesicles by actin when cortical F-actin levels are high, followed by vesicle passage through the cortex when F-actin levels are low, and vesicle fusion with the plasma membrane when Ca(2+) levels subsequently increase. Thus, cells employ oscillating levels of Ca(2+), PtdIns(4,5)P(2) and cortical F-actin to increase secretion efficiency, explaining how the actin cortex can function as a carrier as well as barrier for vesicle secretion.
View details for DOI 10.1038/ncb2614
View details for Web of Science ID 000311890300008
View details for PubMedID 23143397
External push and internal pull forces recruit curvature-sensing N-BAR domain proteins to the plasma membrane
NATURE CELL BIOLOGY
2012; 14 (8): 874-U212
Many of the more than 20 mammalian proteins with N-BAR domains control cell architecture and endocytosis by associating with curved sections of the plasma membrane. It is not well understood whether N-BAR proteins are recruited directly by processes that mechanically curve the plasma membrane or indirectly by plasma-membrane-associated adaptor proteins that recruit proteins with N-BAR domains that then induce membrane curvature. Here, we show that externally induced inward deformation of the plasma membrane by cone-shaped nanostructures (nanocones) and internally induced inward deformation by contracting actin cables both trigger recruitment of isolated N-BAR domains to the curved plasma membrane. Markedly, live-cell imaging in adherent cells showed selective recruitment of full-length N-BAR proteins and isolated N-BAR domains to plasma membrane sub-regions above nanocone stripes. Electron microscopy confirmed that N-BAR domains are recruited to local membrane sites curved by nanocones. We further showed that N-BAR domains are periodically recruited to curved plasma membrane sites during local lamellipodia retraction in the front of migrating cells. Recruitment required myosin-II-generated force applied to plasma-membrane-connected actin cables. Together, our results show that N-BAR domains can be directly recruited to the plasma membrane by external push or internal pull forces that locally curve the plasma membrane.
View details for DOI 10.1038/ncb2533
View details for Web of Science ID 000307115900017
View details for PubMedID 22750946
Cooperative Activation of PI3K by Ras and Rho Family Small GTPases
2012; 47 (2): 281-290
Phosphoinositide 3-kinases (PI3Ks) and Ras and Rho family small GTPases are key regulators of cell polarization, motility, and chemotaxis. They influence each other's activities by direct and indirect feedback processes that are only partially understood. Here, we show that 21 small GTPase homologs activate PI3K. Using a microscopy-based binding assay, we show that K-Ras, H-Ras, and five homologous Ras family small GTPases function upstream of PI3K by directly binding the PI3K catalytic subunit, p110. In contrast, several Rho family small GTPases activated PI3K by an indirect cooperative positive feedback that required a combination of Rac, CDC42, and RhoG small GTPase activities. Thus, a distributed network of Ras and Rho family small GTPases induces and reinforces PI3K activity, explaining past challenges to elucidate the specific relevance of different small GTPases in regulating PI3K and controlling cell polarization and chemotaxis.
View details for DOI 10.1016/j.molcel.2012.05.007
View details for Web of Science ID 000307084000014
View details for PubMedID 22683270
Spatial Positive Feedback at the Onset of Mitosis
2012; 149 (7): 1500-1513
Mitosis is triggered by the activation of Cdk1-cyclin B1 and its translocation from the cytoplasm to the nucleus. Positive feedback loops regulate the activation of Cdk1-cyclin B1 and help make the process irreversible and all-or-none in character. Here we examine whether an analogous process, spatial positive feedback, regulates Cdk1-cyclin B1 redistribution. We used chemical biology approaches and live-cell microscopy to show that nuclear Cdk1-cyclin B1 promotes the translocation of Cdk1-cyclin B1 to the nucleus. Mechanistic studies suggest that cyclin B1 phosphorylation promotes nuclear translocation and, conversely, nuclear translocation promotes cyclin B1 phosphorylation, accounting for the feedback. Interfering with the abruptness of Cdk1-cyclin B1 translocation affects the timing and synchronicity of subsequent mitotic events, underscoring the functional importance of this feedback. We propose that spatial positive feedback ensures a rapid, complete, robust, and irreversible transition from interphase to mitosis and suggest that bistable spatiotemporal switches may be widespread in biological regulation.
View details for DOI 10.1016/j.cell.2012.05.028
View details for Web of Science ID 000305753800016
View details for PubMedID 22726437
Ca2+ Pulses Control Local Cycles of Lamellipodia Retraction and Adhesion along the Front of Migrating Cells
2012; 22 (9): 837-842
Ca(2+) signals regulate polarization, speed, and turning of migrating cells. However, the molecular mechanism by which Ca(2+) acts on moving cells is not understood. Here we show that local Ca(2+) pulses along the front of migrating human endothelial cells trigger cycles of retraction of local lamellipodia and, concomitantly, strengthen local adhesion to the extracellular matrix. These Ca(2+) release pulses had small amplitudes and diameters and were triggered repetitively near the leading plasma membrane with only little coordination between different regions. We show that each Ca(2+) pulse triggers contraction of actin filaments by activating myosin light-chain kinase and myosin II behind the leading edge. The cyclic force generated by myosin II operates locally, causing a partial retraction of the nearby protruding lamellipodia membrane and a strengthening of paxillin-based focal adhesion within the same lamellipodia. Photo release of Ca(2+) demonstrated a direct role of Ca(2+) in triggering local retraction and adhesion. Together, our study suggests that spatial sensing, forward movement, turning, and chemotaxis are in part controlled by confined Ca(2+) pulses that promote local lamellipodia retraction and adhesion cycles along the leading edge of moving cells.
View details for DOI 10.1016/j.cub.2012.03.037
View details for Web of Science ID 000303967600035
View details for PubMedID 22521790
A Two-Dimensional ERK-AKT Signaling Code for an NGF-Triggered Cell-Fate Decision
2012; 45 (2): 196-209
Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.
View details for DOI 10.1016/j.molcel.2011.11.023
View details for Web of Science ID 000299761700008
View details for PubMedID 22206868
Design of experiments to investigate dynamic cell signaling models.
Methods in molecular biology (Clifton, N.J.)
2012; 880: 109-118
This chapter describes approaches to make use of dynamic models of cell signaling systems in order to optimize experiments in cell biology. We are particularly focusing on the question of how small molecule inhibitors or activators can best be used to get the most information out of a limited number of experiments when only a handful of molecular species can be measured. One goal addressed by this chapter is to find time course experiments to discriminate between rivaling molecular mechanisms. The other goal is to find experiments that are useful for inferring rate constants, binding affinities, concentrations, and other model parameters from time course data. Both are treated as optimal control problems in which rapid pharmacological perturbation schemes are identified in silico in order to close an experimental cycle from modeling back to the laboratory bench.
View details for DOI 10.1007/978-1-61779-833-7_7
View details for PubMedID 23361984
Salmonella exploits Arl8B-directed kinesin activity to promote endosome tubulation and cell-to-cell transfer
2011; 13 (11): 1812-1823
The facultative intracellular pathogen Salmonella enterica serovar Typhimurium establishes a replicative niche, the Salmonella-containing vacuole (SCV), in host cells. Here we demonstrate that these bacteria exploit the function of Arl8B, an Arf family GTPase, during infection. Following infection, Arl8B localized to SCVs and to tubulated endosomes that extended along microtubules in the host cell cytoplasm. Arl8B(+) tubules partially colocalized with LAMP1 and SCAMP3. Formation of LAMP1(+) tubules (the Salmonella-induced filaments phenotype; SIFs) required Arl8B expression. SIFs formation is known to require the activity of kinesin-1. Here we find that Arl8B is required for kinesin-1 recruitment to SCVs. We have previously shown that SCVs undergo centrifugal movement to the cell periphery at 24 h post infection and undergo cell-to-cell transfer to infect neighbouring cells, and that both phenotypes require kinesin-1 activity. Here we demonstrate that Arl8B is required for migration of the SCV to the cell periphery 24 h after infection and for cell-to-cell transfer of bacteria to neighbouring cells. These results reveal a novel host factor co-opted by S. Typhimurium to manipulate the host endocytic pathway and to promote the spread of infection within a host.
View details for DOI 10.1111/j.1462-5822.2011.01663.x
View details for Web of Science ID 000296961800014
View details for PubMedID 21824248
Evolutionary origins of STIM1 and STIM2 within ancient Ca2+ signaling systems
TRENDS IN CELL BIOLOGY
2011; 21 (4): 202-211
Human stromal interaction molecule (STIM) proteins are parts of elaborate eukaryotic Ca(2+) signaling systems that include numerous plasma membrane (PM), endoplasmic reticulum (ER), and mitochondrial Ca(2+) transporters, channels and regulators. STIM2 and STIM1 function as Ca(2+) sensors with different sensitivities for ER Ca(2+). They translocate to ER-PM junctions and open PM Orai Ca(2+) influx channels when receptor-mediated Ca(2+) release lowers ER Ca(2+) levels. The resulting increase in cytosolic Ca(2+) leads to the activation of numerous Ca(2+) effector proteins that in turn regulate differentiation, cell contraction, secretion and other cell functions. In this review, we use an evolutionary perspective to survey molecular activation mechanisms in the Ca(2+) signaling system, with a particular focus on regulatory motifs and functions of the two STIM proteins. We discuss the presence and absence of STIM genes in different species, the order of appearance of STIM versus Orai, and the evolutionary addition of new signaling domains to STIM proteins.
View details for DOI 10.1016/j.tcb.2011.01.002
View details for Web of Science ID 000290054000002
View details for PubMedID 21288721
STIM Proteins and the Endoplasmic Reticulum-Plasma Membrane Junctions
ANNUAL REVIEW OF BIOCHEMISTRY, VOL 80
2011; 80: 973-1000
Eukaryotic organelles can interact with each other through stable junctions where the two membranes are kept in close apposition. The junction that connects the endoplasmic reticulum to the plasma membrane (ER-PM junction) is unique in providing a direct communication link between the ER and the PM. In a recently discovered signaling process, STIM (stromal-interacting molecule) proteins sense a drop in ER Ca(2+) levels and directly activate Orai PM Ca(2+) channels across the junction space. In an inverse process, a voltage-gated PM Ca(2+) channel can directly open ER ryanodine-receptor Ca(2+) channels in striated-muscle cells. Although ER-PM junctions were first described 50 years ago, their broad importance in Ca(2+) signaling, as well as in the regulation of cholesterol and phosphatidylinositol lipid transfer, has only recently been realized. Here, we discuss research from different fields to provide a broad perspective on the structures and unique roles of ER-PM junctions in controlling signaling and metabolic processes.
View details for DOI 10.1146/annurev-biochem-061609-165311
View details for Web of Science ID 000292895600038
View details for PubMedID 21548779
- Antibacterial autophagy occurs at PtdIns(3)P-enriched domains of the endoplasmic reticulum and requires Rab1 GTPase AUTOPHAGY 2011; 7 (1): 17-26
Rho small GTPases activates PI3K via a cooperative positive feedback loop
AMER SOC CELL BIOLOGY. 2011
View details for Web of Science ID 000305505504319
A Steering Model of Endothelial Sheet Migration Recapitulates Monolayer Integrity and Directed Collective Migration
MOLECULAR AND CELLULAR BIOLOGY
2011; 31 (2): 342-350
Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.
View details for DOI 10.1128/MCB.00800-10
View details for Web of Science ID 000285621900010
View details for PubMedID 20974808
- CELL BIOLOGY A sensor for calcium uptake NATURE 2010; 467 (7313): 283-283
The NADPH oxidases NOX4 and DUOX2 regulate cell cycle entry via a p53-dependent pathway
2010; 29 (31): 4473-4484
Reactive oxygen species (ROS) are produced in growth factor-signaling pathways leading to cell proliferation, but the mechanisms leading to ROS generation and the targets of ROS signals are not well understood. Using a focused siRNA screen to identify redox-related proteins required for growth factor-induced cell cycle entry, we show that two ROS-generating proteins, the NADPH oxidases NOX4 and DUOX2, are required for platelet-derived growth factor (PDGF) induced retinoblastoma protein (Rb) phosphorylation in normal human fibroblasts. Unexpectedly, NOX4 and DUOX2 knockdown did not inhibit the early signaling pathways leading to cyclin D1 upregulation. However, hours after growth factor stimulation, NOX4 and DUOX2 knockdown reduced ERK1 phosphorylation and increased levels of the tumor suppressor protein p53 and a cell cycle inhibitor protein p21 (Waf1/Cip1) that is transcriptionally regulated by p53. Co-knockdown of NOX4 or DUOX2 with either p53 or with p21 overcame the inhibition of Rb phosphorylation that occurred with NOX4 or DUOX2 knockdown alone. Our results argue that rather than primarily affecting growth factor receptor signaling, NOX4 and DUOX2 regulate cell cycle entry as part of a p53-dependent checkpoint for proliferation.
View details for DOI 10.1038/onc.2010.200
View details for Web of Science ID 000280559100010
View details for PubMedID 20531308
The Phosphoinositide Phosphatase SopB Manipulates Membrane Surface Charge and Trafficking of the Salmonella-Containing Vacuole
CELL HOST & MICROBE
2010; 7 (6): 453-462
Shifts in electrostatic surface charge of membranes have recently been highlighted as a significant factor contributing to protein targeting to the plasma membrane and nascent phagosomes. Intracellular, vacuole-adapted pathogens may also regulate surface charge of their vacuoles to establish a replicative niche. Since Salmonella enterica serovar Typhimurium controls trafficking of the Salmonella-containing vacuole (SCV) and inhibits its fusion with lysosomes, we investigated the contribution of surface charge to this process. Using recently developed fluorescent biosensors, we show that the bacterial phosphoinositide phosphatase SopB controls membrane surface charge of nascent SCVs by reducing levels of negatively charged lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine. This SopB activity results in dissociation of a number of host-cell endocytic trafficking proteins from this compartment and inhibits SCV-lysosome fusion. Moreover, inducible reduction of negative charge rescues DeltasopB bacteria-containing SCVs from fusion with lysosomes. These results reveal a membrane-charge-based mechanism used by S. Typhimurium to control SCV maturation.
View details for DOI 10.1016/j.chom.2010.05.011
View details for Web of Science ID 000279209200006
View details for PubMedID 20542249
An electrostatic switch displaces phosphatidylinositol phosphate kinases from the membrane during phagocytosis
JOURNAL OF CELL BIOLOGY
2009; 187 (5): 701-714
Plasmalemmal phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P(2)) synthesized by PI 4-phosphate (PI4P) 5-kinase (PIP5K) is key to the polymerization of actin that drives chemotaxis and phagocytosis. We investigated the means whereby PIP5K is targeted to the membrane and its fate during phagosome formation. Homology modeling revealed that all PIP5K isoforms feature a positively charged face. Together with the substrate-binding loop, this polycationic surface is proposed to constitute a coincidence detector that targets PIP5Ks to the plasmalemma. Accordingly, manipulation of the surface charge displaced PIP5Ks from the plasma membrane. During particle engulfment, PIP5Ks detached from forming phagosomes as the surface charge at these sites decreased. Precluding the change in surface charge caused the PIP5Ks to remain associated with the phagosomal cup. Chemically induced retention of PIP5K-gamma prevented the disappearance of PI4,5P(2) and aborted phagosome formation. We conclude that a bistable electrostatic switch mechanism regulates the association/dissociation of PIP5Ks from the membrane during phagocytosis and likely other processes.
View details for DOI 10.1083/jcb.200909025
View details for Web of Science ID 000272182900012
View details for PubMedID 19951917
Optimal Experimental Design for Parameter Estimation of a Cell Signaling Model
PLOS COMPUTATIONAL BIOLOGY
2009; 5 (11)
Differential equation models that describe the dynamic changes of biochemical signaling states are important tools to understand cellular behavior. An essential task in building such representations is to infer the affinities, rate constants, and other parameters of a model from actual measurement data. However, intuitive measurement protocols often fail to generate data that restrict the range of possible parameter values. Here we utilized a numerical method to iteratively design optimal live-cell fluorescence microscopy experiments in order to reveal pharmacological and kinetic parameters of a phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) second messenger signaling process that is deregulated in many tumors. The experimental approach included the activation of endogenous phosphoinositide 3-kinase (PI3K) by chemically induced recruitment of a regulatory peptide, reversible inhibition of PI3K using a kinase inhibitor, and monitoring of the PI3K-mediated production of PIP(3) lipids using the pleckstrin homology (PH) domain of Akt. We found that an intuitively planned and established experimental protocol did not yield data from which relevant parameters could be inferred. Starting from a set of poorly defined model parameters derived from the intuitively planned experiment, we calculated concentration-time profiles for both the inducing and the inhibitory compound that would minimize the predicted uncertainty of parameter estimates. Two cycles of optimization and experimentation were sufficient to narrowly confine the model parameters, with the mean variance of estimates dropping more than sixty-fold. Thus, optimal experimental design proved to be a powerful strategy to minimize the number of experiments needed to infer biological parameters from a cell signaling assay.
View details for DOI 10.1371/journal.pcbi.1000558
View details for Web of Science ID 000274228500006
View details for PubMedID 19911077
Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD
2009; 7 (9)
Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)-dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.
View details for DOI 10.1371/journal.pbio.1000187
View details for Web of Science ID 000270820800002
View details for PubMedID 19823667
A Genome-wide siRNA Screen Reveals Diverse Cellular Processes and Pathways that Mediate Genome Stability
2009; 35 (2): 228-239
Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.
View details for DOI 10.1016/j.molcel.2009.06.021
View details for Web of Science ID 000268643700011
View details for PubMedID 19647519
Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors
2009; 8 (7): 1044-1052
Cell cycle analysis typically relies on fixed time-point measurements of cells in particular phases of the cell cycle. The cell cycle, however, is a dynamic process whose subtle shifts are lost by fixed time-point methods. Live-cell fluorescent biosensors and time-lapse microscopy allows the collection of temporal information about real time cell cycle progression and arrest. Using two genetically-encoded biosensors, we measured the precision of the G(1), S, G(2) and M cell cycle phase durations in different cell types and identified a bimodal G(1) phase duration in a fibroblast cell line that is not present in the other cell types. Using a cell line model for neuronal differentiation, we demonstrated that NGF-induced neurite extension occurs independently of NGF-induced cell cycle G(1) phase arrest. Thus, we have begun to use cell cycle fluorescent biosensors to examine the proliferation of cell populations at the resolution of individual cells and neuronal differentiation as a dynamic process of parallel cell cycle arrest and neurite outgrowth.
View details for Web of Science ID 000265593900020
View details for PubMedID 19270522
Calcium Flickers Lighting the Way in Chemotaxis?
2009; 16 (2): 160-161
Recent studies identified local calcium signals at the leading edge of migrating cells, suggesting a new role for calcium in cell polarization and chemotaxis.
View details for DOI 10.1016/j.devcel.2009.01.018
View details for Web of Science ID 000263589700003
View details for PubMedID 19217416
A phosphorylation-dependent intramolecular interaction regulates the membrane association and activity of the tumor suppressor PTEN
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2009; 106 (2): 480-485
The PI 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10), one of the most important tumor suppressors, must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate. Yet the mechanism of membrane binding has received little attention and the key determinants that regulate localization, a phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding motif and a cluster of phosphorylated C-terminal residues, were not included in the crystal structure. We report that membrane binding requires PIP(2) and show that phosphorylation regulates an intramolecular interaction. A truncated version of the enzyme, PTEN(1-351), bound strongly to the membrane, an effect that was reversed by co-expression of the remainder of the molecule, PTEN(352-403). The separate fragments associated in vitro, an interaction dependent on phosphorylation of the C-terminal cluster, a portion of the PIP(2) binding motif, integrity of the phosphatase domain, and the CBR3 loop. Our investigation provides direct evidence for a model in which PTEN switches between open and closed states and phosphorylation favors the closed conformation, thereby regulating localization and function. Small molecules targeting these interactions could potentially serve as therapeutic agents in antagonizing Ras or PI3K-driven tumors. The study also stresses the importance of determining the structure of the native enzyme.
View details for DOI 10.1073/pnas.811212106
View details for Web of Science ID 000262804000024
View details for PubMedID 19114656
Modular control of endothelial sheet migration
GENES & DEVELOPMENT
2008; 22 (23): 3268-3281
Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell-cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell-cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell-cell coordination.
View details for DOI 10.1101/gad.1725808
View details for Web of Science ID 000261349600006
View details for PubMedID 19056882
Feedback loops shape cellular signals in space and time
2008; 322 (5900): 390-395
Positive and negative feedback loops are common regulatory elements in biological signaling systems. We discuss core feedback motifs that have distinct roles in shaping signaling responses in space and time. We also discuss approaches to experimentally investigate feedback loops in signaling systems.
View details for DOI 10.1126/science.1160617
View details for Web of Science ID 000260094500033
View details for PubMedID 18927383
Synthetic Activation of Endogenous PI3K and Rac Identifies an AND-Gate Switch for Cell Polarization and Migration
2008; 3 (8)
Phosphatidylinositol 3-OH kinase (PI3K) has been widely studied as a principal regulator of cell polarization, migration, and chemotaxis. Surprisingly, recent studies showed that mammalian neutrophils and Dictyostelium discoideum cells can polarize and migrate in the absence of PI3K activity. Here we directly probe the roles of PI3K and its downstream effector, Rac, in HL-60 neutrophils by using a chemical biology approach whereby the endogenously present enzymes are synthetically activated in less than one minute. We show that uniform activation of endogenous PI3K is sufficient to polarize previously unpolarized neutrophils and trigger effective cell migration. After a delay following symmetrical phosphatidylinositol (3,4,5)-triphosphate (PIP(3)) production, a polarized distribution of PIP(3) was induced by positive feedback requiring actin polymerization. Pharmacological studies argue that this process does not require receptor-coupled trimeric G proteins. Contrary to the current working model, rapid activation of endogenous Rac proteins triggered effective actin polymerization but failed to feed back to PI3K to generate PIP(3) or induce cell polarization. Thus, the increase in PIP(3) concentration at the leading edge is generated by positive feedback with an AND gate logic with a PI3K-Rac-actin polymerization pathway as a first input and a PI3K initiated non-Rac pathway as a second input. This AND-gate control for cell polarization can explain how Rac can be employed for both PI3K-dependent and -independent signaling pathways coexisting in the same cell.
View details for DOI 10.1371/journal.pone.0003068
View details for Web of Science ID 000264796400008
View details for PubMedID 18728784
A nucleostemin family GTPase, NS3, acts in serotonergic neurons to regulate insulin signaling and control body size
GENES & DEVELOPMENT
2008; 22 (14): 1877-1893
Growth and body size are regulated by the CNS, integrating the genetic developmental program with assessments of an animal's current energy state and environmental conditions. CNS decisions are transmitted to all cells of the animal by insulin/insulin-like signals. The molecular biology of the CNS growth control system has remained, for the most part, elusive. Here we identify NS3, a Drosophila nucleostemin family GTPase, as a powerful regulator of body size. ns3 mutants reach <60% of normal size and have fewer and smaller cells, but exhibit normal body proportions. NS3 does not act cell-autonomously, but instead acts at a distance to control growth. Rescue experiments were performed by expressing wild-type ns3 in many different cells of ns3 mutants. Restoring NS3 to only 106 serotonergic neurons rescued global growth defects. These neurons are closely apposed with those of insulin-producing neurons, suggesting possible communication between the two neuronal systems. In the brains of ns3 mutants, excess serotonin and insulin accumulate, while peripheral insulin pathway activation is low. Peripheral insulin pathway activation rescues the growth defects of ns3 mutants. The findings suggest that NS3 acts in serotonergic neurons to regulate insulin signaling and thus exert global growth control.
View details for DOI 10.1101/gad.1670508
View details for Web of Science ID 000257643400005
View details for PubMedID 18628395
Phospholipase D activity regulates integrin-mediated cell spreading and migration by inducing GTP-Rac translocation to the plasma membrane
MOLECULAR BIOLOGY OF THE CELL
2008; 19 (7): 3111-3123
Small GTPase Rac is a crucial regulator of actin cytoskeletal rearrangement, and it plays an important role in cell spreading, migration, mitogenesis, phagocytosis, superoxide generation, and axonal growth. It is generally accepted that Rac activity is regulated by the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle. But, it is suggested that in addition to Rac-GTP loading, membrane localization is required for the initiation of downstream effector signaling. However, the molecular mechanisms that control the targeting of GTP-Rac to the plasma membrane remain largely unknown. Here, we have uncovered a signaling pathway linking phospholipase D (PLD) to the localized functions of Rac1. We show that PLD product phosphatidic acid (PA) acts as a membrane anchor of Rac1. The C-terminal polybasic motif of Rac1 is responsible for direct interaction with PA, and Rac1 mutated in this region is incapable of translocating to the plasma membrane and of activating downstream target p21-activated kinase upon integrin activation. Finally, we show that PA induces dissociation of Rho-guanine nucleotide dissociation inhibitor from Rac1 and that PA-mediated Rac1 localization is important for integrin-mediated lamellipodia formation, cell spreading, and migration. These results provide a novel molecular mechanism for the GTP-Rac1 localization through the elevating PLD activity, and they suggest a general mechanism for diverse cellular functions that is required localized Rac activation.
View details for DOI 10.1091/mbc.E07-04-0337
View details for Web of Science ID 000259158200037
View details for PubMedID 18480413
Comprehensive identification of PIP3-regulated PH domains from C elegans to H sapiens by model prediction and live imaging
2008; 30 (3): 381-392
Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.
View details for DOI 10.1016/j.molcel.2008.04.008
View details for Web of Science ID 000255761200015
View details for PubMedID 18471983
Suspended-drop electroporation for high-throughput delivery of biomolecules into cells
2008; 5 (5): 393-395
We present a high-throughput method that enables efficient delivery of biomolecules into cells. The device consists of an array of 96 suspended electrode pairs, where small sample volumes are top-loaded, electroporated and bottom-ejected into 96-well plates. We demonstrate the use of this suspended-drop electroporation (SDE) device to effectively introduce fluorescent dextran, small interfering RNA (siRNA) or cDNA into primary neurons, differentiated neutrophils and other cell types with conventionally low transfection rates.
View details for DOI 10.1038/NMETH.1201
View details for Web of Science ID 000255411700012
View details for PubMedID 18408727
Dissecting the role of PtdIns(4,5)P-2 in endocytosis and recycling of the transferrin receptor
JOURNAL OF CELL SCIENCE
2008; 121 (9): 1488-1494
Endocytosis and recycling of membrane proteins are key processes for nutrient uptake, receptor signaling and synaptic transmission. Different steps in these fission and fusion cycles have been proposed to be regulated by physiological changes in plasma membrane (PM) phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P(2)] concentration. Here, we use a chemical enzyme-translocation strategy to rapidly reduce PM PtdIns(4,5)P(2) levels while monitoring clathrin-mediated endocytosis and recycling. PtdIns(4,5)P(2) hydrolysis blocked transferrin receptor endocytosis and led to a marked increase in the concentration of transferrin receptors in the PM, suggesting that endocytosis is more sensitive to changes in PtdIns(4,5)P(2) than recycling. Reduction of PM PtdIns(4,5)P(2) levels led to a near complete dissociation of Adaptor protein 2 (AP-2) from the PM but had only a small effect on clathrin assembly. This argues that receptor-mediated PtdIns(4,5)P(2) reduction preferentially suppresses AP-2-mediated targeting of cargo to endocytic sites rather than the assembly of clathrin coats or recycling of endocytic vesicles.
View details for DOI 10.1242/jcs.020792
View details for Web of Science ID 000255269300016
View details for PubMedID 18411250
Robust neuronal symmetry breaking by Ras-triggered local positive feedback
2008; 18 (1): 44-50
Neuronal polarity is initiated by a symmetry-breaking event whereby one out of multiple minor neurites undergoes rapid outgrowth and becomes the axon . Axon formation is regulated by phosphatidylinositol 3-kinase (PI3K)-related signaling elements [2-10] that drive local actin  and microtubule reorganization [3, 12], but the upstream signaling circuit that causes symmetry breaking and guarantees the formation of a single axon is not known. Here, we use live FRET imaging in hippocampal neurons and show that the activity of the small GTPase HRas, an upstream regulator of PI3K, markedly increases in the nascent axonal growth cone upon symmetry breaking. This local increase in HRas activity results from a positive feedback loop between HRas and PI3K, locally reinforced by vesicular transport of HRas to the axonal growth cone. Recruitment of HRas to the axonal growth cone is paralleled by a decrease in HRas concentration in the remaining neurites, suggesting that competition for a limited pool of HRas guarantees that only one axon forms. Mathematical modeling demonstrates that local positive feedback between HRas and PI3K, coupled to recruitment of a limited pool of HRas, generates robust symmetry breaking and formation of a single axon in the absence of extrinsic spatial cues.
View details for DOI 10.1016/j.cub.2007.11.051
View details for Web of Science ID 000252292400027
View details for PubMedID 18158244
STIM2 is a feedback regulator that stabilizes basal cytosolic and endoplasmic reticulum Ca2+ levels
2007; 131 (7): 1327-1339
Deviations in basal Ca2+ levels interfere with receptor-mediated Ca2+ signaling as well as endoplasmic reticulum (ER) and mitochondrial function. While defective basal Ca2+ regulation has been linked to various diseases, the regulatory mechanism that controls basal Ca2+ is poorly understood. Here we performed an siRNA screen of the human signaling proteome to identify regulators of basal Ca2+ concentration and found STIM2 as the strongest positive regulator. In contrast to STIM1, a recently discovered signal transducer that triggers Ca2+ influx in response to receptor-mediated depletion of ER Ca2+ stores, STIM2 activated Ca2+ influx upon smaller decreases in ER Ca2+. STIM2, like STIM1, caused Ca2+ influx via activation of the plasma membrane Ca2+ channel Orai1. Our study places STIM2 at the center of a feedback module that keeps basal cytosolic and ER Ca2+ concentrations within tight limits.
View details for DOI 10.1016/j.cell.2007.11.039
View details for Web of Science ID 000252217200021
View details for PubMedID 18160041
A transgenic mouse model for high content, cell cycle phenotype screening in live primary cells
2007; 6 (18): 2276-2283
High content cell-based genetic and small molecule library screens are powerful strategies in drug discovery and investigations of disease mechanisms. We report that primary cells derived from a transgenic mouse model expressing a fluorescence mitosis biosensor provide unambiguous phenotype readouts without the need for transfection or immunocytochemistry. Phenotype profiles of cell cycle disruption and of apoptosis are easily detectable at a single time point selected from time-lapse live fluorescence microscopy. Most importantly, this transgenic mouse model may be crossed with cancer mouse models to derive biosensor-expressing primary cancer cells for use in high content screening strategies targeting discovery of tumor-specific chemotherapeutic compounds.
View details for Web of Science ID 000251085600015
View details for PubMedID 17881898
An essential role for the SHIP2-dependent negative feedback loop in neuritogenesis of nerve growth factor-stimulated PC12 cells
JOURNAL OF CELL BIOLOGY
2007; 177 (5): 817-827
The local accumulation of phosphatidylinositol (3,4,5) trisphosphate (PIP(3)) and resulting activation of Rac1/Cdc42 play a critical role in nerve growth factor (NGF)-induced neurite outgrowth. To further explore the mechanism, we visualized PIP(3), phosphatidylinositol (3,4) bisphosphate, and Rac1/Cdc42 activities by fluorescence resonance energy transfer (FRET) imaging in NGF-stimulated PC12 cells. Based on the obtained FRET images, and with the help of in silico kinetic reaction model, we predicted that PI-5-phosphatase negatively regulates PIP(3) upon NGF stimulation. In agreement with this model, depletion of Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2) markedly potentiated NGF-induced Rac1/Cdc42 activation and PIP(3) accumulation and considerably increased the number and the length of neurites in phosphate and tensin homologue-depleted PC12 cells. Further refinement of the computational model predicted Rac1 regulation of PI3-kinase and SHIP2, which was also validated experimentally. We propose that the SHIP2-mediated negative feedback on PIP(3) coordinately works with the PI3-kinase-mediated positive feedback to form an initial protrusive pattern and, later, to punctuate the PIP(3) accumulation to maintain proper neurite outgrowth.
View details for DOI 10.1083/jcb.200609017
View details for Web of Science ID 000247072700008
View details for PubMedID 17535963
Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (22): 9301-9306
Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca(2+) sensor that responds to ER Ca(2+) store depletion and activates Ca(2+) channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca(2+) store depletion. These oligomers rapidly dissociated when ER Ca(2+) stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca(2+) store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 mum to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca(2+) gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca(2+), (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca(2+) channels.
View details for DOI 10.1073/pnas.0702866104
View details for Web of Science ID 000246935700040
View details for PubMedID 17517596
The alliance for cellular signaling plasmid collection: A flexible resource for protein localization studies and signaling pathway analysis
MOLECULAR & CELLULAR PROTEOMICS
2007; 6 (3): 413-424
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
View details for DOI 10.1074/mcp.M600437-MCP200
View details for Web of Science ID 000245214900004
View details for PubMedID 17192258
A network of Rab GTPases controls phagosome maturation and is modulated by Salmonella enterica serovar Typhimurium
JOURNAL OF CELL BIOLOGY
2007; 176 (3): 263-268
Members of the Rab guanosine triphosphatase (GTPase) family are key regulators of membrane traffic. Here we examined the association of 48 Rabs with model phagosomes containing a non-invasive mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium). This mutant traffics to lysosomes and allowed us to determine which Rabs localize to a maturing phagosome. In total, 18 Rabs associated with maturing phagosomes, each with its own kinetics of association. Dominant-negative mutants of Rab23 and 35 inhibited phagosome-lysosome fusion. A large number of Rab GTPases localized to wild-type Salmonella-containing vacuoles (SCVs), which do not fuse with lysosomes. However, some Rabs (8B, 13, 23, 32, and 35) were excluded from wild-type SCVs whereas others (5A, 5B, 5C, 7A, 11A, and 11B) were enriched on this compartment. Our studies demonstrate that a complex network of Rab GTPases controls endocytic progression to lysosomes and that this is modulated by S. Typhimurium to allow its intracellular growth.
View details for DOI 10.1083/jcb.200611056
View details for Web of Science ID 000243997700003
View details for PubMedID 17261845
Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1
2007; 17 (1): 85-91
Mitosis is thought to be triggered by the activation of Cdk-cyclin complexes. Here we have used RNA interference (RNAi) to assess the roles of three mitotic cyclins, cyclins A2, B1, and B2, in the regulation of centrosome separation and nuclear-envelope breakdown (NEB) in HeLa cells. We found that the timing of NEB was affected very little by knocking down cyclins B1 and B2 alone or in combination. However, knocking down cyclin A2 markedly delayed NEB, and knocking down both cyclins A2 and B1 delayed NEB further. The timing of cyclin B1-Cdk1 activation was normal in cyclin A2 knockdown cells, and there was no delay in centrosome separation, an event apparently controlled by the activation of cytoplasmic cyclin B1-Cdk1. However, nuclear accumulation of cyclin B1-Cdk1 was markedly delayed in cyclin A2 knockdown cells. Finally, a constitutively nuclear cyclin B1, but not wild-type cyclin B1, restored normal NEB timing in cyclin A2 knockdown cells. These findings show that cyclin A2 is required for timely NEB, whereas cyclins B1 and B2 are not. Nevertheless cyclin B1 translocates to the nucleus just prior to NEB in a cyclin A2-dependent fashion and is capable of supporting NEB if rendered constitutively nuclear.
View details for DOI 10.1016/j.cub.2006.11.066
View details for Web of Science ID 000243461300030
View details for PubMedID 17208191
siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5) P-3-mTOR signaling pathway as a primary regulator of transferrin uptake
2007; 8 (7)
Iron uptake via endocytosis of iron-transferrin-transferrin receptor complexes is a rate-limiting step for cell growth, viability and proliferation in tumor cells as well as non-transformed cells such as activated lymphocytes. Signaling pathways that regulate transferrin uptake have not yet been identified.We surveyed the human signaling proteome for regulators that increase or decrease transferrin uptake by screening 1,804 dicer-generated signaling small interfering RNAs using automated quantitative imaging. In addition to known transport proteins, we identified 11 signaling proteins that included a striking signature set for the phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)-target of rapamycin (mTOR) signaling pathway. We show that the PI3K-mTOR signaling pathway is a positive regulator of transferrin uptake that increases the number of transferrin receptors per endocytic vesicle without affecting endocytosis or recycling rates.Our study identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a new regulator of iron-transferrin uptake and serves as a proof-of-concept that targeted RNA interference screens of the signaling proteome provide a powerful and unbiased approach to discover or rank signaling pathways that regulate a particular cell function.
View details for DOI 10.1186/gb-2007-8-7-r142
View details for Web of Science ID 000249416400022
View details for PubMedID 17640392
Activation mechanisms of the ER Ca2+ sensor STIM1
CELL PRESS. 2007: 365A-366A
View details for Web of Science ID 000243972402257
PI(3,4,5)P-3 and PI(4,5)P-2 lipids target proteins with polybasic clusters to the plasma membrane
2006; 314 (5804): 1458-1461
Many signaling, cytoskeletal, and transport proteins have to be localized to the plasma membrane (PM) in order to carry out their function. We surveyed PM-targeting mechanisms by imaging the subcellular localization of 125 fluorescent protein-conjugated Ras, Rab, Arf, and Rho proteins. Out of 48 proteins that were PM-localized, 37 contained clusters of positively charged amino acids. To test whether these polybasic clusters bind negatively charged phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] lipids, we developed a chemical phosphatase activation method to deplete PM PI(4,5)P2. Unexpectedly, proteins with polybasic clusters dissociated from the PM only when both PI(4,5)P2 and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] were depleted, arguing that both lipid second messengers jointly regulate PM targeting.
View details for DOI 10.1126/science.1134389
View details for Web of Science ID 000242406100043
View details for PubMedID 17095657
Rapid chemically induced changes of Ptdlns(4,5)P-2 gate KCNQ ion channels
2006; 314 (5804): 1454-1457
To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C-mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.
View details for DOI 10.1126/science.1131163
View details for Web of Science ID 000242406100042
View details for PubMedID 16990515
A high throughput approach to the live cell monitoring of the mitotic cell cycle.
ELSEVIER SCIENCE INC. 2006: S218-S218
View details for Web of Science ID 000241038500524
Synergistic control of protein kinase C gamma activity by ionotropic and metabotropic glutamate receptor inputs in hippocampal neurons
JOURNAL OF NEUROSCIENCE
2006; 26 (13): 3404-3411
Conventional protein kinase C (PKC) isoforms are abundant neuronal signaling proteins with important roles in regulating synaptic plasticity and other neuronal processes. Here, we investigate the role of ionotropic and metabotropic glutamate receptor (iGluR and mGluR, respectively) activation on the generation of Ca2+ and diacylglycerol (DAG) signals and the subsequent activation of the neuron-specific PKCgamma isoform in hippocampal neurons. By combining Ca2+ imaging with total internal reflection microscopy analysis of specific biosensors, we show that elevation of both Ca2+ and DAG is necessary for sustained translocation and activation of EGFP (enhanced green fluorescent protein)-PKCgamma. Both DAG production and PKCgamma translocation were localized processes, typically observed within discrete microdomains along the dendritic branches. Markedly, intermediate-strength NMDA receptor (NMDAR) activation or moderate electrical stimulation generated Ca2+ but no DAG signals, whereas mGluR activation generated DAG but no Ca2+ signals. Both receptors were needed for PKCgamma activation. This suggests that a coincidence detection process exists between iGluRs and mGluRs that relies on a molecular coincidence detection process based on the corequirement of Ca2+ and DAG for PKCgamma activation. Nevertheless, the requirement for costimulation with mGluRs could be overcome for maximal NMDAR stimulation through a direct production of DAG via activation of the Ca2+-sensitive PLCdelta (phospholipase Cdelta) isoform. In a second important exception, mGluRs were sufficient for PKCgamma activation in neurons in which Ca2+ stores were loaded by previous electrical activity. Together, the dual activation requirement for PKCgamma provides a plausible molecular interpretation for different synergistic contributions of mGluRs to long-term potentiation and other synaptic plasticity processes.
View details for DOI 10.1523/JNEUROSCI.0478-06.2006
View details for Web of Science ID 000236363400005
View details for PubMedID 16571747
Interlinked fast and slow positive feedback loops drive reliable cell decisions
2005; 310 (5747): 496-498
Positive feedback is a ubiquitous signal transduction motif that allows systems to convert graded inputs into decisive, all-or-none outputs. Here we investigate why the positive feedback switches that regulate polarization of budding yeast, calcium signaling, Xenopus oocyte maturation, and various other processes use multiple interlinked loops rather than single positive feedback loops. Mathematical simulations revealed that linking fast and slow positive feedback loops creates a "dual-time" switch that is both rapidly inducible and resistant to noise in the upstream signaling system.
View details for DOI 10.1126/science.1113834
View details for Web of Science ID 000232786000048
View details for PubMedID 16239477
Spines and neurite branches function as geometric attractors that enhance protein kinase C action
JOURNAL OF CELL BIOLOGY
2005; 170 (7): 1147-1158
Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.
View details for DOI 10.1083/jcb.200503118
View details for Web of Science ID 000232120700015
View details for PubMedID 16186260
Reversible intracellular translocation of KRas but not HRas in hippocampal neurons regulated by Ca2+/calmodulin
JOURNAL OF CELL BIOLOGY
2005; 170 (3): 429-441
The Ras/MAPK pathway regulates synaptic plasticity and cell survival in neurons of the central nervous system. Here, we show that KRas, but not HRas, acutely translocates from the plasma membrane (PM) to the Golgi complex and early/recycling endosomes in response to neuronal activity. Translocation is reversible and mediated by the polybasic-prenyl membrane targeting motif of KRas. We provide evidence that KRas translocation occurs through sequestration of the polybasic-prenyl motif by Ca2+/calmodulin (Ca2+/CaM) and subsequent release of KRas from the PM, in a process reminiscent of GDP dissociation inhibitor-mediated membrane recycling of Rab and Rho GTPases. KRas translocation was accompanied by partial intracellular redistribution of its activity. We conclude that the polybasic-prenyl motif acts as a Ca2+/CaM-regulated molecular switch that controls PM concentration of KRas and redistributes its activity to internal sites. Our data thus define a novel signaling mechanism that differentially regulates KRas and HRas localization and activity in neurons.
View details for DOI 10.1083/jcb.200409157
View details for Web of Science ID 000230901600012
View details for PubMedID 16043511
STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx
2005; 15 (13): 1235-1241
Ca(2+) signaling in nonexcitable cells is typically initiated by receptor-triggered production of inositol-1,4,5-trisphosphate and the release of Ca(2+) from intracellular stores. An elusive signaling process senses the Ca(2+) store depletion and triggers the opening of plasma membrane Ca(2+) channels. The resulting sustained Ca(2+) signals are required for many physiological responses, such as T cell activation and differentiation. Here, we monitored receptor-triggered Ca(2+) signals in cells transfected with siRNAs against 2,304 human signaling proteins, and we identified two proteins required for Ca(2+)-store-depletion-mediated Ca(2+) influx, STIM1 and STIM2. These proteins have a single transmembrane region with a putative Ca(2+) binding domain in the lumen of the endoplasmic reticulum. Ca(2+) store depletion led to a rapid translocation of STIM1 into puncta that accumulated near the plasma membrane. Introducing a point mutation in the STIM1 Ca(2+) binding domain resulted in prelocalization of the protein in puncta, and this mutant failed to respond to store depletion. Our study suggests that STIM proteins function as Ca(2+) store sensors in the signaling pathway connecting Ca(2+) store depletion to Ca(2+) influx.
View details for DOI 10.1016/j.cub.2005.05.055
View details for Web of Science ID 000230662400030
View details for PubMedID 16005298
An inducible translocation strategy to rapidly activate and inhibit small GTPase signaling pathways
2005; 2 (6): 415-418
We made substantial advances in the implementation of a rapamycin-triggered heterodimerization strategy. Using molecular engineering of different targeting and enzymatic fusion constructs and a new rapamycin analog, Rho GTPases were directly activated or inactivated on a timescale of seconds, which was followed by pronounced cell morphological changes. As signaling processes often occur within minutes, such rapid perturbations provide a powerful tool to investigate the role, selectivity and timing of Rho GTPase-mediated signaling processes.
View details for DOI 10.1038/NMETH763
View details for Web of Science ID 000229424300011
View details for PubMedID 15908919
Efficient chromosome capture requires a bias in the 'search-and-capture' process during mitotic-spindle assembly
2005; 15 (9): 828-832
The mitotic spindle assembles into a bipolar, microtubule-based protein machine during prometaphase. One proposed mechanism for this process is "search-and-capture," in which dynamically unstable microtubules (MTs) search space to capture chromosomes. Although existing theoretical estimates suggest that dynamic instability is efficient enough to allow capture within characteristic mitotic timescales, they are limited in scope and do not address the capture times for realistic numbers of chromosomes. Here we used mathematical modeling to explore this issue. We show that without any bias toward the chromosomes, search-and-capture is not efficient enough to explain the typical observed duration of prometaphase. We further analyze search-and-capture in the presence of a spatial gradient of a stabilizing factor that biases MT dynamics toward the chromosomes. We show theoretically that such biased search-and-capture is efficient enough to account for chromosome capture. We also show that additional factors must contribute to accelerate the spindle assembly for cells with large nuclear volumes. We discuss the possibility that a RanGTP gradient introduces a spatial bias into microtubule dynamics and thus improves the efficiency of search-and-capture as a mechanism for spindle assembly.
View details for DOI 10.1016/j.cub.2005.03.019
View details for Web of Science ID 000229298100023
View details for PubMedID 15886100
A local coupling model and compass parameter for eukaryotic chemotaxis
2005; 8 (2): 215-227
Chemotaxis is a cellular sensing mechanism that guides immune cells to sites of infection and leads fibroblasts to sites of injury. Here, we show in migrating primary dendritic cells and fibroblasts that the leading edge is not a uniform signaling entity, but instead consists of independent coupling units in which transient activation of PI3-kinase links to local lamellipod extension and small discrete turns in the direction of migration. These findings led to a model in which global cell polarization is independent from the chemotaxis mechanism. In this model, chemotaxis does not require spatial integration but is instead a stochastic process in which each receptor binding event within the leading edge triggers a local lamellipod extension and a small turn in the direction of migration. We show that this model and a derived "compass parameter" are sufficient to simulate the observed random migration, biased random walk, and persistent chemotactic behaviors of eukaryotic cells.
View details for DOI 10.1016/j.devcel.2004.12.007
View details for Web of Science ID 000226875500012
View details for PubMedID 15691763
Protein localization studies in the age of 'Omics'
CURRENT OPINION IN CHEMICAL BIOLOGY
2005; 9 (1): 82-87
As thousands of new genes are identified in genomics efforts, the rush is on to learn something about the functional roles of the proteins encoded by those genes. Clues to protein functions, activation states and protein-protein interactions have been revealed in focused studies of protein localization. With technical breakthroughs such as GFP protein tagging and recombinase cloning systems, large-scale screens of protein localization are now being undertaken.
View details for DOI 10.1016/j.cbpa.2004.12.002
View details for Web of Science ID 000227483800014
View details for PubMedID 15701458
Simplified analogs of bryostatin with anticancer activity display greater potency for translocation of PKC delta-GFP
CHEMISTRY & BIOLOGY
2004; 11 (9): 1261-1267
Structurally simplified analogs of bryostatin 1, a marine natural product in clinical trials for the treatment of cancer, have been shown to be up to 50 times more potent than bryostatin 1 at inducing the translocation of PKCdelta-GFP from the cytosol of rat basophilic leukemia (RBL) cells. The end distribution of the protein is similar for all three compounds, despite a significant difference in translocation kinetics. The potency of the compounds for inducing the translocation response appears to be only qualitatively related to their binding affinity for PKC, highlighting the importance of using binding affinity in conjunction with real-time measurements of protein localization for the pharmacological profiling of biologically active agents.
View details for DOI 10.1016/j.chembiol.2004.06.014
View details for Web of Science ID 000224228900009
View details for PubMedID 15380186
A critical intramolecular interaction for protein kinase C epsilon translocation
JOURNAL OF BIOLOGICAL CHEMISTRY
2004; 279 (16): 15831-15840
Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.
View details for DOI 10.1074/jbc.M310696200
View details for Web of Science ID 000220747900019
View details for PubMedID 14739299
The neural F-box protein NFB42 mediates the nuclear export of the herpes simplex virus type 1 replication initiator protein (UL9 protein) after viral infection
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (12): 4036-4040
The neural F-box 42-kDa protein (NFB42) is a component of the SCF(NFB42) E3 ubiquitin ligase that is expressed in all major areas of the brain; it is not detected in nonneuronal tissues. We previously identified NFB42 as a binding partner for the herpes simplex virus 1 (HSV-1) UL9 protein, the viral replication-initiator, and showed that coexpression of NFB42 and UL9 in human embryonic kidney (293T) cells led to a significant decrease in the level of UL9 protein. We have now found that HSV-1 infection promotes the shuttling of NFB42 between the cytosol and the nucleus in both 293T cells and primary hippocampal neurons, permitting NFB42 to bind to the phosphorylated UL9 protein, which is localized in the nucleus. This interaction mediates the export of the UL9 protein from the nucleus to the cytosol, leading to its ubiquitination and degradation via the 26S proteasome. Because the intranuclear localization of the UL9 protein, along with other viral and cellular factors, is an essential step in viral DNA replication, degradation of the UL9 protein in neurons by means of nuclear export through its specific interaction with NFB42 may prevent active replication and promote neuronal latency of HSV-1.
View details for DOI 10.1073/pnas.0400738101
View details for Web of Science ID 000220472200011
View details for PubMedID 15010529
Probing the precision of the mitotic clock with a live-cell fluorescent biosensor
2004; 22 (3): 306-312
Precise timing of mitosis is essential for high-fidelity cell duplication. However, temporal measurements of the mitotic clock have been challenging. Here we present a fluorescent mitosis biosensor that monitors the time between nuclear envelope breakdown (NEB) and re-formation using parallel total internal reflection fluorescence (TIRF) microscopy. By tracking tens to hundreds of mitotic events per experiment, we found that the mitotic clock of unsynchronized rat basophilic leukemia cells has a marked precision with 80% of cells completing mitosis in 32 +/- 6 min. This assay further allowed us to observe delays in mitotic timing at Taxol concentrations 100 times lower than previous minimal effective doses, explaining why Taxol is clinically active at low concentrations. Inactivation of the spindle checkpoint by targeting the regulator Mad2 with RNAi consistently shortened mitosis, providing direct evidence that the internal mitotic timing mechanism is much faster in cells that lack the checkpoint.
View details for DOI 10.1038/nbt941
View details for Web of Science ID 000189297200023
View details for PubMedID 14990952
Function oriented synthesis: the design, synthesis, PKC binding and translocation activity of a new bryostatin analog.
Current drug discovery technologies
2004; 1 (1): 1-11
Bryostatin 1 represents a novel and potent therapeutic lead with a unique activity profile. Its natural and synthetic availability is severely limited. Function oriented synthesis provides a means to address this supply problem through the design of synthetically more accessible simplified structures that at the same time incorporate improved functional activity. Pharmacophore searching and a new computer aided visualization of a possible binding mode are combined with an understanding of function and knowledge of synthesis to design and prepare a new and simplified compound with bryostatin-like function in biological systems. This new compound is a potent ligand for protein kinase C in vitro (K(i) = 8.0 nM). More significantly, the described molecule retains the functional ability to translocate a PKCdelta-GFP fusion protein in RBL cells. The extent of protein translocation and the sub-cellular localization induced by this new compound is similar to that seen in response to bryostatin 1, indicating that the new molecule retains the functional activity of the natural product but is simpler and can be synthesized in a practical fashion.
View details for PubMedID 16472215
Specific localization and timing in neuronal signal transduction mediated by protein-lipid interactions
2003; 40 (2): 319-330
A large number of signaling proteins translocate from the cytosol to the plasma membrane in response to receptor and electrical stimuli. The site of translocation to the plasma membrane and the "on" and "off" rates of the translocation process are critical for defining the specificity of the signaling response. In addition to targeting mechanisms based on protein-protein interactions, signaling proteins have evolved a large repertoire of covalent lipid modifications and lipid binding protein modules that regulate reversible membrane association. The time constants of these membrane interactions range from milliseconds to several hours. Here we discuss how diversity in lipid-based membrane anchoring and targeting motifs contributes to plasticity in neuronal signaling by providing local and regional control mechanisms as well as a means to transduce and integrate signals over a broad range of different time scales.
View details for Web of Science ID 000185875800009
View details for PubMedID 14556711
An ultrasensitive Ca2+/calmodulin-dependent protein kinase II-protein phosphatase 1 switch facilitates specificity in postsynaptic calcium signaling
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2003; 100 (18): 10512-10517
The strength of hippocampal synapses can be persistently increased by signals that activate Ca2+/calmodulin-dependent protein kinase II (CaMKII). This CaMKII-dependent long-term potentiation is important for hippocampal learning and memory. In this work we show that CaMKII exhibits an intriguing switch-like activation that likely is important for changes in synaptic strength. We found that autophosphorylation of CaMKII by itself showed a steep dependence on Ca2+ concentration [Hill coefficient (nH) approximately 5]. However, an even steeper Ca2+ dependence (nH approximately 8) was observed when autophosphorylation is balanced by the dephosphorylation activity of protein phosphatase 1 (PP1). This autophosphorylation-dephosphorylation switch was found to be reversible because PP1 dephosphorylates CaMKII when Ca2+ is lowered to a basal level. The switch-like response of a CaMKII-PP1 system suggests that CaMKII and PP1 may function together as a simple molecular device that specifically translates only strong Ca2+ signals into all-or-none potentiation of individual hippocampal synapses.
View details for DOI 10.1073/pnas.1932759100
View details for Web of Science ID 000185119300068
View details for PubMedID 12928489
Selective regulation of neurite extension and synapse formation by the beta but not the of isoform of CaMKII
2003; 39 (2): 283-297
Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.
View details for Web of Science ID 000184256800009
View details for PubMedID 12873385
Switch-of-function mutants based on morphology classification of Ras superfamily small GTPases
2003; 113 (3): 315-328
Signaling proteins from the same family can have markedly different roles in a given cellular context. Here, we show that expression of one hundred constitutively active human small GTPases induced cell morphologies that fell into nine distinct classes. We developed an algorithm for pairs of classes that predicted amino acid positions that can be exchanged to create mutants with switched functionality. The algorithm was validated by creating switch-of-function mutants for Rac1, CDC42, H-Ras, RalA, Rap2B, and R-Ras3. Contrary to expectations, the relevant residues were mostly outside known interaction surfaces and were structurally far apart from each other. Our study shows that specificity in protein families can be explored by combining genome-wide experimental functional classification with the creation of switch-of-function mutants.
View details for Web of Science ID 000182640800007
View details for PubMedID 12732140
Recombinant Dicer efficiently converts large dsRNAs into siRNAs suitable for gene silencing
2003; 21 (3): 324-328
RNA interference (RNAi) is a powerful method for specifically silencing gene expression in diverse cell types. RNAi is mediated by approximately 21-nucleotide small interfering RNAs (siRNAs), which are produced from larger double-stranded RNAs (dsRNAs) in vivo through the action of Dicer, an RNase III-family enzyme. Transfecting cells with siRNAs rather than larger dsRNAs avoids the nonspecific gene silencing of the interferon response, underscoring the importance of developing efficient methods for producing reliable siRNAs. Here we show that pools of 20- to 21-base pair (bp) siRNAs can be produced enzymatically in vitro using active recombinant Dicer. Yields of < or = 70% are obtained, and the siRNAs can be easily separated from any residual large dsRNA by a series of spin columns or gel purification. Dicer-generated siRNAs (d-siRNAs) are effective in silencing transiently transfected reporter genes and endogenous genes, making in vitro dicing a useful, practical alternative for the production of siRNAs.
View details for DOI 10.1038/nbt792
View details for Web of Science ID 000181312500032
View details for PubMedID 12592410
Single cell imaging of PI3K activity and glucose transporter insertion into the plasma membrane by dual color evanescent wave microscopy.
Science's STKE : signal transduction knowledge environment
2003; 2003 (169): PL4-?
Many signaling events involve the translocation of signaling molecules to or from the plasma membrane; however, suitable techniques to quantify the temporal relationships between such signaling events are lacking. Here, we describe an evanescent wave microscopy technique that allows parallel measurement of the recruitment and dissociation of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) labeled proteins to and from the plasma membrane in individual living cells. The selective excitation of fluorescence in a zone less than 100 nm above a cover glass enables selective imaging within the plasma membrane of adherent cells, with markedly improved resolution, lower background, and minimal phototoxicity compared to confocal microscopy and other microscopy-based assays. In the microscope design we have developed, the beams from helium-cadmium (442 nm) and argon (514 nm) lasers are merged and focused through a dove prism at an angle that yields total internal reflection. In this configuration, evanescent wave-excited fluorescence at the glass-water interface can be detected with either high or low magnification, to allow for high-resolution imaging or the study of many cells in parallel. We applied this technique to make parallel measurements of the time-course of insulin-triggered activation of phosphatidylinositol 3-kinase (PI3K) and GLUT4 glucose transporter insertion into the plasma membrane of individual differentiated 3T3L1 adipocytes using a phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P(3)]-binding pleckstrin homology domain fused to CFP, and GLUT4 conjugated to YFP. The technique should have wide applicability to various cell types and diverse signaling processes.
View details for PubMedID 12582202
Fluorescence imaging of signaling networks
TRENDS IN CELL BIOLOGY
2003; 13 (2): 101-106
Receptor-triggered signaling processes exhibit complex cross-talk and feedback interactions, with many signaling proteins and second messengers acting locally within the cell. The flow of information in this input-output system can only be understood by tracking where and when local signaling activities are induced. Systematic strategies are therefore needed to measure the localization and translocation of all signaling proteins, and to develop fluorescent biosensors that can track local signaling activities in individual cells. Such a biosensor tool chest can be based on two types of green fluorescent protein constructs that either translocate or undergo fluorescence-resonance-energy transfer when local signaling occurs. Broad strategies to measure quantitative, dynamic parameters in signaling networks, together with perturbation approaches, are needed to develop comprehensive models of signaling networks*.
View details for Web of Science ID 000180957300008
View details for PubMedID 12559761
A PI3-kinase signaling code for insulin-triggered insertion of glucose transporters into the plasma membrane
2002; 12 (21): 1871-1876
Activation of phosphatidyl-inositol-3'-OH-kinase (PI3K) and the resulting production of phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) are ubiquitous signaling steps that link various cell surface receptors to multiple intracellular targets. In fat and muscle cells, the same PI3K pathway that regulates metabolic enzymes, proliferation, and differentiation has also been shown to be involved in insulin-triggered insertion of glucose transporter GLUT4 into the plasma membrane. The multiple PI3K functions raise the question of how the same PI3K pathway can be selectively used for different cell functions. Here we developed a dual-color evanescent wave microscopy method to simultaneously measure PIP3 production and GLUT4 insertion in individual 3T3L1 adipocytes. Activation of PI3K was found to be both necessary and sufficient for triggering GLUT4 insertion, but transporter insertion was markedly suppressed for small-amplitude, persistent PIP3 signals and for large-amplitude, short PIP3 signals. The rejection of these common PI3K signaling responses may explain the selective advantage of insulin over platelet-derived growth factor and other stimuli for inducing GLUT4 insertion. Our study suggests that the same PI3K pathway can control specific cell functions by relying on effector systems that respond to particular receptor-encoded time courses and amplitudes of PIP3 signals.
View details for Web of Science ID 000179169800028
View details for PubMedID 12419189
Elimination of host cell PtdIns(4,5)P-2 by bacterial SigD promotes membrane fission during invasion by Salmonella
NATURE CELL BIOLOGY
2002; 4 (10): 766-773
Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.
View details for DOI 10.1038/ncb854
View details for Web of Science ID 000178316500013
View details for PubMedID 12360287
Molecular mechanisms of CaMKII activation in neuronal plasticity
CURRENT OPINION IN NEUROBIOLOGY
2002; 12 (3): 293-299
Calcium/calmodulin-dependent protein kinase II (CaMKII) is thought to be a critical mediator of neuronal plasticity that links transiently triggered Ca(2+) signals to persistent changes in neuronal physiology. In one of its roles, CaMKII is an essential player in the N-methyl-D-aspartate receptor-mediated increase in conductance at glutamatergic synapses, a process described as long-term potentiation, which serves as a common model for neuronal plasticity and memory. Recent studies have used genetic, biochemical, live cell imaging and mathematical modeling approaches to investigate neuronal CaMKII and have led to a model of the molecular steps of CaMKII translocation and activation that can explain its role in neuronal plasticity.
View details for DOI 10.1016/S0959-4388(02)00327-6
View details for Web of Science ID 000176180800009
View details for PubMedID 12049936
Parallel single-cell monitoring of receptor-triggered membrane translocation of a calcium-sensing protein module
2002; 295 (5561): 1910-1912
Time courses of translocation of fluorescently conjugated proteins to the plasma membrane were simultaneously measured in thousands of individual rat basophilic leukemia cells. We found that the C2 domain---a calcium-sensing, lipid-binding protein module that is an essential regulator of protein kinase C and numerous other proteins---targeted proteins to the plasma membrane transiently if calcium was released from internal stores, and persistently in response to entry of extracellular calcium across the plasma membrane. The C2 domain translocation time courses of stimulated cells clustered into only two primary modes. Hence, the reversible recruitment of families of signaling proteins from one cellular compartment to another is a rapid bifurcation mechanism for inducing discrete states of cellular signaling networks.
View details for Web of Science ID 000174299500045
View details for PubMedID 11884760
Active EGF receptors have limited access to Ptdlns(4,5)P-2 in endosomes: implications for phospholipase C and PI 3-kinase signaling
JOURNAL OF CELL SCIENCE
2002; 115 (2): 303-310
Although prolonged cell signaling is attenuated by internalization and downregulation of active receptors, it is now appreciated that many receptors continue to signal in intracellular compartments. Employing enhanced green fluorescent protein fusion probes, we have investigated the hypothesis that multiple signaling pathways are affected by the differential trafficking of membrane substrates such as PtdIns(4,5)P(2). A phosphotyrosine-specific probe, but not a PtdIns(4,5)P(2)-specific probe, colocalized with internalized EGF as well as transferrin in EGF-stimulated living cells expressing autophosphorylation-competent EGF receptors. Neither probe colocalized with transferrin in the absence of EGF, demonstrating that the reduced level of accessible PtdIns(4,5)P(2) in endosomes is constitutive. Finally, a PtdIns(3,4,5)P(3)-specific probe, which monitors phosphorylation of PtdIns(4,5)P(2) by phosphoinositide 3-kinases, was recruited to the plasma membrane but not to EGF- or transferrin-containing endosomes in response to EGF stimulation. These results suggest that while many internalized receptors continue to engage intracellular enzymes, the phospholipase C and phosphoinositide 3-kinase signaling pathways are abrogated by the constitutive lack of accessible PtdIns(4,5)P(2) in endosomes.
View details for Web of Science ID 000173768800008
View details for PubMedID 11839782
Control of astrocyte Ca2+ oscillations and waves by oscillating translocation and activation of protein kinase C
2001; 11 (14): 1089-1097
Glutamate-induced Ca2+ oscillations and waves coordinate astrocyte signaling responses, which in turn regulate neuronal excitability. Recent studies have suggested that the generation of these Ca2+ oscillations requires a negative feedback that involves the activation of conventional protein kinase C (cPKC). Here, we use total internal reflection fluorescence (TIRF) microscopy to investigate if and how periodic plasma membrane translocation of cPKC is used to generate Ca2+ oscillations and waves.Glutamate stimulation of astrocytes triggered highly localized GFP-PKCgamma plasma membrane translocation events, induced rapid oscillations in GFP-PKCgamma translocation, and generated GFP-PKCgamma translocation waves that propagated across and between cells. These translocation responses were primarily mediated by the Ca2+-sensitive C2 domains of PKCgamma and were driven by localized Ca2+ spikes, by oscillations in Ca2+ concentration, and by propagating Ca(2+) waves, respectively. Interestingly, GFP-conjugated C1 domains from PKCgamma or PKCdelta that have been shown to bind diacylglycerol (DAG) also oscillated between the cytosol and the plasma membrane after glutamate stimulation, suggesting that PKC is repetitively activated by combined oscillating increases in Ca(2+) and DAG concentrations. The expression of C1 domains, which increases the DAG buffering capacity and thereby delays changes in DAG concentrations, led to a marked prolongation of Ca(2+) spikes, suggesting that PKC activation is involved in terminating individual Ca(2+) spikes and waves and in defining the time period between Ca(2+) spikes.Our study suggests that cPKCs have a negative feedback role on Ca(2+) oscillations and waves that is mediated by their repetitive activation by oscillating DAG and Ca(2+) concentrations. Periodic translocation and activation of cPKC can be a rapid and markedly localized signaling event that can limit the duration of individual Ca(2+) spikes and waves and can define the Ca(2+) spike and wave frequencies.
View details for Web of Science ID 000170093700018
View details for PubMedID 11509231
Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis
JOURNAL OF CELL BIOLOGY
2001; 153 (7): 1369-1380
Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.
View details for Web of Science ID 000169583700004
View details for PubMedID 11425868
Subcellular targeting by membrane lipids
CURRENT OPINION IN CELL BIOLOGY
2001; 13 (2): 146-152
The reversible localization of signaling proteins to both the plasma and the internal membranes of cells is critical for the selective activation of downstream functions and depends on interactions with both proteins and membrane lipids. New structural and biochemical analyses of C1, C2, PH, FYVE, FERM and other domains have led to an unprecedented amount of information on the molecular interactions of these signaling proteins with regulatory lipids. A wave of studies using GFP-tagged membrane binding domains as reporters has led to new quantitative insights into the kinetics of these signaling mechanisms.
View details for Web of Science ID 000167619200005
View details for PubMedID 11248547
Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis
JOURNAL OF CELL BIOLOGY
2000; 151 (7): 1353-1367
Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.
View details for Web of Science ID 000166237200001
View details for PubMedID 11134066
Spatial sensing in fibroblasts mediated by 3 ' phosphoinositides
JOURNAL OF CELL BIOLOGY
2000; 151 (6): 1269-1279
The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3' PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein-tagged Akt pleckstrin homology domain (GFP-AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3' PI lipids in the adhesion zone of fibroblasts. Polar GFP-AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3' PI production and rapid membrane spreading implicates 3' PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3' PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 microm(2)/s) and short lifetime of 3' PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3' PI second messengers.
View details for Web of Science ID 000165851300016
View details for PubMedID 11121441
- Translocation and reversible localization of signaling proteins: A dynamic future for signal transduction CELL 2000; 103 (2): 181-184
Molecular memory by reversible translocation of calcium/calmodulin-dependent protein kinase II
2000; 3 (9): 881-886
Synaptic plasticity is thought to be a key process for learning, memory and other cognitive functions of the nervous system. The initial events of plasticity require the conversion of brief electrical signals into alterations of the biochemical properties of synapses that last for much longer than the initial stimuli. Here we show that a regulator of synaptic plasticity, calcium/calmodulin-dependent protein kinase IIalpha (CaMKII), sequentially translocates to postsynaptic sites, undergoes autophosphorylation and gets trapped for several minutes until its dissociation is induced by secondary autophosphorylation and phosphatase 1 action. Once dissociated, CaMKII shows facilitated translocation for several minutes. This suggests that trapping of CaMKII by its targets and priming of CaMKII translocation may function as biochemical memory mechanisms that change the signaling capacity of synapses.
View details for Web of Science ID 000167177400014
View details for PubMedID 10966618
In and out of the postsynaptic region: signalling proteins on the move
TRENDS IN CELL BIOLOGY
2000; 10 (6): 238-244
Reversible translocation of signalling proteins to and from their sites of action has emerged as an important theme in signal transduction. The recent findings of the stimulus-induced translocation of Ca2+-calmodulin-dependent protein kinase II (CaMKII) and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor to and from the postsynaptic region are model cases for understanding how the dynamic localization of signalling proteins is used to regulate signal transduction.
View details for Web of Science ID 000087147000004
View details for PubMedID 10802539
Differential codes for free Ca2+-calmodulin signals in nucleus and cytosol
2000; 10 (2): 86-94
Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration.Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol.Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.
View details for Web of Science ID 000085042100019
View details for PubMedID 10662666
Phosphatidylinositol 4,5-bisphoshate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion
2000; 100 (2): 221-228
Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.
View details for Web of Science ID 000084932200006
View details for PubMedID 10660045
- Studies of signal transduction events using chimeras to green fluorescent protein APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS PT B 2000; 327: 500-513
A versatile microporation technique for the transfection of cultured CNS neurons
JOURNAL OF NEUROSCIENCE METHODS
1999; 93 (1): 37-48
The application of molecular techniques to cultured central nervous system (CNS) neurons has been limited by a lack of simple and efficient methods to introduce macromolecules into their cytosol. We have developed an electroporation technique that efficiently transfers RNA, DNA and other large membrane-impermeant molecules into adherent hippocampal neurons. Microporation allowed the use of either in vitro transcribed RNA or cDNA to transfect neurons. While RNA transfection yielded a higher percentage of transfected neurons and produced quantitative co-expression of two proteins, DNA transfection yielded higher levels of protein expression. Dextran-based calcium indicators also were efficiently delivered into the cytosol. Microporated neurons appear to survive poration quite well, as indicated by their morphological integrity, electrical excitability, ability to produce action potential-evoked calcium signals, and intact synaptic transmission. Furthermore, green fluorescent protein (GFP)-tagged marker proteins were expressed and correctly localized to the cytosol, plasma membrane, or endoplasmic reticulum. The microporation method is efficient, convenient, and inexpensive: macromolecules can be introduced into most adherent neurons in a 3 mm2 surface area while requiring as little as 1 microl of the material to be introduced. We conclude that the microporation of macromolecules is a versatile approach to investigate signaling, secretion, and other processes in CNS neurons.
View details for Web of Science ID 000084033900005
View details for PubMedID 10598863
Estrogen-induced activation of mitogen-activated protein kinase requires mobilization of intracellular calcium
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1999; 96 (8): 4686-4691
Estrogens and growth factors such as epidermal growth factor (EGF) act as mitogens promoting cellular proliferation in the breast and in the reproductive tract. Although it was considered originally that these agents manifested their mitogenic actions through separate pathways, there is a growing body of evidence suggesting that the EGF and estrogen-mediated signaling pathways are intertwined. Indeed, it has been demonstrated recently that 17beta-estradiol (E2) can induce a rapid activation of mitogen-activated protein kinase (MAPK) in mammalian cells, an event that is independent of both transcription and protein synthesis. In this study, we have used a pharmacological approach to dissect this novel pathway in MCF-7 breast cancer cells and have determined that in the presence of endogenous estrogen receptor, activation of MAPK by E2 is preceded by a rapid increase in cytosolic calcium. The involvement of intracellular calcium in this process was supported by the finding that the presence of EGTA and Ca2+-free medium did not affect the activation of MAPK by E2 and, additionally, that this response was blocked by the addition of the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate. Cumulatively, these data indicate that the estrogen receptor, in addition to functioning as a transcription factor, is also involved, through a nongenomic mechanism, in the regulation of both intracellular calcium homeostasis and MAPK-signaling pathways. Although nongenomic actions of estrogens have been suggested by numerous studies in the past, the ability to link estradiol and the estrogen receptor to a well defined signaling pathway strongly supports a physiological role for this activity.
View details for Web of Science ID 000079766500086
View details for PubMedID 10200323
Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation
1999; 284 (5411): 162-166
Calcium-calmodulin-dependent protein kinase II (CaMKII) is thought to increase synaptic strength by phosphorylating postsynaptic density (PSD) ion channels and signaling proteins. It is shown that N-methyl-D-aspartate (NMDA) receptor stimulation reversibly translocates green fluorescent protein-tagged CaMKII from an F-actin-bound to a PSD-bound state. The translocation time was controlled by the ratio of expressed beta-CaMKII to alpha-CaMKII isoforms. Although F-actin dissociation into the cytosol required autophosphorylation of or calcium-calmodulin binding to beta-CaMKII, PSD translocation required binding of calcium-calmodulin to either the alpha- or beta-CaMKII subunits. Autophosphorylation of CaMKII indirectly prolongs its PSD localization by increasing the calmodulin-binding affinity.
View details for Web of Science ID 000079509000059
View details for PubMedID 10102820
Characterization of a dextran-based bifunctional calcium indicator immobilized in cells by the enzymatic addition of isoprenoid lipids
1999; 25 (1): 1-7
Cellular processes can be controlled by cell-wide increases in the cytosolic Ca2+ concentration or, alternatively, by localized Ca2+ signals in micro- and nano-domains. The experimental characterization of such localized Ca2+ signals would be facilitated using an immobilized Ca2+ indicator, which could prevent the accelerated spatial spreading of Ca2+ ions that is mediated by binding to diffusible indicators. Here we characterize a dextran-based Ca2+ indicator (CAAX-green) that becomes immobilized in the cytosol by an enzyme-mediated addition of a geranylgeranyl lipid group. CAAX-green consists of a dextran backbone with an attached Ca(2+)-green as well as an 11 residue peptide ending in a C-terminal CAAX-motif. Once introduced into cells by microporation, geranylgeranyl lipid groups are attached to the CAAX peptides by cytosolic enzymes. Measurements in tumor mastcells, myocytes and fibroblasts showed that the indicator becomes membrane attached between 30 min and 1 h following incorporation into the cytoplasm. A time-dependent 10-fold reduction of the diffusion coefficient and a parallel increase in the cytosolic retention after permeabilization indicates that at least 90% of cellular CAAX-green is immobilized. The KD of the indicator in permeabilized cells is 0.65 microM. Overall, these properties make CAAX-green well suited for the investigation of localized Ca2+ signals in a variety of cell types.
View details for Web of Science ID 000078609900001
View details for PubMedID 10191955
Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals
1998; 95 (3): 307-318
The specificity of many signal transduction pathways relies on the temporal coordination of different second messenger signals. Here we found a molecular mechanism which guarantees that conventional protein kinase C (PKC) isoforms are sequentially activated by calcium and diacylglycerol signals. Receptor stimuli that triggered repetitive calcium spikes induced a parallel repetitive translocation of GFP-tagged PKCgamma to the plasma membrane. While calcium acted rapidly, diacylglycerol binding to PKCgamma was initially prevented by a pseudosubstrate clamp, which kept the diacylglycerol-binding site inaccessible and delayed calcium- and diacylglycerol-mediated kinase activation. After termination of calcium signals, bound diacylglycerol prolonged kinase activity. The properties of this molecular decoding machine make PKCgamma responsive to persistent diacylglycerol increases combined with high- but not low-frequency calcium spikes.
View details for Web of Science ID 000076789600005
View details for PubMedID 9814702
CaMKII beta functions as an F-actin targeting module that localizes CaMKII alpha/beta heterooligomers to dendritic spines
1998; 21 (3): 593-606
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine protein kinase that regulates long-term potentiation and other forms of neuronal plasticity. Functional differences between the neuronal CaMKIIalpha and CaMKIIbeta isoforms are not yet known. Here, we use green fluorescent protein-tagged (GFP-tagged) CaMKII isoforms and show that CaMKIIbeta is bound to F-actin in dendritic spines and cell cortex while CaMKIIalpha is largely a cytosolic enzyme. When expressed together, the two isoforms form large heterooligomers, and a small fraction of CaMKIIbeta is sufficient to dock the predominant CaMKIIalpha to the actin cytoskeleton. Thus, CaMKIIbeta functions as a targeting module that localizes a much larger number of CaMKIIalpha isozymes to synaptic and cytoskeletal sites of action.
View details for Web of Science ID 000076196400015
View details for PubMedID 9768845
Tyrosine 1101 of Tie2 is the major site of association of p85 and is required for activation of phosphatidylinositol 3-kinase and Akt
MOLECULAR AND CELLULAR BIOLOGY
1998; 18 (7): 4131-4140
Tie2 is an endothelium-specific receptor tyrosine kinase that is required for both normal embryonic vascular development and tumor angiogenesis and is thought to play a role in vascular maintenance. However, the signaling pathways responsible for the function of Tie2 remain unknown. In this report, we demonstrate that the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase) associates with Tie2 and that this association confers functional lipid kinase activity. Mutation of tyrosine 1101 of Tie2 abrogated p85 association both in vitro and in vivo in yeast. Tie2 was found to activate PI3-kinase in vivo as demonstrated by direct measurement of increases in cellular phosphatidylinositol 3-phosphate and phosphatidylinositol 3, 4-bisphosphate, by plasma membrane translocation of a green fluorescent protein-Akt pleckstrin homology domain fusion protein, and by downstream activation of the Akt kinase. Activation of PI3-kinase was abrogated in these assays by mutation of Y1101 to phenylalanine, consistent with a requirement for this residue for p85 association with Tie2. These results suggest that activation of PI3-kinase and Akt may in part account for Tie2's role in both embryonic vascular development and pathologic angiogenesis, and they are consistent with a role for Tie2 in endothelial cell survival.
View details for Web of Science ID 000074380100051
View details for PubMedID 9632797
Visualization of dynamic trafficking of a protein kinase C beta II green fluorescent protein conjugate reveals differences in G protein-coupled receptor activation and desensitization
JOURNAL OF BIOLOGICAL CHEMISTRY
1998; 273 (17): 10755-10762
Protein kinase C (PKC) links various extracellular signals to intracellular responses and is activated by diverse intracellular factors including diacylglycerol, Ca2+, and arachidonic acid. In this study, using a fully functional green fluorescent protein conjugated PKCbetaII (GFP-PKCbetaII), we demonstrate a novel approach to study the dynamic redistribution of PKC in live cells in response to G protein-coupled receptor activation. Agonist-induced PKC translocation was rapid, transient, and selectively mediated by the activation of Gqalpha- but not Gsalpha- or Gialpha-coupled receptors. Interestingly, although the stimuli were continuously present, only one brief peak of PKC membrane translocation was observed, consistent with rapid desensitization of the signaling pathway. Moreover, when GFP-PKCbetaII was used to examine cross-talk between two Gqalpha-coupled receptors, angiotensin II type 1A receptor (AT1AR) and endothelin A receptor (ETAR), activation of ETARs resulted in a subsequent loss of AT1AR responsiveness, whereas stimulation of AT1ARs did not cause desensitization of the ETAR signaling. The development of GFP-PKCbetaII has allowed not only the real time visualization of the dynamic PKC trafficking in live cells in response to physiological stimuli but has also provided a direct and sensitive means in the assessment of activation and desensitization of receptors implicated in the phospholipase C signaling pathway.
View details for Web of Science ID 000073224200090
View details for PubMedID 9553141
Receptor-induced transient reduction in plasma membrane PtdIns(4,5)P-2 concentration monitored in living cells
1998; 8 (6): 343-346
Although phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is a well-characterized precursor for the second messengers inositol 1,4,5-trisphosphate, diacylglycerol  and phosphatidylinositol 3,4,5-trisphosphate , it also interacts with the actin-binding proteins profilin and gelsolin , as well as with many signaling molecules that contain pleckstrin homology (PH) domains . It is conceivable that stimuli received by receptors in the plasma membrane could be sufficiently strong to decrease the PtdIns(4,5)P2 concentration; this decrease could alter the structure of the cortical cytoskeleton and modulate the activity of signaling molecules that have PH domains. Here, we tested this hypothesis by using an in vivo fluorescent indicator for PtdIns(4,5)P2, by tagging the PH domain of phospholipase C delta 1 (PLC-delta 1) with the green fluorescent protein (GFP-PH). When expressed in cells, GFP-PH was found to be enriched at the plasma membrane. Binding studies in vitro and mutant analysis suggested that GFP-PH bound PtdIns(4,5)P2 selectively over other phosphatidylinositol lipids. Strikingly, receptor stimulation induced a transient dissociation of GFP-PH from the plasma membrane, suggesting that the concentration of PtdIns(4,5)P2 in the plasma membrane was effectively lowered. This transient dissociation was blocked by the PLC inhibitor U73122 but was not affected by the phosphoinositide (PI) 3-kinase inhibitor wortmannin, suggesting that it is mostly mediated by PLC and not by PI 3-kinase activation. Overall, our studies show that PtdIns(4,5)P2 can have second messenger functions of its own, by mediating a transient dissociation of proteins anchored in the plasma membrane.
View details for Web of Science ID 000072600000021
View details for PubMedID 9512420
Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells
JOURNAL OF CELL BIOLOGY
1998; 140 (3): 485-498
Cysteine-rich domains (Cys-domains) are approximately 50-amino acid-long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-gamma (Cys1-GFP). Strikingly, stimulation of G-protein or tyrosine kinase-coupled receptors induced a transient translocation of cytosolic Cys1-GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1-GFP in the membrane, whereas DiC8 left Cys1-GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1-GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-gamma also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2-GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester-mediated translocation of proteins to selective lipid membranes.
View details for Web of Science ID 000072026300003
View details for PubMedID 9456311
In vivo and in vitro characterization of the sequence requirement for oligomer formation of Ca2+/calmodulin-dependent protein kinase II alpha
JOURNAL OF NEUROCHEMISTRY
1998; 70 (1): 96-104
Ca2+/calmodulin-dependent protein kinase II (CaMK II) is a multifunctional serine/threonine protein kinase that regulates ion channels, metabolic enzymes, cytoskeletal proteins, and possibly transcription factors. CaMK II holoenzymes have been shown to be large oligomers built from eight or more subunits. The oligomeric structure of CaMK II is required for a critical trans-autophosphorylation step by which each subunit traps bound calmodulin and renders the enzyme partially active in the absence of Ca2+/calmodulin. Here we define a minimal C-terminal domain of alphaCaMK II that is necessary and sufficient for oligomerization. The oligomeric structure of alphaCaMK II deletion mutants was investigated in vitro by gel filtration chromatography and in living cells by measuring nuclear exclusion and diffusion coefficients of green fluorescent protein-tagged mutants. These studies showed that a C-terminal region of alphaCaMK II of 135 amino acids is sufficient for oligomer formation. Oligomer formation was abolished by further C-terminal and N-terminal deletions, indicating that the same region is not only sufficient but also necessary for oligomerization. Thus, an oligomerization domain of only 15 kDa is sufficient to build the circular structure of CaMK II holoenzyme.
View details for Web of Science ID 000071146400011
View details for PubMedID 9422351
Compartmentalized IgE receptor-mediated signal transduction in living cells
JOURNAL OF CELL BIOLOGY
1997; 139 (6): 1447-1454
Several receptor-mediated signal transduction pathways, including EGF and IgE receptor pathways, have been proposed to be spatially restricted to plasma membrane microdomains. However, the experimental evidence for signaling events in these microdomains is largely based on biochemical fractionation and immunocytochemical studies and only little is known about their spatial dynamics in living cells. Here we constructed green fluorescent protein-tagged SH2 domains to investigate where and when IgE receptor (FcepsilonRI)-mediated tyrosine phosphorylation occurs in living tumor mast cells. Strikingly, within minutes after antigen addition, tandem SH2 domains from Syk or PLC-gamma1 translocated from a uniform cytosolic distribution to punctuate plasma membrane microdomains. Colocalization experiments showed that the microdomains where tyrosine phosphorylation occurred were indistinguishable from those stained by cholera toxin B, a marker for glycosphingolipids. Competitive binding studies with coelectroporated unlabeled Syk, PLC-gamma1, and other SH2 domains selectively suppressed the induction of IgE receptor-mediated calcium signals as well as the binding of the fluorescent SH2 domains. This supports the hypothesis that PLC-gamma1 and Syk SH2 domains selectively bind to Syk and IgE receptors, respectively. Unlike the predicted prelocalization of EGF receptors to caveolae microdomains, fluorescently labeled IgE receptors were found to be uniformly distributed in the plasma membrane of unstimulated cells and only transiently translocated to glycosphingolipid rich microdomains after antigen addition. Thus, these in vivo studies support a plasma membrane signaling mechanism by which IgE receptors transiently associate with microdomains and induce the spatially restricted activation of Syk and PLC-gamma1.
View details for Web of Science ID 000071147400008
View details for PubMedID 9396750
Prenylation-dependent association of Ki-Ras with microtubules - Evidence for a role in subcellular trafficking
JOURNAL OF BIOLOGICAL CHEMISTRY
1997; 272 (48): 30362-30370
We recently identified a prenyl peptide-binding protein in microsomal membranes from bovine brain (Thissen, J. A., and Casey, P. J. (1993) J. Biol. Chem. 268, 13780-13783). Through a variety of approaches, this binding protein has been identified as the cytoskeletal protein tubulin. Prenyl peptides bind to purified tubulin with a Kd of 40 nM and also bind to tubulin polymerized into microtubules. Microtubule affinity chromatography of extracts from cells in which the prenyl protein pool was metabolically labeled revealed that prenyl proteins bound to the immobilized microtubules; one, a 24-kDa protein, was tentatively identified as a GTP-binding protein. Of several prenylated GTP-binding proteins tested, including Ki-Ras4B, Ha-Ras, RhoB, RhoA, and Rap1B, only Ki-Ras was found to bind significantly to microtubules, and this was in a prenylation-dependent fashion. A potential significance of the interaction of Ki-Ras4B with microtubules was indicated from analysis of the localization of newly synthesized Ki-Ras4B and Ha-Ras, each tagged with green fluorescence protein (GFP). Treatment of NIH-3T3 cells expressing GFP-Ki-Ras with Taxol (paclitaxel) resulted in accumulation of the expressed protein in intracellular locations, whereas in control cells the protein was correctly targeted to the plasma membrane. Importantly, such treatment with paclitaxel did not affect the cellular localization of expressed GFP-Ha-Ras. These results indicate that an intact microtubule network may be directly involved in Ki-Ras processing and/or targeting and provide direct evidence for a physiological distinction between Ki-Ras and Ha-Ras in cells. Additionally, the finding that paclitaxel treatment of cells disrupts Ki-Ras trafficking suggests an additional mechanism for the anti-proliferative effects of this drug.
View details for Web of Science ID A1997YH61300058
View details for PubMedID 9374526
Electroporation-induced formation of individual calcium entry sites in the cell body and processes of adherent cells
1997; 73 (4): 1785-1796
Electroporation is a widely used method for introducing macromolecules into cells. We developed an electroporation device that requires only 1 microl of sample to load adherent cells in a 10-mm2 surface area while retaining greater than 90% cell survivability. To better understand this device, field-induced permeabilization of adherent rat basophilic leukemia and neocortical neuroblastoma cells was investigated by using fluorescent calcium and voltage indicators. Rectangular field pulses led to the formation of only a few calcium entry sites, preferentially in the hyperpolarized parts of the cell body and processes. Individual entry sites were formed at the same locations when field pulses were repeated. Before calcium entry, a partial breakdown of the membrane potential was observed in both polar regions. Based on our results, a model is proposed for the formation and closure of macromolecule entry sites in adherent cells. First, the rapid formation of a large number of small pores leads to a partial membrane potential breakdown in both polar regions of the cell. Second, over tens of milliseconds, a few entry sites for macromolecules are formed, preferentially in the hyperpolarized part of cell body and processes, at locations defined by the local membrane structure. These entry sites reseal on a time scale of 50 ms to several seconds, with residual small pores remaining open for several minutes.
View details for Web of Science ID A1997XY95500008
View details for PubMedID 9336174
Inhibition of Lyn function in mast cell activation by SH3 domain binding peptides
1997; 36 (31): 9388-9394
While Lyn tyrosine kinase has been shown to be necessary for IgE-receptor (FcepsilonRI)-mediated mast cell activation, the mechanism of Lyn activation is not yet understood. Using a micro-electroporation technique to quantitatively introduce peptides into the cytosol of tumor mast cells, we show that proline-rich peptides that preferentially bind Src family SH3 domains block receptor-induced repetitive calcium spikes in a concentration dependent manner. The Src family member Lyn was the likely target, since a series of phage displaying derived peptides with increased Lyn SH3 domain binding specificity inhibited FcepsilonRI-mediated calcium signaling at concentrations consistent with binding to Lyn rather than other Src-type kinases. Furthermore, SH3 binding peptides prevented the plasma membrane translocation of a fluorescently labeled Syk tandem SH2 domain, which binds to phosphorylated FcepsilonRI, suggesting that the peptides specifically block the Lyn-mediated step by which FcepsilonRI cross-linking leads to receptor phosphorylation. Our study suggests that the binding of proline-rich peptides, or corresponding cellular interaction partners, to Lyn SH3 domain suppresses the Lyn-mediated phosphorylatation of FcepsilonRI and calcium signaling.
View details for Web of Science ID A1997XP63100014
View details for PubMedID 9235982
Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores
1997; 89 (6): 963-971
The spatial organization of endoplasmic reticulum (ER) and nuclear envelope (NE) calcium stores is important for the regulation of localized calcium signals and sustained calcium gradients. Here, we have used a lumenal GFP fusion protein and shown that, in resting cells, large molecules can rapidly diffuse across the cell within the lumenal storage space defined by the ER and NE membranes. Increases in cytosolic calcium concentration reversibly fragmented ER tubules and prevented lumenal diffusion. However, the integrity of the NE was maintained, and a significant fraction of NE lumenal protein accumulated in an NE-associated vesicle. These dynamic properties of ER-NE calcium stores provide insights into the spatiotemporal control of calcium signaling.
View details for Web of Science ID A1997XE35700016
View details for PubMedID 9200614
Elementary calcium-release units induced by inositol trisphosphate
1997; 276 (5319): 1690-1693
The extent to which inositol 1,4,5-trisphosphate (InsP3)-induced calcium signals are localized is a critical parameter for understanding the mechanism of effector activation. The spatial characteristics of InsP3-mediated calcium signals were determined by targeting a dextran-based calcium indicator to intracellular membranes through the in situ addition of a geranylgeranyl lipid group. Elementary calcium-release events observed with this indicator typically lasted less than 33 milliseconds, had diameters less than 2 micrometers, and were uncoupled from each other by the calcium buffer EGTA. Cellwide calcium transients are likely to result from synchronized triggering of such local release events, suggesting that calcium-dependent effector proteins could be selectively activated by localization near sites of local calcium release.
View details for Web of Science ID A1997XD94700040
View details for PubMedID 9180077
Control of action potential-induced Ca2+ signaling in the soma of hippocampal neurons by Ca2+ release from intracellular stores
JOURNAL OF NEUROSCIENCE
1997; 17 (11): 4129-4135
Stimulus-induced increases in neuronal Ca2+ concentration are important signaling events for transcriptional regulation and neuronal plasticity. Electrical inputs are thought to mediate Ca2+ responses in the soma by triggering action potentials, which in turn open voltage-gated Ca2+ channels in the somatic plasma membrane. It is not yet known to what extent internal Ca2+ amplification contributes to the somatic Ca2+ responses. Here we used fluorescent Ca2+ measurements in cultured hippocampal neurons and report that the amplitude of the somatic Ca2+ increase triggered by field stimulation is independent of the extracellular Ca2+ concentration as long as the concentration is greater than 50 microM. Furthermore, significantly more La3+ has to be added extracellularly for blocking Ca2+ responses, as predicted from the reported La3+ dependence of voltage-gated Ca2+ channels. These measurements suggest that field stimulation-induced somatic Ca2+ responses in hippocampal neurons are largely attributable to Ca2+ release from intracellular stores. Only a small number of Ca2+ ions have to enter across the plasma membrane for this intracellular Ca2+ amplification process to occur. Rapid fluorescence-imaging measurements showed that the internal Ca2+ amplification occurs over 10-15 msec and linearly increases intracellular Ca2+ concentrations for up to 40 action potentials. At a fixed number of field pulses, frequencies of 40 Hz were optimal for somatic Ca2+ increases. Our studies suggest that the opening of intracellular Ca2+ release channels plays a crucial part in shaping the action potential-induced neuronal Ca2+ response.
View details for Web of Science ID A1997XA05600017
View details for PubMedID 9151730
Internal trafficking and surface mobility of a functionally intact beta(2)-adrenergic receptor-green fluorescent protein conjugate
1997; 51 (2): 177-184
The beta2-adrenergic receptor (beta2AR) is prototypic of the large family of G protein-coupled receptors (GPCRs) whose desensitization and resensitization are regulated by intracellular kinases, arrestin proteins, phosphatases, and ill-defined components of the cellular endocytic machinery. The study of beta2AR signal transduction and behavior in living cells is technically difficult because of the relatively low cellular expression of the receptor and a lack of useful biological reagents. Availability of a functional beta2AR tagged with the highly sensitive Green Fluorescent Protein (GFP) could allow measurements of the various properties of the beta2AR. We demonstrate that a fully functional beta2AR/GFP can be engineered. In mammalian cells, beta2AR/S65T/GFP demonstrates strong, diffuse plasma membrane fluorescence when observed with 480 nm excitation. The fluorescent receptor binds agonist and antagonist, stimulates adenylyl cyclase, undergoes phosphorylation, and is internalized in a manner indistinguishable from wild-type receptor. We then show that its internal trafficking and surface mobility can be determined by measuring only the endogenous fluorescence of the conjugate. beta2AR/S65T/GFP was found to be localized on endosomal membranes in living cells within minutes of agonist treatment, and within 15 min it is observed in more complicated structures formed from fusion of multiple endosomes. Finally, its free diffusion (diffusion coefficient, 4.0-12 x 10(-9) cm2/sec) was assessed on living cells using photobleaching recovery measurements. This approach and the fidelity of the biochemical properties of the beta2AR/S65T/GFP demonstrate that real-time optical measurements of beta2AR (as well as other GPCR) interactions and dynamics on living cells are feasible.
View details for Web of Science ID A1997WJ44600002
View details for PubMedID 9203621
Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement
1996; 14 (10): 1252-1256
We describe a method of monitoring the spatial dynamics of proteins in intact cells by locally enhancing the blue excited fluorescence of green fluorescent protein (GFP) using a spatially focused ultraviolet-laser pulse. GFP fusion proteins were efficiently expressed by micro-electroporation of in vitro synthesized mRNA into adherent mammalian cells. We found that the diffusion coefficient of cycle 3 mutant GFP was 43 microns2/sec, compared to 4 microns2/sec for wild-type GFP, suggesting that cycle 3 GFP diffuses freely in mammalian cells and is ideally suited as a fusion tag. The local fluorescence enhancement method was used to study the membrane dissociation rate of GFP-tagged K-ras, a small GTP binding protein that localizes to plasma membranes by a farnesyl lipid group and a polybasic region. Our data suggest that K-ras exists in a dynamic equilibrium and rapidly switches between a plasma membrane bound form and a cytosolic form with a plasma membrane dissociation time constant of 1.5 sec.
View details for Web of Science ID A1996VK61800022
View details for PubMedID 9631088
Reversible desensitization of inositol trisphosphate-induced calcium release provides a mechanism for repetitive calcium spikes
JOURNAL OF BIOLOGICAL CHEMISTRY
1996; 271 (29): 17253-17260
Repetitive transient increases in cytosolic calcium concentration (calcium spikes or calcium oscillations) are a common mode of signal transduction in receptor-mediated cell activation. Repetitive calcium spikes are initiated by phospholipase C-mediated production of inositol 1,4,5-trisphosphate (InsP3) and are thought to be generated by a positive feedback mechanism in which calcium potentiates its own release, a negative feedback mechanism by which calcium release is terminated, and a slow recovery process that defines the time interval between calcium spikes. The molecular mechanisms that terminate each calcium spike and define the spike frequency are not yet known. Here we show, in intact rat basophilic leukemia cells, that calcium responses induced by InsP3 are diminished for a period of 30-60 s following an InsP3-induced calcium spike. The sensitivity of calcium release for InsP3 was probed by UV laser-mediated photorelease of InsP3, and calcium responses were monitored by fluorescence calcium imaging. A maximal loss in sensitivity (desensitization) was observed for InsP3 increases that resulted in a near maximal calcium spike and was expressed as an 80-100% reduction in the calcium response to an equal amount of InsP3, released 10 s after the first UV pulse. When the amount of released InsP3 in the second pulse was increased 2-3-fold, desensitization was overcome and a second calcium response of equal amplitude to the first was produced. A power dependence of 3.2 was measured between the amount of released InsP3 and the amplitude of the triggered calcium response, explaining how a small decrease in InsP3 sensitivity can lead to a nearly complete reduction in the calcium response. Desensitization was abolished by the addition of the calcium buffers BAPTA and EGTA and could be induced by microinjection of calcium, suggesting that it is a calcium-dependent process. Half-maximal desensitization was observed at a free calcium concentration of 290 nM and increased with a power of 3.7 with peak calcium concentration. These studies suggest that reversible desensitization of InsP3-induced calcium release serves as a "saw-tooth" parameter that controls the termination of each spike and the frequency of calcium spikes.
View details for Web of Science ID A1996UX94300044
View details for PubMedID 8663416
LUMINAL CALCIUM REGULATES THE INOSITOL TRISPHOSPHATE RECEPTOR OF RAT BASOPHILIC LEUKEMIA-CELLS AT A CYTOSOLIC SITE
1995; 34 (39): 12738-12746
Hormones, growth factors, and other stimuli can generate Ca2+ spikes and waves by activation of the phosphoinositide (PI) pathway. The sources of these Ca2+ signals are inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ stores. Here we use a rapid perfusion apparatus to measure the release of 45Ca2+ from permeabilized rat basophilic leukemia (RBL) cells to investigate the regulation of IP3-mediated Ca2+ release by cytosolic and luminal Ca2+. At 200 nM IP3, Ca2+ release was potentiated by an increase in the cytosolic Ca2+ concentration. This potentiation by Ca2+ was nearly absent at 500 nM IP3. Previous studies in smooth muscle cells and neurons showed an inhibition of Ca2+ release above 300 nM Ca2+. In contrast, no such inhibition was observed in RBL cells. When assayed at low cytosolic Ca2+ concentrations, IP3-mediated release was steeply dependent upon luminal Ca2+ concentration. At high luminal Ca2+ concentration, 1 microM IP3 released most of the stored Ca2+ even in the complete absence of cytosolic Ca2+. However, at high cytosolic Ca2+ concentrations (890 nM), IP3-mediated release was no longer steeply dependent upon the luminal Ca2+ concentration. Furthermore, high concentrations of BAPTA inhibited IP3-mediated release in the absence of cytosolic Ca2+. This suggests that a rapid and local luminal Ca2+ feedback is generated by luminal Ca2+ ions binding to cytosolic sites of the same channel or closely associated channels. This "luminal Ca2+ feedback" can be initiated by an increase in the concentration either of IP3, of cytosolic Ca2+, or of luminal Ca2+. It is likely that "luminal Ca2+ feedback" is exploited by cells in both the initiation and termination of Ca2+ spikes.
View details for Web of Science ID A1995RY46000033
View details for PubMedID 7548027
LOCALIZED CALCIUM SIGNALS IN EARLY ZEBRAFISH DEVELOPMENT
1995; 170 (1): 50-61
Activation of the phosphoinositide (PI) pathway has been shown to be involved in the compaction of blastomeres in mouse embryos and in embryonic axis formation in Xenopus and in zebrafish embryos. Here we investigate Ca2+ signals in individual blastomeres of zebrafish embryos with the goal to better understand the role of PI and Ca2+ signaling for early vertebrate embryogenesis. Initial studies showed that the inositol 1,4,5-trisphosphate (IP3) concentration increases after the 32-cell stage of development, suggesting that IP3-mediated Ca2+ signals may be present during the blastula stage. Ca2+ signals were measured by identifying individual cells using confocal imaging of a nuclear localized Ca2+ indicator. Using this in situ indicator, changes in Ca2+ concentration were measured over several hours in each cell of a series of sections through the developing embryo. Transient increases in Ca2+ concentration that lasted 20-50 sec (Ca2+ spikes) were first triggered during the 32- to 128-cell stage in cells of the outer embryonic cell layer. These cells develop epithelial characteristics and specialize into the enveloping layer (EVL). No Ca2+ activity was observed during the earlier cleavage cycles or in deep blastomeres. Ca2+ spikes remained restricted to the EVL until the end of the blastula stage. Ca2+ spikes in neighboring EVL cells often occurred in the same short time interval, indicating that small groups of EVL cells can synchronize their activity. When averaged over several cell cycles, Ca2+ activity showed an even distribution in the EVL and did not indicate future polarities.
View details for Web of Science ID A1995RH86300005
View details for PubMedID 7541377
REGULATION OF NUCLEAR CALCIUM-CONCENTRATION
JOHN WILEY & SONS LTD. 1995: 252-262
Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.
View details for Web of Science ID A1995BD06E00016
View details for PubMedID 7587621
SOURCE OF NUCLEAR CALCIUM SIGNALS
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1994; 91 (26): 12458-12462
Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.
View details for Web of Science ID A1994PY29400022
View details for PubMedID 7809059
DUAL ROLE OF CALMODULIN IN AUTOPHOSPHORYLATION OF MULTIFUNCTIONAL CAM KINASE MAY UNDERLIE DECODING OF CALCIUM SIGNALS
1994; 12 (5): 943-956
Autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase makes it Ca2+ independent by trapping bound calmodulin and by enabling the kinase to remain partially active even after calmodulin dissociates. We show that autophosphorylation is an intersubunit reaction between neighbors in the multimeric kinase which requires two molecules of calmodulin. Ca2+/calmodulin acts not only to activate the "kinase" subunit but also to present effectively the "substrate" subunit for autophosphorylation. Conversion of the kinase to the potentiated or trapped state is a cooperative process that is inefficient at low occupancy of calmodulin. Simulations show that repetitive Ca2+ pulses at limiting calmodulin lead to the recruitment of calmodulin to the holoenzyme, which further stimulates autophosphorylation and trapping. This cooperative, positive feedback loop will potentiate the response of the kinase to sequential Ca2+ transients and establish a threshold frequency at which the enzyme becomes highly active.
View details for Web of Science ID A1994NM83100002
View details for PubMedID 8185953
LOCALIZED CALCIUM SPIKES AND PROPAGATING CALCIUM WAVES
1993; 14 (10): 691-697
Ca2+ signals control or modulate diverse cellular processes such as cell growth, muscle contraction, hormone secretion, and neuronal plasticity. Elevations in intracellular Ca2+ concentrations can be highly localized to micron and submicron domains or propagated as intra- and intercellular waves over distances as large as 1 mm. Localized, subcellular Ca2+ spikes are thought to selectively activate effector systems such as Ca2+ activated chloride currents in pancreatic acinar cells, neurotransmitter release in synaptic nerve terminals, and morphological changes in neural growth cones. In contrast, long-ranged Ca2+ waves synchronize the activities of different cytoplasmic regions of a single cell, such as cortical granule exocytosis after egg fertilization or coordinate the activities of many cells, such as ciliary beating in pulmonary epithelium. The purpose of this review is to delineate the role of Ca2+ in the generation of localized, subcellular Ca2+ spikes and long-ranged intracellular and intercellular Ca2+ waves.
View details for Web of Science ID A1993MH25500003
View details for PubMedID 8131187
USE OF INTRACELLULAR CA2+ STORES FROM RAT BASOPHILIC LEUKEMIA-CELLS TO STUDY THE MOLECULAR MECHANISM LEADING TO QUANTAL CA2+ RELEASE BY INOSITOL 1,4,5-TRISPHOSPHATE
1993; 32 (5): 1270-1277
Quantal Ca2+ release is a novel motif for the mediation of signal transduction in which the amplitude of a biological response following multiple stepwise increases in agonist concentration is retained. The release of Ca2+ from permeabilized cells in response to the second messenger inositol 1,4,5-trisphosphate (InsP3) proceeds in this fashion. The mechanisms leading to quantal Ca2+ release are unknown. InsP3 releases 50-90% of the Ca2+ sequestered within the intracellular stores of mammalian cells permeabilized with saponin. However, preparation of microsomes results in the loss of this sensitivity. In this report, functionally intact intracellular Ca2+ stores were isolated from rat basophilic leukemia (RBL) cells by osmotic lysis followed by differential and sucrose density gradient centrifugation. From this preparation, 64% of the stored Ca2+ is released by InsP3. We demonstrate that quantal Ca2+ release is retained by isolated Ca2+ stores and is identical to that observed in permeabilized cells. Addition of a subsaturating (28 nM) concentration of InsP3 to permeabilized cells at 37 degrees C results in the release of only a small fraction of the sequestered Ca2+. When the cells are cooled to 11 degrees C, the remaining Ca2+ is rapidly released. Hence, the mechanism leading to the quantal nature of Ca2+ release is reversible and is thus not likely to be the result of a covalent modification of the channel protein or of the Ca2+ store.(ABSTRACT TRUNCATED AT 250 WORDS)
View details for Web of Science ID A1993KL45800011
View details for PubMedID 8448137
RANGE OF MESSENGER ACTION OF CALCIUM-ION AND INOSITOL 1,4,5-TRISPHOSPHATE
1992; 258 (5089): 1812-1815
The range of messenger action of a point source of Ca2+ or inositol 1,4,5-trisphosphate (IP3) was determined from measurements of their diffusion coefficients in a cytosolic extract from Xenopus laevis oocytes. The diffusion coefficient (D) of [3H]IP3 injected into an extract was 283 microns 2/s. D for Ca2+ increased from 13 to 65 microns 2/s when the free calcium concentration was raised from about 90 nM to 1 microM. The slow diffusion of Ca2+ in the physiologic concentration range results from its binding to slowly mobile or immobile buffers. The calculated effective ranges of free Ca2+ before it is buffered, buffered Ca2+, and IP3 determined from their diffusion coefficients and lifetimes were 0.1 micron, 5 microns, and 24 microns, respectively. Thus, for a transient point source of messenger in cells smaller than 20 microns, IP3 is a global messenger, whereas Ca2+ acts in restricted domains.
View details for Web of Science ID A1992KB96400044
View details for PubMedID 1465619
DECODING CALCIUM SIGNALS BY MULTIFUNCTIONAL CAM KINASE
1992; 13 (6-7): 401-411
Multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) is one of the three major protein kinases coordinating cellular responses to hormones and neurotransmitters. It mediates the action of Ca2+ on neurotransmitter synthesis and release, on carbohydrate metabolism and on the cytoskeleton. CaM kinase has structural/functional properties that facilitate its response to distinctive attributes of Ca2+ signals which often involve transient increases that span a narrow concentration range and increases that are pulsatile rather than persistent. The kinase responds to the narrow working range of Ca2+ signals by the use of calmodulin as the Ca2+ sensor. It is activated by the binding of calmodulin to an autoinhibitory domain that keeps the kinase inactive in the basal state. The transient nature of the signal is accommodated by autophosphorylation of this autoinhibitory domain which allows the kinase to remain partially active after calmodulin dissociates and thereby switches it to a Ca(2+)-independent species. The pulsatile nature of Ca2+ signals may also be decoded by CaM kinase. Autophosphorylation traps calmodulin on autophosphorylated subunits by greatly reducing its off-rate. At high frequency of stimulation, calmodulin would remain trapped during the brief interval between Ca2+ oscillations and each successive rise in Ca2+ would recruit more calmodulin. This may enable a stimulus frequency dependent activation of CaM kinase.
View details for Web of Science ID A1992JF05300006
View details for PubMedID 1324121
CALMODULIN TRAPPING BY CALCIUM-CALMODULIN DEPENDENT PROTEIN-KINASE
1992; 256 (5060): 1199-1202
Multifunctional calcium-calmodulin-dependent protein kinase (CaM kinase) transduces transient elevations in intracellular calcium into changes in the phosphorylation state and activity of target proteins. By fluorescence emission anisotropy, the affinity of CaM kinase for dansylated calmodulin was measured and found to increase 1000 times after autophosphorylation of the threonine at position 286 of the protein. Autophosphorylation markedly slowed the release of bound calcium-calmodulin; the release time increased from less than a second to several hundred seconds. In essence, calmodulin is trapped by autophosphorylation. The shift in affinity does not occur in a site-directed mutant in which threonine at position 286 has been replaced by a non-phosphorylatable amino acid. These experiments demonstrate the existence of a new state in which calmodulin is bound to CaM kinase even though the concentration of calcium is basal. Calmodulin trapping provides for molecular potentiation of calcium transients and may enable detection of their frequency.
View details for Web of Science ID A1992HV19200035
View details for PubMedID 1317063
ASSOCIATION OF THE BETA ISOFORM OF PROTEIN-KINASE-C WITH VIMENTIN FILAMENTS
CELL MOTILITY AND THE CYTOSKELETON
1992; 22 (4): 250-256
Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform.
View details for Web of Science ID A1992JF18900004
View details for PubMedID 1516148
- CELL SIGNALING BY 2ND MESSENGER WAVES CELL 1991; 64 (4): 675-678
- CALCIUM SPIKING ANNUAL REVIEW OF BIOPHYSICS AND BIOPHYSICAL CHEMISTRY 1991; 20: 153-174
TRANSIENT CALCIUM RELEASE INDUCED BY SUCCESSIVE INCREMENTS OF INOSITOL 1,4,5-TRISPHOSPHATE
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1990; 87 (10): 3841-3845
Many hormonal, neurotransmitter, and sensory stimuli trigger the formation of inositol 1,4,5-trisphosphate, which in turn releases calcium from intracellular stores. We report here that inositol 1,4,5-trisphosphate-induced calcium release from saponin-permeabilized rat basophilic leukemia cells at 37 degrees C is markedly biphasic, in contrast with nearly monophasic release kinetics at 11 degrees C. Hepatoma, PC-12 neuronal cells, and several other cell types exhibit similar biphasic release at 37 degrees C. The biphasic kinetics are not due to degradation of inositol 1,4,5-trisphosphate or to increased Ca2(+)-ATPase pump activity. Biphasic calcium release was also seen when ATP was quenched to less than 0.4 microM by adding hexokinase and glucose, suggesting that phosphorylation is not involved. External calcium (100 nM-600 nM) range had little influence on the biphasic kinetics. Rapid-mixing experiments revealed that rapid efflux of calcium is followed in approximately 0.5 s by a 30-fold slower efflux. Most striking, successive additions of the same amount of inositol 1,4,5-trisphosphate induced short bursts of calcium release of similar size. This retention of responsiveness, which we term increment detection, may be a distinct mode of signal transduction. Like inactivation and adaptation, increment detection gives rise to transient responses to sustained stimuli. Systems exhibiting inactivation, adaptation, and increment detection differ in their responsiveness (none, partial, and full, respectively) to stepwise increases in stimulus intensity. Increment detection could be advantageous in generating receptor-triggered calcium oscillations.
View details for Web of Science ID A1990DD87300043
View details for PubMedID 2339124
KINETICS OF CALCIUM-CHANNEL OPENING BY INOSITOL 1,4,5-TRISPHOSPHATE
1990; 29 (1): 32-37
The subsecond mobilization of intracellular Ca2+ by IP3 was measured with rapid mixing techniques to determine how cells achieve rapid rises in cytosolic [Ca2+] during receptor-triggered calcium spiking. In permeabilized rat basophilic leukemia cells at 11 degrees C, more than 80% of the 0.7 fmol of Ca2+/cell sequestered by the ATP-driven pump could be released by IP3. Half of the stored Ca2+ was released within 200 ms after addition of saturating (1 microM) IP3. The flux rate was half-maximal at 120 nM IP3. Ca2+ release from fully loaded stores was highly cooperative; the Hill coefficient over the 2-40 nM range was greater than 3. The delay time of channel opening was inversely proportional to [IP3], increasing from 150 ms at 100 nM IP3 to 1 s at 15 nM, indicating that the rate-limiting step in channel opening is IP3 binding. Multiple binding steps are required to account for the observed delay and nonexponential character of channel opening. A simple model is proposed in which the binding of four IP3 molecules to identical and independent sites leads to channel opening. The model agrees well with the data for KD = 18 nM, kon = 1.2 X 10(8) M-1 s-1, and koff = 2.2 s-1. The approximately 1-s exchange time of bound IP3 indicates that the channel gating sites are distinct from binding sites having approximately 100-s exchange times that were previously found with radiolabeled IP3. The approximately 1-1s response time of [Ca2+] to a rapid increase in IP3 level can account for observed rise times of calcium spikes.
View details for Web of Science ID A1990CH52900004
View details for PubMedID 1691015
PARTICLE COUNTING BY FLUORESCENCE CORRELATION SPECTROSCOPY - SIMULTANEOUS MEASUREMENT OF AGGREGATION AND DIFFUSION OF MOLECULES IN SOLUTIONS AND IN MEMBRANES
1988; 54 (6): 983-993
A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described. Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot. For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s. The time for a determination is approximately 1 min. Aggregation can be followed by changes of either M or D. This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers. Essential parameters for the development of the method are described. Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived. The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values.
View details for Web of Science ID A1988R365500002
View details for PubMedID 3233275
MOLECULAR-MODEL FOR RECEPTOR-STIMULATED CALCIUM SPIKING
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
1988; 85 (14): 5051-5055
Many cells exhibit periodic transient increases in cytosolic calcium levels rather than a sustained rise when stimulated by a hormone or growth factor. We propose here a molecular model that accounts for periodic calcium spiking induced by a constant stimulus. Four elements give rise to repetitive calcium transients: cooperativity and positive feedback between a pair of reciprocally coupled (crosscoupled) messengers, followed by deactivation and then by reactivation. The crosscoupled messengers in our model are inositol 1,4,5-trisphosphate (InsP3) and cytosolic calcium ions. The opening of calcium channels in the endoplasmic reticulum by the binding of multiple molecules of InsP3 provides the required cooperativity. The stimulation of receptor-activated phospholipase C by released calcium ions leads to positive feedback. InsP3 is destroyed by a phosphatase, and calcium ion is pumped back into the endoplasmic reticulum. These processes generate bistability: the cytosolic calcium concentration abruptly increases from a basal level to a stimulated level at a threshold degree of activation of phospholipase C. Spiking further requires slow deactivation and subsequent reactivation. In our model, mitochondrial sequestration of calcium ion prevents the cytosolic level from increasing above several micromolar and enables the system to return to the basal state. When the endoplasmic reticulum calcium store is refilled to a critical level by the Ca2+-ATPase pump, cooperative positive feedback between the InsP3-gated channel and phospholipase C begins again to give the next calcium spike. The time required for the calcium level in the endoplasmic reticulum to reach a threshold sets the interval between spikes. The amplitude, shape, and period of calcium spikes calculated for this model are like those observed experimentally.
View details for Web of Science ID A1988P362000022
View details for PubMedID 2455890
HIGHLY COOPERATIVE OPENING OF CALCIUM CHANNELS BY INOSITOL 1,4,5-TRISPHOSPHATE
1988; 240 (4852): 653-656
The kinetics of calcium release by inositol 1,4,5-trisphosphate (IP3) in permeabilized rat basophilic leukemia cells were studied to obtain insight into the molecular mechanism of action of this intracellular messenger of the phosphoinositide cascade. Calcium release from intracellular storage sites was monitored with fura-2, a fluorescent indicator. The dependence of the rate of calcium release on the concentration of added IP3 in the 4 to 40 nM range showed that channel opening requires the binding of at least three molecules of IP3. Channel opening occurred in the absence of added adenosine triphosphate, indicating that IP3 acts directly on the channel or on a protein that gates it. The channels were opened by IP3 in less than 4 seconds. The highly cooperative opening of calcium channels by nanomolar concentrations of IP3 enables cells to detect and amplify very small changes in the concentration of this messenger in response to hormonal, sensory, and growth control stimuli.
View details for Web of Science ID A1988N126500033
View details for PubMedID 2452482