Chair, Stanford Neuroscience Institute (SNI) Interdisciplinary Scholars Program (2014 - Present)
Associate Chair, Molecular and Cellular Physiology (2010 - 2013)
Chair, Molecular and Cellular Physiology (2017 - Present)
Deputy Director, Stanford Neuroscience Institute (2013 - 2017)
Honors & Awards
Excellence in Diversity and Inclusion, Stanford University School of Medicine (2015)
Excellence in Graduate Teaching, Stanford University School of Medicine (2014)
MIchael and Kate Barany Award for Young Investiators, Biophysical Society (2014)
Excellence in Graduate Teaching, Stanford University School of Medicine (2011)
Klingenstein Fellow in Neuroscience, The Klingenstein Fund (2005-2008)
McKnight Scholar Award, McKnight Endowment (2005-2008)
Prize in Neurobiology, Eppendorf & Science (2004)
Terman Fellow, Stanford University (2002-2005)
Alfred P. Sloan Fellow, Alfred P. Sloan Foundation (2002-2004)
Baxter Fellow, Donald B. and Delia E. Baxter Foundation (2002)
Terman Fellow, Stanford University (2002)
Boards, Advisory Committees, Professional Organizations
Academic Editor, eNeuro (2017 - Present)
Editorial Board Member, Section on Ion Channels & Transporters, Biophysical Journal (2013 - Present)
Editorial Advisory Board, Journal of General Physiology (2011 - Present)
Academic Editor, PloS Genetics (2009 - 2013)
Ph.D., The University of Chicago, Neurobiology (1995)
Sc.B., Brown University, Biochemistry (1986)
Current Research and Scholarly Interests
We study the molecular events that give rise to the sensation of touch and temperature using C. elegans nematodes as a model system. To do this, we use a combination of quantitative behavioral analysis, genetics, in vivo electrophysiology, and heterologous expression of ion channels. We also collaborate with Pruitt's group in Mechanical Engineering (http://microsystems.stanford.edu) to develop and fabricate novel devices for the study of sensory transduction.
BIOS 254 (Spr)
- Diversity and Inclusion in Science
BIOS 225 (Win)
- Promises and Pitfalls: A Critical Evaluation of Neuroscience Techniques
BIOS 255 (Win)
Independent Studies (9)
- Directed Reading in Biophysics
BIOPHYS 399 (Aut, Win, Spr, Sum)
- Directed Reading in Molecular and Cellular Physiology
MCP 299 (Aut, Win, Spr, Sum)
- Directed Reading in Neurosciences
NEPR 299 (Aut, Win, Spr, Sum)
- Graduate Research
BIOPHYS 300 (Aut, Win, Spr, Sum)
- Graduate Research
MCP 399 (Aut, Win, Spr, Sum)
- Graduate Research
NEPR 399 (Aut, Win, Spr, Sum)
- Medical Scholars Research
MCP 370 (Aut, Win, Spr, Sum)
- Out-of-Department Graduate Research
BIO 300X (Aut)
- Undergraduate Research
MCP 199 (Aut, Win, Spr, Sum)
- Directed Reading in Biophysics
Prior Year Courses
BIOS 254 (Spr)
- Diversity and Inclusion in Science
BIOS 225 (Win)
- Promises and Pitfalls: A Critical Evaluation of Neuroscience Techniques
BIOS 255 (Win)
Forces applied during classical touch assays for Caenorhabditis elegans
2017; 12 (5)
For decades, Caenorhabditis elegans roundworms have been used to study the sense of touch, and this work has been facilitated by a simple behavioral assay for touch sensation. To perform this classical assay, an experimenter uses an eyebrow hair to gently touch a moving worm and observes whether or not the worm reverses direction. We used two experimental approaches to determine the manner and moment of contact between the eyebrow hair tool and freely moving animals and the forces delivered by the classical assay. Using high-speed video (2500 frames/second), we found that typical stimulus delivery events include a brief moment when the hair is contact with the worm's body and not the agar substrate. To measure the applied forces, we measured forces generated by volunteers mimicking the classical touch assay by touching a calibrated microcantilever. The mean (61 μN) and median forces (26 μN) were more than ten times higher than the 2-μN force known to saturate the probability of evoking a reversal in adult C. elegans. We also considered the eyebrow hairs as an additional source of variation. The stiffness of the sampled eyebrow hairs varied between 0.07 and 0.41 N/m and was correlated with the free length of hair. Collectively, this work establishes that the classical touch assay applies enough force to saturate the probability of evoking reversals in adult C. elegans in spite of its variability among trials and experimenters and that increasing the free length of the hair can decrease the applied force.
View details for DOI 10.1371/journal.pone.0178080
View details for Web of Science ID 000401672600044
View details for PubMedID 28542494
View details for PubMedCentralID PMC5438190
Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.
Lab on a chip
New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm's cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons.
View details for DOI 10.1039/c6lc01165a
View details for PubMedID 28207921
View details for PubMedCentralID PMC5360562
- Genetic defects in beta-spectrin and tau sensitize C.elegans axons to movement-induced damage via torque-tension coupling ELIFE 2017; 6
Molecules empowering animals to sense and respond to temperature in changing environments
CURRENT OPINION IN NEUROBIOLOGY
2016; 41: 92-98
Adapting behavior to thermal cues is essential for animal growth and survival. Indeed, each and every biological and biochemical process is profoundly affected by temperature and its extremes can cause irreversible damage. Hence, animals have developed thermotransduction mechanisms to detect and encode thermal information in the nervous system and acclimation mechanisms to finely tune their response over different timescales. While temperature-gated TRP channels are the best described class of temperature sensors, recent studies highlight many new candidates, including ionotropic and metabotropic receptors. Here, we review recent findings in vertebrate and invertebrate models, which highlight and substantiate the role of new candidate molecular thermometers and reveal intracellular signaling mechanisms implicated in thermal acclimation at the behavioral and cellular levels.
View details for DOI 10.1016/j.conb.2016.09.006
View details for Web of Science ID 000389292300013
View details for PubMedID 27657982
- The tubulin repertoire of Caenorhabditis elegans sensory neurons and its context-dependent role in process outgrowth MOLECULAR BIOLOGY OF THE CELL 2016; 27 (23): 3717-3728
- Tissue mechanics govern the rapidly adapting and symmetrical response to touch PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2015; 112 (50): E6955-E6963
Mechanical systems biology of C. elegans touch sensation.
2015; 37 (3): 335-344
The sense of touch informs us of the physical properties of our surroundings and is a critical aspect of communication. Before touches are perceived, mechanical signals are transmitted quickly and reliably from the skin's surface to mechano-electrical transduction channels embedded within specialized sensory neurons. We are just beginning to understand how soft tissues participate in force transmission and how they are deformed. Here, we review empirical and theoretical studies of single molecules and molecular ensembles thought to be involved in mechanotransmission and apply the concepts emerging from this work to the sense of touch. We focus on the nematode Caenorhabditis elegans as a well-studied model for touch sensation in which mechanics can be studied on the molecular, cellular, and systems level. Finally, we conclude that force transmission is an emergent property of macromolecular cellular structures that mutually stabilize one another.
View details for DOI 10.1002/bies.201400154
View details for PubMedID 25597279
FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis.
During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development.
View details for DOI 10.7554/eLife.06565
View details for PubMedID 25798732
- Feeling Force: Physical and Physiological Principles Enabling Sensory Mechanotransduction ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 31 2015; 31: 347-371
CaMKI-Dependent Regulation of Sensory Gene Expression Mediates Experience-Dependent Plasticity in the Operating Range of a Thermosensory Neuron
2014; 84 (5): 919-926
Sensory adaptation represents a form of experience-dependent plasticity that allows neurons to retain high sensitivity over a broad dynamic range. The mechanisms by which sensory neuron responses are altered on different timescales during adaptation are unclear. The threshold for temperature-evoked activity in the AFD thermosensory neurons (T*(AFD)) in C. elegans is set by the cultivation temperature (T(c)) and regulated by intracellular cGMP levels. We find that T*(AFD) adapts on both short and long timescales upon exposure to temperatures warmer than T(c), and that prolonged exposure to warmer temperatures alters expression of AFD-specific receptor guanylyl cyclase genes. These temperature-regulated changes in gene expression are mediated by the CMK-1 CaMKI enzyme, which exhibits T(c)-dependent nucleocytoplasmic shuttling in AFD. Our results indicate that CaMKI-mediated changes in sensory gene expression contribute to long-term adaptation of T*(AFD), and suggest that similar temporally and mechanistically distinct phases may regulate the operating ranges of other sensory neurons.
View details for DOI 10.1016/j.neuron.2014.10.046
View details for Web of Science ID 000346574300006
View details for PubMedID 25467978
The Balance between Cytoplasmic and Nuclear CaM Kinase-1 Signaling Controls the Operating Range of Noxious Heat Avoidance
2014; 84 (5): 983-996
Through encounters with predators, competitors, and noxious stimuli, animals have evolved defensive responses that minimize injury and are essential for survival. Physiological adaptation modulates the stimulus intensities that trigger such nocifensive behaviors, but the molecular networks that define their operating range are largely unknown. Here, we identify a gain-of-function allele of the cmk-1 CaMKI gene in C. elegans and show that loss of the regulatory domain of the CaMKI enzyme produces thermal analgesia and shifts the operating range for nocifensive heat avoidance to higher temperatures. Such analgesia depends on nuclear CMK-1 signaling, while cytoplasmic CMK-1 signaling lowers the threshold for thermal avoidance. CMK-1 acts downstream of heat detection in thermal receptor neurons and controls neuropeptide release. Our results establish CaMKI as a key regulator of the operating range for nocifensive behaviors and suggest strategies for producing thermal analgesia through the regulation of CaMKI-dependent signaling.
View details for DOI 10.1016/j.neuron.2014.10.039
View details for Web of Science ID 000346574300011
View details for PubMedID 25467982
Sensory biology: it takes Piezo2 to tango.
2014; 24 (12): R566-9
A trio of papers has resolved an outstanding controversy regarding the function of Merkel cells and their afferent nerve fiber partners. Merkel cells sense mechanical stimuli (through Piezo2), fire action potentials, and are sufficient to activate downstream sensory neurons.
View details for DOI 10.1016/j.cub.2014.05.011
View details for PubMedID 24937283
Mechanical control of the sense of touch by ß-spectrin.
Nature cell biology
2014; 16 (3): 224-233
The ability to sense and respond to mechanical stimuli emanates from sensory neurons and is shared by most, if not all, animals. Exactly how such neurons receive and distribute mechanical signals during touch sensation remains mysterious. Here, we show that sensation of mechanical forces depends on a continuous, pre-stressed spectrin cytoskeleton inside neurons. Mutations in the tetramerization domain of Caenorhabditis elegans β-spectrin (UNC-70), an actin-membrane crosslinker, cause defects in sensory neuron morphology under compressive stress in moving animals. Through atomic force spectroscopy experiments on isolated neurons, in vivo laser axotomy and fluorescence resonance energy transfer imaging to measure force across single cells and molecules, we show that spectrin is held under constitutive tension in living animals, which contributes to elevated pre-stress in touch receptor neurons. Genetic manipulations that decrease such spectrin-dependent tension also selectively impair touch sensation, suggesting that such pre-tension is essential for efficient responses to external mechanical stimuli.
View details for DOI 10.1038/ncb2915
View details for PubMedID 24561618
- Mechanical control of the sense of touch by ß-spectrin. Nature cell biology 2014; 16 (3): 224-233
Bidirectional thermotaxis in Caenorhabditis elegans is mediated by distinct sensorimotor strategies driven by the AFD thermosensory neurons
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (7): 2776-2781
The nematode Caenorhabditis elegans navigates toward a preferred temperature setpoint (Ts) determined by long-term temperature exposure. During thermotaxis, the worm migrates down temperature gradients at temperatures above Ts (negative thermotaxis) and performs isothermal tracking near Ts. Under some conditions, the worm migrates up temperature gradients below Ts (positive thermotaxis). Here, we analyze positive and negative thermotaxis toward Ts to study the role of specific neurons that have been proposed to be involved in thermotaxis using genetic ablation, behavioral tracking, and calcium imaging. We find differences in the strategies for positive and negative thermotaxis. Negative thermotaxis is achieved through biasing the frequency of reorientation maneuvers (turns and reversal turns) and biasing the direction of reorientation maneuvers toward colder temperatures. Positive thermotaxis, in contrast, biases only the direction of reorientation maneuvers toward warmer temperatures. We find that the AFD thermosensory neuron drives both positive and negative thermotaxis. The AIY interneuron, which is postsynaptic to AFD, may mediate the switch from negative to positive thermotaxis below Ts. We propose that multiple thermotactic behaviors, each defined by a distinct set of sensorimotor transformations, emanate from the AFD thermosensory neurons. AFD learns and stores the memory of preferred temperatures, detects temperature gradients, and drives the appropriate thermotactic behavior in each temperature regime by the flexible use of downstream circuits.
View details for DOI 10.1073/pnas.1315205111
View details for Web of Science ID 000331396500075
View details for PubMedID 24550307
View details for PubMedCentralID PMC3932917
Phospholipids that Contain Polyunsaturated Fatty Acids Enhance Neuronal Cell Mechanics and Touch Sensation.
2014; 6 (1): 70-80
Mechanoelectrical transduction (MeT) channels embedded in neuronal cell membranes are essential for touch and proprioception. Little is understood about the interplay between native MeT channels and membrane phospholipids, in part because few techniques are available for altering plasma membrane composition in vivo. Here, we leverage genetic dissection, chemical complementation, and optogenetics to establish that arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, enhances touch sensation and mechanoelectrical transduction activity while incorporated into membrane phospholipids in C. elegans touch receptor neurons (TRNs). Because dynamic force spectroscopy reveals that AA modulates the mechanical properties of TRN plasma membranes, we propose that this polyunsaturated fatty acid (PUFA) is needed for MeT channel activity. These findings establish that polyunsaturated phospholipids are crucial determinants of both the biochemistry and mechanics of mechanoreceptor neurons and reinforce the idea that sensory mechanotransduction in animals relies on a cellular machine composed of both proteins and membrane lipids.
View details for DOI 10.1016/j.celrep.2013.12.012
View details for PubMedID 24388754
Thermotaxis navigation behavior.
WormBook : the online review of C. elegans biology
This chapter describes four different protocols used to assay thermotaxis navigation behavior of single, or populations of, C. elegans hermaphrodites on spatial thermal gradients within the physiological temperature range (15-25°C). A method to assay avoidance of noxious temperatures is also described.
View details for DOI 10.1895/wormbook.1.168.1
View details for PubMedID 24563245
- PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in Caenorhabditis elegans neurons. eLife 2014; 3
WormBook : the online review of C. elegans biology
C. elegans detect and respond to diverse mechanical stimuli using neuronal circuitry that has been defined by decades of work by C. elegans researchers. In this WormMethods chapter, we review and comment on the techniques currently used to assess mechanosensory response. This methods review is intended both as an introduction for those new to the field and a convenient compendium for the expert. A brief discussion of commonly used mechanosensory assays is provided, along with a discussion of the neural circuits involved, consideration of critical protocol details, and references to the primary literature.
View details for DOI 10.1895/wormbook.1.172.1
View details for PubMedID 25093996
PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in Caenorhabditis elegans neurons.
In neuronal processes, microtubules (MTs) provide structural support and serve as tracks for molecular motors. While it is known that neuronal MTs are more stable than MTs in non-neuronal cells, the molecular mechanisms underlying this stability are not fully understood. In this study, we used live fluorescence microscopy to show that the C. elegans CAMSAP protein PTRN-1 localizes to puncta along neuronal processes, stabilizes MT foci, and promotes MT polymerization in neurites. Electron microscopy revealed that ptrn-1 null mutants have fewer MTs and abnormal MT organization in the PLM neuron. Animals grown with a MT depolymerizing drug caused synthetic defects in neurite branching in the absence of ptrn-1 function, indicating that PTRN-1 promotes MT stability. Further, ptrn-1 null mutants exhibited aberrant neurite morphology and synaptic vesicle localization that is partially dependent on dlk-1. Our results suggest that PTRN-1 represents an important mechanism for promoting MT stability in neurons. DOI: http://dx.doi.org/10.7554/eLife.01498.001.
View details for DOI 10.7554/eLife.01498
View details for PubMedID 24569477
Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis
2013; 9 (10)
The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the 'metazoanome'), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan biology that contains a number of neuronally expressed and disease-related genes, and reveal a key role for TBCEL/coel-1 in regulating microtubule function during metazoan development and neuronal differentiation.
View details for DOI 10.1371/journal.pgen.1003804
View details for Web of Science ID 000330367200009
View details for PubMedID 24098140
GCY-8, PDE-2, and NCS-1 are critical elements of the cGMP-dependent thermotransduction cascade in the AFD neurons responsible for C. elegans thermotaxis
JOURNAL OF GENERAL PHYSIOLOGY
2013; 142 (4): 437-449
Certain thermoreceptor neurons are sensitive to tiny thermal fluctuations (0.01°C or less) and maintain their sensitivity across a wide range of ambient temperatures through a process of adaptation, but understanding of the biochemical basis for this performance is rudimentary. Prior studies of the AFD thermoreceptor in Caenorhabditis elegans revealed a signaling cascade that depends on a trio of receptor guanylate cyclases (rGCs), GCY-8, GCY-18, and GCY-23, and gives rise to warming-activated thermoreceptor currents (ThRCs) carried by cyclic GMP-gated ion channels. The threshold for ThRC activation adapts to the ambient temperature through an unknown calcium-dependent process. Here, we use in vivo whole-cell patch-clamp recording from AFD to show that loss of GCY-8, but not of GCY-18 or GCY-23, reduces or eliminates ThRCs, identifying this rGC as a crucial signaling element. To learn more about thermotransduction and adaptation, we used behavioral screens and analysis of gene expression patterns to identify phosphodiesterases (PDEs) likely to contribute to thermotransduction. Deleting PDE-2 decouples the threshold for ThRC activation from ambient temperature, altering adaptation. We provide evidence that the conserved neuronal calcium sensor 1 protein also regulates the threshold for ThRC activation and propose a signaling network to account for ThRC activation and adaptation. Because PDEs play essential roles in diverse biological processes, including vertebrate phototransduction and olfaction, and regulation of smooth muscle contractility and cardiovascular function, this study has broad implications for understanding how extraordinary sensitivity and dynamic range is achieved in cyclic nucleotide-based signaling networks.
View details for DOI 10.1085/jgp.201310959
View details for Web of Science ID 000325278400010
View details for PubMedID 24081984
MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation.
2013; 5 (6): 853-864
Touch is enabled by mechanoreceptor neurons in the skin and plays an essential role in our everyday lives, but is among the least understood of our five basic senses. Force applied to the skin deforms these neurons and activates ion channels within them. Despite the importance of the mechanics of the skin in determining mechanoreceptor neuron deformation and ultimately touch sensation, the role of mechanics in touch sensitivity is poorly understood. Here, we use the model organism Caenorhabditis elegans to directly test the hypothesis that body mechanics modulate touch sensitivity. We demonstrate a microelectromechanical system (MEMS)-based force clamp that can apply calibrated forces to freely crawling C. elegans worms and measure touch-evoked avoidance responses. This approach reveals that wild-type animals sense forces <1 μN and indentation depths <1 μm. We use both genetic manipulation of the skin and optogenetic modulation of body wall muscles to alter body mechanics. We find that small changes in body stiffness dramatically affect force sensitivity, while having only modest effects on indentation sensitivity. We investigate the theoretical body deformation predicted under applied force and conclude that local mechanical loads induce inward bending deformation of the skin to drive touch sensation in C. elegans.
View details for DOI 10.1039/c3ib20293c
View details for PubMedID 23598612
View details for PubMedCentralID PMC3701114
The doublecortin-related gene zyg-8 is a microtubule organizer in Caenorhabditis elegans neurons
JOURNAL OF CELL SCIENCE
2012; 125 (22): 5417-5427
Doublecortin-domain containing (DCDC) genes play key roles in the normal and pathological development of the human brain cortex. The origin of the cellular specialisation and the functional redundancy of these microtubule (MT)-associated proteins (MAPs), especially those of Doublecortin (DCX) and Doublecortin-like kinase (DCLKs) genes, is still unclear. The DCX domain has the ability to control MT architecture and bundling. However, the physiological significance of such properties is not fully understood. To address these issues, we sought post-mitotic roles for zyg-8, the sole representative of the DCX-DCLK subfamily of genes in C. elegans. Previously, zyg-8 has been shown to control anaphase-spindle positioning in one-cell stage embryos, but functions of the gene later in development have not been investigated. Here we show that wild-type zyg-8 is required beyond early embryonic divisions for proper development, spontaneous locomotion and touch sensitivity of adult worms. Consistently, we find zyg-8 expression in the six touch receptor neurons (TRNs), as well as in a subset of other neuronal and non-neuronal cells. In TRNs and motoneurons, zyg-8 controls cell body shape/polarity and process outgrowth and morphology. Ultrastructural analysis of mutant animals reveals that zyg-8 promotes structural integrity, length and number of individual MTs, as well as their bundled organisation in TRNs, with no impact on MT architecture.
View details for DOI 10.1242/jcs.108381
View details for Web of Science ID 000314511900016
View details for PubMedID 22956537
Insight into DEG/ENaC Channel Gating from Genetics and Structure
2012; 27 (5): 282-290
The founding members of the superfamily of DEG/ENaC ion channel proteins are C. elegans proteins that form mechanosensitive channels in touch and pain receptors. For more than a decade, the research community has used mutagenesis to identify motifs that regulate gating. This review integrates insight derived from unbiased in vivo mutagenesis screens with recent crystal structures to develop new models for activation of mechanically gated DEGs.
View details for DOI 10.1152/physiol.00006.2012
View details for Web of Science ID 000309512000002
View details for PubMedID 23026751
Posttranslational Acetylation of alpha-Tubulin Constrains Protofilament Number in Native Microtubules
2012; 22 (12): 1066-1074
Microtubules are built from linear polymers of α-β tubulin dimers (protofilaments) that form a tubular quinary structure. Microtubules assembled from purified tubulin in vitro contain between 10 and 16 protofilaments; however, such structural polymorphisms are not found in cells. This discrepancy implies that factors other than tubulin constrain microtubule protofilament number, but the nature of these constraints is unknown.Here, we show that acetylation of MEC-12 α-tubulin constrains protofilament number in C. elegans touch receptor neurons (TRNs). Whereas the sensory dendrite of wild-type TRNs is packed with a cross-linked bundle of long, 15-protofilament microtubules, mec-17;atat-2 mutants lacking α-tubulin acetyltransferase activity have short microtubules, rampant lattice defects, and variable protofilament number both between and within microtubules. All-atom molecular dynamics simulations suggest a model in which acetylation of lysine 40 promotes the formation of interprotofilament salt bridges, stabilizing lateral interactions between protofilaments and constraining quinary structure to produce stable, structurally uniform microtubules in vivo.Acetylation of α-tubulin is an essential constraint on protofilament number in vivo. We propose a structural model in which this posttranslational modification promotes the formation of lateral salt bridges that fine-tune the association between adjacent protofilaments and enable the formation of uniform microtubule populations in vivo.
View details for DOI 10.1016/j.cub.2012.05.012
View details for Web of Science ID 000305766900020
View details for PubMedID 22658592
How We Feel: Ion Channel Partnerships that Detect Mechanical Inputs and Give Rise to Touch and Pain Perception
2012; 74 (4): 609-619
Every moment of every day, our skin and its embedded sensory neurons are bombarded with mechanical cues that we experience as pleasant or painful. Knowing the difference between innocuous and noxious mechanical stimuli is critical for survival and relies on the function of mechanoreceptor neurons that vary in their size, shape, and sensitivity. Their function is poorly understood at the molecular level. This review emphasizes the importance of integrating analysis at the molecular and cellular levels and focuses on the discovery of ion channel proteins coexpressed in the mechanoreceptors of worms, flies, and mice.
View details for DOI 10.1016/j.neuron.2012.04.023
View details for Web of Science ID 000304747200005
View details for PubMedID 22632719
Electrophysiological Methods for Caenorhabditis elegans Neurobiology
CAENORHABDITIS ELEGANS: CELL BIOLOGY AND PHYSIOLOGY, SECOND EDITION
2012; 107: 409-436
Patch-clamp electrophysiology is a technique of choice for the biophysical analysis of the function of nerve, muscle, and synapse in Caenorhabditis elegans nematodes. Considerable technical progress has been made in C. elegans electrophysiology in the decade since the initial publication of this technique. Today, most, if not all, electrophysiological studies that can be done in larger animal preparations can also be done in C. elegans. This chapter has two main goals. The first is to present to a broad audience the many techniques available for patch-clamp analysis of neurons, muscles, and synapses in C. elegans. The second is to provide a methodological introduction to the techniques for patch clamping C. elegans neurons and body-wall muscles in vivo, including emerging methods for optogenetic stimulation coupled with postsynaptic recording. We also present samples of the cell-intrinsic and postsynaptic ionic currents that can be measured in C. elegans nerves and muscles.
View details for DOI 10.1016/B978-0-12-394620-1.00014-X
View details for Web of Science ID 000299611000014
View details for PubMedID 22226532
Intragenic alternative splicing coordination is essential for Caenorhabditis elegans slo-1 gene function
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (51): 20790-20795
Alternative splicing is critical for diversifying eukaryotic proteomes, but the rules governing and coordinating splicing events among multiple alternate splice sites within individual genes are not well understood. We developed a quantitative PCR-based strategy to quantify the expression of the 12 transcripts encoded by the Caenorhabditis elegans slo-1 gene, containing three alternate splice sites. Using conditional probability-based models, we show that splicing events are coordinated across these sites. Further, we identify a point mutation in an intron adjacent to one alternate splice site that disrupts alternative splicing at all three sites. This mutation leads to aberrant synaptic transmission at the neuromuscular junction. In a genomic survey, we found that a UAAAUC element disrupted by this mutation is enriched in introns flanking alternate exons in genes with multiple alternate splice sites. These results establish that proper coordination of intragenic alternative splicing is essential for normal physiology of slo-1 in vivo and identify putative specialized cis-regulatory elements that regulate the coordination of intragenic alternative splicing.
View details for DOI 10.1073/pnas.1116712108
View details for Web of Science ID 000298289400102
View details for PubMedID 22084100
Alternatively spliced domains interact to regulate BK potassium channel gating
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2011; 108 (51): 20784-20789
Most human genes contain multiple alternative splice sites believed to extend the complexity and diversity of the proteome. However, little is known about how interactions among alternative exons regulate protein function. We used the Caenorhabditis elegans slo-1 large-conductance calcium and voltage-activated potassium (BK) channel gene, which contains three alternative splice sites (A, B, and C) and encodes at least 12 splice variants, to investigate the functional consequences of alternative splicing. These splice sites enable the insertion of exons encoding part of the regulator of K(+) conductance (RCK)1 Ca(2+) coordination domain (exons A1 and A2) and portions of the RCK1-RCK2 linker (exons B0, B1, B2, C0, and C1). Exons A1 and A2 are used in a mutually exclusive manner and are 67% identical. The other exons can extend the RCK1-RCK2 linker by up to 41 residues. Electrophysiological recordings of all isoforms show that the A1 and A2 exons regulate activation kinetics and Ca(2+) sensitivity, but only if alternate exons are inserted at site B or C. Thus, RCK1 interacts with the RCK1-RCK2 linker, and the effect of exon variation on gating depends on the combination of alternate exons present in each isoform.
View details for DOI 10.1073/pnas.1116795108
View details for Web of Science ID 000298289400101
View details for PubMedID 22049343
DEG/ENaC but Not TRP Channels Are the Major Mechanoelectrical Transduction Channels in a C. elegans Nociceptor
2011; 71 (5): 845-857
Many nociceptors detect mechanical cues, but the ion channels responsible for mechanotransduction in these sensory neurons remain obscure. Using in vivo recordings and genetic dissection, we identified the DEG/ENaC protein, DEG-1, as the major mechanotransduction channel in ASH, a polymodal nociceptor in Caenorhabditis elegans. But DEG-1 is not the only mechanotransduction channel in ASH: loss of deg-1 revealed a minor current whose properties differ from those expected of DEG/ENaC channels. This current was independent of two TRPV channels expressed in ASH. Although loss of these TRPV channels inhibits behavioral responses to noxious stimuli, we found that both mechanoreceptor currents and potentials were essentially wild-type in TRPV mutants. We propose that ASH nociceptors rely on two genetically distinct mechanotransduction channels and that TRPV channels contribute to encoding and transmitting information. Because mammalian and insect nociceptors also coexpress DEG/ENaCs and TRPVs, the cellular functions elaborated here for these ion channels may be conserved.
View details for DOI 10.1016/j.neuron.2011.06.038
View details for Web of Science ID 000294877900010
View details for PubMedID 21903078
The DEG/ENaC Protein MEC-10 Regulates the Transduction Channel Complex in Caenorhabditis elegans Touch Receptor Neurons
JOURNAL OF NEUROSCIENCE
2011; 31 (35): 12695-12704
Gentle touch sensation in Caenorhabditis elegans is mediated by the MEC-4/MEC-10 channel complex, which is expressed exclusively in six touch receptor neurons (TRNs). The complex contains two pore-forming subunits, MEC-4 and MEC-10, as well as the accessory subunits MEC-2, MEC-6, and UNC-24. MEC-4 is essential for channel function, but beyond its role as a pore-forming subunit, the functional contribution of MEC-10 to the channel complex and to touch sensation is unclear. We addressed this question using behavioral assays, in vivo electrophysiological recordings from TRNs, and heterologous expression of mutant MEC-10 isoforms. Animals with a deletion in mec-10 showed only a partial loss of touch sensitivity and a modest decrease in the size of the mechanoreceptor current (MRC). In contrast, five previously identified mec-10 alleles acted as recessive gain-of-function alleles that resulted in complete touch insensitivity. Each of these alleles produced a substantial decrease in MRC size and a shift in the reversal potential in vivo. The latter finding indicates that these mec-10 mutations alter the ionic selectivity of the transduction channel in vivo. All mec-10 mutant animals had properly localized channel complexes, indicating that the loss of MRCs was not attributable to a dramatic mislocalization of transduction channels. Finally, electrophysiological examination of heterologously expressed complexes suggests that mutant MEC-10 proteins may affect channel current via MEC-2.
View details for DOI 10.1523/JNEUROSCI.4580-10.2011
View details for Web of Science ID 000294451900032
View details for PubMedID 21880930
Heat Avoidance Is Regulated by Transient Receptor Potential (TRP) Channels and a Neuropeptide Signaling Pathway in Caenorhabditis elegans
2011; 188 (1): 91-U150
The ability to avoid noxious extremes of hot and cold is critical for survival and depends on thermal nociception. The TRPV subset of transient receptor potential (TRP) channels is heat activated and proposed to be responsible for heat detection in vertebrates and fruit flies. To gain insight into the genetic and neural basis of thermal nociception, we developed assays that quantify noxious heat avoidance in the nematode Caenorhabditis elegans and used them to investigate the genetic basis of this behavior. First, we screened mutants for 18 TRP channel genes (including all TRPV orthologs) and found only minor defects in heat avoidance in single and selected double and triple mutants, indicating that other genes are involved. Next, we compared two wild isolates of C. elegans that diverge in their threshold for heat avoidance and linked this phenotypic variation to a polymorphism in the neuropeptide receptor gene npr-1. Further analysis revealed that loss of either the NPR-1 receptor or its ligand, FLP-21, increases the threshold for heat avoidance. Cell-specific rescue of npr-1 implicates the interneuron RMG in the circuit regulating heat avoidance. This neuropeptide signaling pathway operates independently of the TRPV genes, osm-9 and ocr-2, since mutants lacking npr-1 and both TRPV channels had more severe defects in heat avoidance than mutants lacking only npr-1 or both osm-9 and ocr-2. Our results show that TRPV channels and the FLP-21/NPR-1 neuropeptide signaling pathway determine the threshold for heat avoidance in C. elegans.
View details for DOI 10.1534/genetics.111.127100
View details for Web of Science ID 000290276900009
View details for PubMedID 21368276
Caenorhabditis elegans Body Mechanics Are Regulated by Body Wall Muscle Tone
2011; 100 (8): 1977-1985
Body mechanics in the nematode Caenorhabditis elegans are central to both mechanosensation and locomotion. Previous work revealed that the mechanics of the outer shell, rather than internal hydrostatic pressure, dominates stiffness. This shell is comprised of the cuticle and the body wall muscles, either of which could contribute to the body mechanics. Here, we tested the hypothesis that the muscles are an important contributor by modulating muscle tone using optogenetic and pharmacological tools, and measuring animal stiffness using piezoresistive microcantilevers. As a proxy for muscle tone, we measured changes in animal length under the same treatments. We found that treatments that induce muscle contraction generally resulted in body shortening and stiffening. Conversely, methods to relax the muscles more modestly increased length and decreased stiffness. The results support the idea that body wall muscle activation contributes significantly to and can modulate C. elegans body mechanics. Modulation of body stiffness would enable nematodes to tune locomotion or swimming gaits and may have implications in touch sensation.
View details for DOI 10.1016/j.bpj.2011.02.035
View details for Web of Science ID 000289864100017
View details for PubMedID 21504734
View details for PubMedCentralID PMC3077690
Piezoresistive cantilever force-clamp system
REVIEW OF SCIENTIFIC INSTRUMENTS
2011; 82 (4)
We present a microelectromechanical device-based tool, namely, a force-clamp system that sets or "clamps" the scaled force and can apply designed loading profiles (e.g., constant, sinusoidal) of a desired magnitude. The system implements a piezoresistive cantilever as a force sensor and the built-in capacitive sensor of a piezoelectric actuator as a displacement sensor, such that sample indentation depth can be directly calculated from the force and displacement signals. A programmable real-time controller operating at 100 kHz feedback calculates the driving voltage of the actuator. The system has two distinct modes: a force-clamp mode that controls the force applied to a sample and a displacement-clamp mode that controls the moving distance of the actuator. We demonstrate that the system has a large dynamic range (sub-nN up to tens of μN force and nm up to tens of μm displacement) in both air and water, and excellent dynamic response (fast response time, <2 ms and large bandwidth, 1 Hz up to 1 kHz). In addition, the system has been specifically designed to be integrated with other instruments such as a microscope with patch-clamp electronics. We demonstrate the capabilities of the system by using it to calibrate the stiffness and sensitivity of an electrostatic actuator and to measure the mechanics of a living, freely moving Caenorhabditis elegans nematode.
View details for DOI 10.1063/1.3574362
View details for Web of Science ID 000290051500022
View details for PubMedID 21529009
View details for PubMedCentralID PMC3091308
The major alpha-tubulin K40 acetyltransferase alpha TAT1 promotes rapid ciliogenesis and efficient mechanosensation
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2010; 107 (50): 21517-21522
Long-lived microtubules found in ciliary axonemes, neuronal processes, and migrating cells are marked by α-tubulin acetylation on lysine 40, a modification that takes place inside the microtubule lumen. The physiological importance of microtubule acetylation remains elusive. Here, we identify a BBSome-associated protein that we name αTAT1, with a highly specific α-tubulin K40 acetyltransferase activity and a catalytic preference for microtubules over free tubulin. In mammalian cells, the catalytic activity of αTAT1 is necessary and sufficient for α-tubulin K40 acetylation. Remarkably, αTAT1 is universally and exclusively conserved in ciliated organisms, and is required for the acetylation of axonemal microtubules and for the normal kinetics of primary cilium assembly. In Caenorhabditis elegans, microtubule acetylation is most prominent in touch receptor neurons (TRNs) and MEC-17, a homolog of αTAT1, and its paralog αTAT-2 are required for α-tubulin acetylation and for two distinct types of touch sensation. Furthermore, in animals lacking MEC-17, αTAT-2, and the sole C. elegans K40α-tubulin MEC-12, touch sensation can be restored by expression of an acetyl-mimic MEC-12[K40Q]. We conclude that αTAT1 is the major and possibly the sole α-tubulin K40 acetyltransferase in mammals and nematodes, and that tubulin acetylation plays a conserved role in several microtubule-based processes.
View details for DOI 10.1073/pnas.1013728107
View details for Web of Science ID 000285521500055
View details for PubMedID 21068373
Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis in C. elegans and Drosophila
GENES & DEVELOPMENT
2010; 24 (21): 2365-2382
Like other ectotherms, the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster rely on behavioral strategies to stabilize their body temperature. Both animals use specialized sensory neurons to detect small changes in temperature, and the activity of these thermosensors governs the neural circuits that control migration and accumulation at preferred temperatures. Despite these similarities, the underlying molecular, neuronal, and computational mechanisms responsible for thermotaxis are distinct in these organisms. Here, we discuss the role of thermosensation in the development and survival of C. elegans and Drosophila, and review the behavioral strategies, neuronal circuits, and molecular networks responsible for thermotaxis behavior.
View details for DOI 10.1101/gad.1953710
View details for Web of Science ID 000283656400002
View details for PubMedID 21041406
An Arf-like Small G Protein, ARL-8, Promotes the Axonal Transport of Presynaptic Cargoes by Suppressing Vesicle Aggregation
2010; 66 (5): 710-723
Presynaptic assembly requires the packaging of requisite proteins into vesicular cargoes in the cell soma, their long-distance microtubule-dependent transport down the axon, and, finally, their reconstitution into functional complexes at prespecified sites. Despite the identification of several molecules that contribute to these events, the regulatory mechanisms defining such discrete states remain elusive. We report the characterization of an Arf-like small G protein, ARL-8, required during this process. arl-8 mutants prematurely accumulate presynaptic cargoes within the proximal axon of several neuronal classes, with a corresponding failure to assemble presynapses distally. This proximal accumulation requires the activity of several molecules known to catalyze presynaptic assembly. Dynamic imaging studies reveal that arl-8 mutant vesicles exhibit an increased tendency to form immotile aggregates during transport. Together, these results suggest that arl-8 promotes a trafficking identity for presynaptic cargoes, facilitating their efficient transport by repressing premature self-association.
View details for DOI 10.1016/j.neuron.2010.04.033
View details for Web of Science ID 000278941900011
View details for PubMedID 20547129
View details for PubMedCentralID PMC3168544
- Neuropeptides strike back NATURE NEUROSCIENCE 2010; 13 (5): 528-529
The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans
2009; 5 (12)
Genetic defects in the dystrophin-associated protein complex (DAPC) are responsible for a variety of pathological conditions including muscular dystrophy, cardiomyopathy, and vasospasm. Conserved DAPC components from humans to Caenorhabditis elegans suggest a similar molecular function. C. elegans DAPC mutants exhibit a unique locomotory deficit resulting from prolonged muscle excitation and contraction. Here we show that the C. elegans DAPC is essential for proper localization of SLO-1, the large conductance, voltage-, and calcium-dependent potassium (BK) channel, which conducts a major outward rectifying current in muscle under the normal physiological condition. Through analysis of mutants with the same phenotype as the DAPC mutants, we identified the novel islo-1 gene that encodes a protein with two predicted transmembrane domains. We demonstrate that ISLO-1 acts as a novel adapter molecule that links the DAPC to SLO-1 in muscle. We show that a defect in either the DAPC or ISLO-1 disrupts normal SLO-1 localization in muscle. Consistent with observations that SLO-1 requires a high calcium concentration for full activation, we find that SLO-1 is localized near L-type calcium channels in muscle, thereby providing a mechanism coupling calcium influx with the outward rectifying current. Our results indicate that the DAPC modulates muscle excitability by localizing the SLO-1 channel to calcium-rich regions of C. elegans muscle.
View details for DOI 10.1371/journal.pgen.1000780
View details for Web of Science ID 000273469700030
View details for PubMedID 20019812
SU-8 force sensing pillar arrays for biological measurements
LAB ON A CHIP
2009; 9 (10): 1449-1454
The generation and sensation of mechanical force plays a role in many dynamic biological processes, including touch sensation. This paper presents a two-axis micro strain gauge force sensor constructed from multiple layers of SU-8 and metal on quartz substrates. The sensor was designed to meet requirements for measuring tactile sensitivity and interaction forces exerted during locomotion by small organisms such as the nematode Caenorhabditis elegans. The device is transparent and compatible with light microscopes, allowing behavioral experiments to be combined with quantitative force measurements. For the first time, we have characterized the scale of interaction forces generated in wild-type C. elegans in probing and responding to their environment during locomotion. The device features sub-microN force resolution from 1 Hz to 1 kHz, >25 microN range, kHz acquisition rates and biocompatibility.
View details for DOI 10.1039/b818622g
View details for Web of Science ID 000268227400019
View details for PubMedID 19417913
The quest for action potentials in C. elegans neurons hits a plateau
2009; 12 (4): 377-378
The small size and high resistance of C. elegans neurons makes them sensitive to the random opening of single ion channels, probably rendering codes that are based on classical, all-or-none action potentials unworkable. The recent discovery in C. elegans of a special class of regenerative events known as plateau potentials introduces the possibility of digital neural codes. Such codes would solve the problem of representing information in nervous systems in which action potentials are unreliable.
View details for DOI 10.1038/nn0409-377
View details for Web of Science ID 000264563100008
View details for PubMedID 19322241
PIEZORESISTIVE CANTILEVER-BASED FORCE-CLAMP SYSTEM FOR THE STUDY OF MECHANOTRANSDUCTION IN C. ELEGANS
22nd International Conference on Micro Electro Mechanical Systems (MEMS)
IEEE. 2009: 188–191
View details for Web of Science ID 000341431500047
Thermotaxis is a Robust Mechanism for Thermoregulation in Caenorhabditis elegans Nematodes
JOURNAL OF NEUROSCIENCE
2008; 28 (47): 12546-12557
Many biochemical networks are robust to variations in network or stimulus parameters. Although robustness is considered an important design principle of such networks, it is not known whether this principle also applies to higher-level biological processes such as animal behavior. In thermal gradients, Caenorhabditis elegans uses thermotaxis to bias its movement along the direction of the gradient. Here we develop a detailed, quantitative map of C. elegans thermotaxis and use these data to derive a computational model of thermotaxis in the soil, a natural environment of C. elegans. This computational analysis indicates that thermotaxis enables animals to avoid temperatures at which they cannot reproduce, to limit excursions from their adapted temperature, and to remain relatively close to the surface of the soil, where oxygen is abundant. Furthermore, our analysis reveals that this mechanism is robust to large variations in the parameters governing both worm locomotion and temperature fluctuations in the soil. We suggest that, similar to biochemical networks, animals evolve behavioral strategies that are robust, rather than strategies that rely on fine tuning of specific behavioral parameters.
View details for DOI 10.1523/JNEUROSCI.2857-08.2008
View details for Web of Science ID 000261191000040
View details for PubMedID 19020047
The C-elegans EMAP-like protein, ELP-1 is required for touch sensation and associates with microtubules and adhesion complexes
BMC DEVELOPMENTAL BIOLOGY
The founding member of the EMAP-like protein family is the Echinoderm Microtubule-Associated Protein (EMAP), so-named for its abundance in sea urchin, starfish, and sand dollar eggs. The EMAP-like protein family has five members in mammals (EML1 through EML5) and only one in both Drosophila (ELP-1) and C. elegans (ELP-1). Biochemical studies of sea urchin EMAP and vertebrate EMLs implicate these proteins in the regulation of microtubule stability. So far, however, the physiological function of this protein family remains unknown.We examined the expression pattern of C. elegans ELP-1 by means of transgenic gene expression in living embryos and adults, and by immunolocalization with an ELP-1-specific antibody in fixed tissues. In embryos, ELP-1 is expressed in the hypodermis. In larvae and adults, ELP-1 is expressed in the body wall, spermatheca and vulval muscles, intestine, and hypodermal seam cells. In muscle, ELP-1 is associated with adhesion complexes near the cell surface and is bound to a criss-crossing network of microtubules in the cytoplasm. ELP-1 is also expressed in a subset of mechanoreceptor neurons, including the ray neurons in the male tail, microtubule-rich touch receptor neurons, and the six ciliated IL1 neurons. This restricted localization in the nervous system implies that ELP-1 plays a role in mechanotransmission. Consistent with this idea, decreasing ELP-1 expression decreases sensitivity to gentle touch applied to the body wall.These data imply that ELP-1 may play an important role during the transmission of forces and signals between the body surface and both muscle cells and touch receptor neurons.
View details for DOI 10.1186/1471-213X-8-110
View details for Web of Science ID 000264068400001
View details for PubMedID 19014691
Bidirectional temperature-sensing by a single thermosensory neuron in C-elegans
2008; 11 (8): 908-915
Humans and other animals can sense temperature changes as small as 0.1 degree C. How animals achieve such exquisite sensitivity is poorly understood. By recording from the C. elegans thermosensory neurons AFD in vivo, we found that cooling closes and warming opens ion channels. We found that AFD thermosensitivity, which exceeds that of most biological processes by many orders of magnitude, is achieved by nonlinear signal amplification. Mutations in genes encoding subunits of a cyclic guanosine monophosphate (cGMP)-gated ion channel (tax-4 and tax-2) and transmembrane guanylate cyclases (gcy-8, gcy-18 and gcy-23) eliminated both cooling- and warming-activated thermoreceptor currents, indicating that a cGMP-mediated pathway links variations in temperature to changes in ionic currents. The resemblance of C. elegans thermosensation to vertebrate photosensation and the sequence similarity between TAX-4 and TAX-2 and subunits of the rod phototransduction channel raise the possibility that nematode thermosensation and vertebrate vision are linked by conserved evolution.
View details for DOI 10.1038/nn.2157
View details for Web of Science ID 000257969100016
View details for PubMedID 18660808
Artificial dirt: Microfluidic substrates for nematode neurobiology and behavior
JOURNAL OF NEUROPHYSIOLOGY
2008; 99 (6): 3136-3143
With a nervous system of only 302 neurons, the free-living nematode Caenorhabditis elegans is a powerful experimental organism for neurobiology. However, the laboratory substrate commonly used in C. elegans research, a planar agarose surface, fails to reflect the complexity of this organism's natural environment, complicates stimulus delivery, and is incompatible with high-resolution optophysiology experiments. Here we present a new class of microfluidic devices for C. elegans neurobiology and behavior: agarose-free, micron-scale chambers and channels that allow the animals to crawl as they would on agarose. One such device mimics a moist soil matrix and facilitates rapid delivery of fluid-borne stimuli. A second device consists of sinusoidal channels that can be used to regulate the waveform and trajectory of crawling worms. Both devices are thin and transparent, rendering them compatible with high-resolution microscope objectives for neuronal imaging and optical recording. Together, the new devices are likely to accelerate studies of the neuronal basis of behavior in C. elegans.
View details for Web of Science ID 000256632600035
View details for PubMedID 18337372
MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: Auxiliary Subunits that enable channel activity
JOURNAL OF GENERAL PHYSIOLOGY
2008; 131 (6): 605-616
The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.
View details for DOI 10.1085/jgp.200709910
View details for Web of Science ID 000256625300010
View details for PubMedID 18504316
The Parallel Worm Tracker: A Platform for Measuring Average Speed and Drug-Induced Paralysis in Nematodes
2008; 3 (5)
Caenorhabditis elegans locomotion is a simple behavior that has been widely used to dissect genetic components of behavior, synaptic transmission, and muscle function. Many of the paradigms that have been created to study C. elegans locomotion rely on qualitative experimenter observation. Here we report the implementation of an automated tracking system developed to quantify the locomotion of multiple individual worms in parallel.Our tracking system generates a consistent measurement of locomotion that allows direct comparison of results across experiments and experimenters and provides a standard method to share data between laboratories. The tracker utilizes a video camera attached to a zoom lens and a software package implemented in MATLAB. We demonstrate several proof-of-principle applications for the tracker including measuring speed in the absence and presence of food and in the presence of serotonin. We further use the tracker to automatically quantify the time course of paralysis of worms exposed to aldicarb and levamisole and show that tracker performance compares favorably to data generated using a hand-scored metric.Although this is not the first automated tracking system developed to measure C. elegans locomotion, our tracking software package is freely available and provides a simple interface that includes tools for rapid data collection and analysis. By contrast with other tools, it is not dependent on a specific set of hardware. We propose that the tracker may be used for a broad range of additional worm locomotion applications including genetic and chemical screening.
View details for DOI 10.1371/journal.pone.0002208
View details for Web of Science ID 000262258700017
View details for PubMedID 18493300
Patch clamp recording of ion channels expressed in Xenopus oocytes.
Journal of visualized experiments : JoVE
Since its development by Sakmann and Neher (1, 2), the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.
View details for DOI 10.3791/936
View details for PubMedID 19078941
Making patch-pipettes and sharp electrodes with a programmable puller.
Journal of visualized experiments : JoVE
Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass <1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.
View details for DOI 10.3791/939
View details for PubMedID 19078940
Pressure-polishing pipettes for improved patch-clamp recording.
Journal of visualized experiments : JoVE
Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (<3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (<5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.
View details for DOI 10.3791/964
View details for PubMedID 19078936
Nanoscale organization of the MEC-4 DEG/ENaC sensory mechanotransduction channel in Caenorhabditis elegans touch receptor neurons
JOURNAL OF NEUROSCIENCE
2007; 27 (51): 14089-14098
Hearing, touch and proprioception are thought to involve direct activation of mechano-electrical transduction (MeT) channels. In Caenorhabditis elegans touch receptor neurons (TRNs), such channels contain two pore-forming subunits (MEC-4 and MEC-10) and two auxiliary subunits (MEC-2 and MEC-6). MEC-4 and MEC-10 belong to a large superfamily of ion channel proteins (DEG/ENaCs) that form nonvoltage-gated, amiloride-sensitive Na+ channels. In TRNs, unique 15-protofilament microtubules and an electron-dense extracellular matrix have been proposed to serve as gating tethers critical for MeT channel activation. We combined high-pressure freezing and serial-section immunoelectron microscopy to determine the position of MeT channels relative to putative gating tethers. MeT channels were visualized using antibodies against MEC-4 and MEC-2. This nanometer-resolution view of a sensory MeT channel establishes structural constraints on the mechanics of channel gating. We show here that MEC-2 and MEC-5 collagen, a putative extracellular tether, occupy overlapping but distinct domains in TRN neurites. Although channels decorate all sides of TRN neurites; they are not associated with the distal endpoints of 15-protofilament microtubules hypothesized to be gating tethers. These specialized microtubules, which are unique to TRNs, assemble into a cross-linked bundle connected by a network of kinked filaments to the neurite membrane. We speculate that the microtubule bundle converts external point loads into membrane stretch which, in turn, facilitates MeT channel activation.
View details for DOI 10.1523/JNEUROSCI.4179-07.2007
View details for Web of Science ID 000251910800021
View details for PubMedID 18094248
Dissecting a circuit for olfactory behaviour in Caenorhabditis elegans
2007; 450 (7166): 63-?
Although many properties of the nervous system are shared among animals and systems, it is not known whether different neuronal circuits use common strategies to guide behaviour. Here we characterize information processing by Caenorhabditis elegans olfactory neurons (AWC) and interneurons (AIB and AIY) that control food- and odour-evoked behaviours. Using calcium imaging and mutations that affect specific neuronal connections, we show that AWC neurons are activated by odour removal and activate the AIB interneurons through AMPA-type glutamate receptors. The level of calcium in AIB interneurons is elevated for several minutes after odour removal, a neuronal correlate to the prolonged behavioural response to odour withdrawal. The AWC neuron inhibits AIY interneurons through glutamate-gated chloride channels; odour presentation relieves this inhibition and results in activation of AIY interneurons. The opposite regulation of AIY and AIB interneurons generates a coordinated behavioural response. Information processing by this circuit resembles information flow from vertebrate photoreceptors to 'OFF' bipolar and 'ON' bipolar neurons, indicating a conserved or convergent strategy for sensory information processing.
View details for DOI 10.1038/nature06292
View details for Web of Science ID 000250585800036
View details for PubMedID 17972877
Analysis of nematode mechanics by piezoresistive displacement clamp
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (44): 17376-17381
Studying animal mechanics is critical for understanding how signals in the neuromuscular system give rise to behavior and how force-sensing organs and sensory neurons work. Few techniques exist to provide forces and displacements appropriate for such studies. To address this technological gap, we developed a metrology using piezoresistive cantilevers as force-displacement sensors coupled to a feedback system to apply and maintain defined load profiles to micrometer-scale animals. We show that this system can deliver forces between 10(-8) and 10(-3) N across distances of up to 100 mum with a resolution of 12 nN between 0.1 Hz and 100 kHz. We use this new metrology to show that force-displacement curves of wild-type nematodes (Caenorhabditis elegans) are linear. Because nematodes have approximately cylindrical bodies, this finding demonstrates that nematode body mechanics can be modeled as a cylindrical shell under pressure. Little is known about the relative importance of hydrostatic pressure and shell mechanics, however. We show that dissipating pressure by cuticle puncture or decreasing it by hyperosmotic shock has only a modest effect on stiffness, whereas defects in the dpy-5 and lon-2 genes, which alter body shape and cuticle proteins, decrease and increase stiffness by 25% and 50%, respectively. This initial analysis of C. elegans body mechanics suggests that shell mechanics dominates stiffness and is a first step in understanding how body mechanics affect locomotion and force sensing.
View details for DOI 10.1073/pnas.0702138104
View details for Web of Science ID 000250638400028
View details for PubMedID 17962419
View details for PubMedCentralID PMC2077264
Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotranscluction channel alter gating and drug blockade
JOURNAL OF GENERAL PHYSIOLOGY
2007; 129 (2): 161-173
MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43-50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039-1042). To gain insight into the structure-function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit-subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.
View details for DOI 10.1085/jgp.200609672
View details for Web of Science ID 000244376400007
View details for PubMedID 17261841
WormBook : the online review of C. elegans biology
Wild C. elegans and other nematodes live in dirt and eat bacteria, relying on mechanoreceptor neurons (MRNs) to detect collisions with soil particles and other animals as well as forces generated by their own movement. MRNs may also help animals detect bacterial food sources. Hermaphrodites and males have 22 putative MRNs; males have an additional 46 MRNs, most, if not all of which are needed for mating. This chapter reviews key aspects of C. elegans mechanosensation, including MRN anatomy, what is known about their contributions to behavior as well as the neural circuits linking MRNs to movement. Emerging models of the mechanisms used to convert mechanical energy into electrical signals are also discussed. Prospects for future research include expanding our understanding of the molecular basis of mechanotransduction and how activation of MRNs guides and modulates behavior.
View details for PubMedID 18050466
The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals
2005; 8 (1): 43-50
Transformation of mechanical energy into ionic currents is essential for touch, hearing and nociception. Although DEG/ENaC proteins are believed to form sensory mechanotransduction channels, the evidence for this role remains indirect. By recording from C. elegans touch receptor neurons in vivo, we found that external force evokes rapidly activating mechanoreceptor currents (MRCs) carried mostly by Na(+) and blocked by amiloride-characteristics consistent with direct mechanical gating of a DEG/ENaC channel. Like mammalian Pacinian corpuscles, these neurons depolarized with both positive and negative changes in external force but not with sustained force. Null mutations in the DEG/ENaC gene mec-4 and in the accessory ion channel subunit genes mec-2 and mec-6 eliminated MRCs. In contrast, the genetic elimination of touch neuron-specific microtubules reduced, but did not abolish, MRCs. Our findings link the application of external force to the activation of a molecularly defined metazoan sensory transduction channel.
View details for DOI 10.1038/nn1362
View details for Web of Science ID 000225967600014
View details for PubMedID 15580270
Measurement of mechanical properties of Caenorhabditis elegans with a piezoresistive microcantilever system
3rd IEEE/EMBS Special Topic Conference on Microtechnology in Medicine and Biology
IEEE. 2005: 400–403
View details for Web of Science ID 000234204800125
- Molecules and mechanisms of mechanotransduction 34th Annual Meeting of the Society-for-Neuroscience SOC NEUROSCIENCE. 2004: 9220–22
Deconstructing C-elegans sensory mechanotransduction
2004; 306 (5695): 427-428
View details for Web of Science ID 000224626500036
Sensation is painless
TRENDS IN NEUROSCIENCES
2003; 26 (12): 643-645
Emily Dickinson declared: 'After great pain, a formal feeling comes'. This formal feeling begins when sensory neurons are activated by noxious stimuli, such as stepping on a tack. Recently, Seymour Benzer's group identified sensory neurons in Drosophila larvae that mediate aversive responses to noxious heat and mechanical stimuli. Thresholds for behavioral and nerve responses are elevated by mutations in the painless gene, which encodes a TRP ion channel protein. Painless thus joins an elite group of TRPs implicated in sensory transduction in insects, nematodes, mammals and fish.
View details for DOI 10.1016/j.tins.2003.09.013
View details for Web of Science ID 000187078200002
View details for PubMedID 14624845
Transducing touch in Caenorhabditis elegans
ANNUAL REVIEW OF PHYSIOLOGY
2003; 65: 429-452
Mechanosensation has been studied for decades, but understanding of its molecular mechanism is only now emerging from studies in Caenorhabditis elegans and Drosophila melanogaster. In both cases, the entry point proved to be genetic screens that allowed molecules needed for mechanosensation to be identified without any prior understanding of the likely components. In C. elegans, genetic screens revealed molecules needed for touch sensation along the body wall and other regions of force sensitivity. Members of two extensive membrane protein families have emerged as candidate sensory mechanotransduction channels: mec-4 and mec-10, which encode amiloride-sensitive channels (ASCs or DEG/ENaCs), and osm-9, which encodes a TRP ion channel. There are roughly 50 other members of these families whose functions in C. elegans are unknown. This article classifies these channels in C. elegans, with an emphasis on insights into their function derived from mutation. We also review the neuronal cell types in which these channels might be expressed and mediate mechanotransduction.
View details for DOI 10.1146/annurev.physiol.65.092101.142659
View details for Web of Science ID 000182523700019
View details for PubMedID 12524464
The mechanosensory protein MEC-6 is a subunit of the C-elegans touch-cell degenerin channel
2002; 420 (6916): 669-673
Mechanosensory transduction in touch receptor neurons is believed to be mediated by DEG/ENaC (degenerin/epithelial Na+ channel) proteins in nematodes and mammals. In the nematode Caenorhabditis elegans, gain-of-function mutations in the degenerin genes mec-4 and mec-10 (denoted mec-4(d) and mec-10(d), respectively) cause degeneration of the touch cells. This phenotype is completely suppressed by mutation in a third gene, mec-6 (refs 3, 4), that is needed for touch sensitivity. This last gene is also required for the function of other degenerins. Here we show that mec-6 encodes a single-pass membrane-spanning protein with limited similarity to paraoxonases, which are implicated in human coronary heart disease. This gene is expressed in muscle cells and in many neurons, including the six touch receptor neurons. MEC-6 increases amiloride-sensitive Na+ currents produced by MEC-4(d)/MEC-10(d) by approximately 30-fold, and functions synergistically with MEC-2 (a stomatin-like protein that regulates MEC-4(d)/MEC-10(d) channel activity) to increase the currents by 200-fold. MEC-6 physically interacts with all three channel proteins. In vivo, MEC-6 co-localizes with MEC-4, and is required for punctate MEC-4 expression along touch-neuron processes. We propose that MEC-6 is a part of the degenerin channel complex that may mediate mechanotransduction in touch cells.
View details for DOI 10.1038/nature01205
View details for Web of Science ID 000179751800046
View details for PubMedID 12478294
MEC-2 regulates C-elegans DEG/ENaC channels needed for mechanosensation
2002; 415 (6875): 1039-1042
Touch sensitivity in animals relies on nerve endings in the skin that convert mechanical force into electrical signals. In the nematode Caenorhabditis elegans, gentle touch to the body wall is sensed by six mechanosensory neurons that express two amiloride-sensitive Na+ channel proteins (DEG/ENaC). These proteins, MEC-4 and MEC-10, are required for touch sensation and can mutate to cause neuronal degeneration. Here we show that these mutant or 'd' forms of MEC-4 and MEC-10 produce a constitutively active, amiloride-sensitive ionic current when co-expressed in Xenopus oocytes, but not on their own. MEC-2, a stomatin-related protein needed for touch sensitivity, increased the activity of mutant channels about 40-fold and allowed currents to be detected with wild-type MEC-4 and MEC-10. Whereas neither the central, stomatin-like domain of MEC-2 nor human stomatin retained the activity of full-length MEC-2, both produced amiloride-sensitive currents with MEC-4d. Our findings indicate that MEC-2 regulates MEC-4/MEC-10 ion channels and raise the possibility that similar ion channels may be formed by stomatin-like proteins and DEG/ENaC proteins that are co-expressed in both vertebrates and invertebrates. Some of these channels may mediate mechanosensory responses.
View details for Web of Science ID 000174075000049
View details for PubMedID 11875573
Pressure polishing: a method for re-shaping patch pipettes during fire polishing
JOURNAL OF NEUROSCIENCE METHODS
2000; 100 (1-2): 13-15
The resolution of patch-clamp recordings is limited by the geometrical and electrical properties of patch pipettes. The ideal whole-cell patch pipette has a blunt, cone-shaped tip and a low resistance. The best glasses for making patch pipettes are low noise, low capacitance glasses such as borosilicate and aluminasilicate glasses. Regrettably, nearly all borosilicate glasses form pipettes with sharp, cone-shaped tips and relatively high resistance. It is possible, however, to reshape the tip during fire polishing by pressurizing the pipette lumen during fire polishing, a technique we call 'pressure polishing.' We find that this technique works with pipettes made from virtually any type of glass, including thick-walled aluminasilicate glass. We routinely use this technique to make pipettes suitable for whole-cell patch-clamp recording of tiny neurons (1-3 microm in diameter). Our pipettes are made from thick-walled, borosilicate glass and have submicron tip openings and resistances <10 MOmega. Similar pipettes could be used to record from subcellular neuronal structures such as axons, dendrites and dendritic spines. Pressure polishing should also be useful in patch-clamp applications that benefit from using pipettes with blunt tips, such as perforated-patch whole-cell recordings, low-noise single channel recordings and experiments that require internal perfusion of the pipette.
View details for Web of Science ID 000089039900002
View details for PubMedID 11040361
Active currents regulate sensitivity and dynamic range in C-elegans neurons
1998; 20 (4): 763-772
Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.
View details for Web of Science ID 000073346500014
View details for PubMedID 9581767
- Tight-seal whole-cell patch clamping of Caenorhabditis elegans neurons ION CHANNELS, PT B 1998; 293: 201-217
Variations in the ensemble of potassium currents underlying resonance in turtle hair cells
JOURNAL OF PHYSIOLOGY-LONDON
1996; 497 (2): 395-412
1. Potassium currents were characterized in turtle cochlear hair cells by whole-cell voltage clamp during superfusion with the potassium channel antagonists, tetraethylammonium (TEA) and 4-aminopyridine (4-AP). The estimated resonant frequency, f0, was inferred from tau, the time constant of deactivation of outward current upon repolarization to -50 mV, according to the empirical relation, f0 = k1 tau-1/2 + k2. 2. Dose-response relations for TEA and 4-AP were obtained by exposing single cells to ten concentrations exponentially distributed over four orders of magnitude. Potassium current in cells tuned to low frequencies was carried by a single class of channels with an apparent affinity constant, K1, for TEA of 35.9 mM. Half-blocking concentrations of 4-AP were correlated with the time constant of deactivation and varied between 26.2 and 102 microM. In cells tuned to higher frequencies, K+ current was carried by a single class of channels with high affinity for TEA (K1 = 0.215 mM) and low affinity for 4-AP (K1 = 12.3 mM). This pharmacological profile suggests that K+ current in low frequency cells is purely voltage gated and in high frequency cells, it is gated by both Ca2+ and voltage. 3. For each current type, the voltage dependence of activation was determined from tail current amplitude at -50 mV. The purely voltage-gated current, IK(V), was found to increase e-fold in 4.0 +/- 0.3 mV (n = 3) in low frequency cells exposed to TEA (25 mM). The Ca(2+)- and voltage-gated current, IK(Ca), was more steeply voltage dependent, increasing e-fold in 1.9 mV (n = 2) in high frequency cells exposed to 4-AP (0.8 mM). 4. IK(V) was found to inactivate slowly during prolonged voltage steps (approximately 10 s). Steady-state inactivation increased with depolarization from -70 mV and was incomplete such that on average IK(v) did not fall below approximately 0.39 of its maximum value. 5. Superfusion of 4-AP (0.8 mM) reversibly depolarized a low frequency cell and eliminated steady voltage oscillations, while TEA (6 mM) had no effect. In a high frequency cell, voltage oscillations were abolished by TEA, but not by 4-AP. 6. The differential pharmacology of IK(V) and IK(Ca) was used to measure their contribution to K+ current in cells tuned to different frequencies. Both currents exhibited a frequency-dependent increase in maximum conductance. IK(V) accounted for nearly all K+ current in cells tuned to less than 60 Hz, while IK(Ca) was the dominant current in higher frequency cells. 7. Mapping resonant frequency onto epithelial position suggests an exponential relation between K+ current size and position. IK(V) appeared to be limited to the apical or low frequency portion of the basilar papilla and coincided with maximal expression of a K(+)-selective inward rectifier, IK(IR). This finding is consistent with the notion that low frequency resonance is produced by interaction of IK(V) and IK(IR) with the voltage-gated Ca2+ current, ICa, and the cell's capacitance. The ionic events underlying higher frequency resonance are dominated by the action of IK(Ca) and ICa and include a contribution from IK(IR).
View details for Web of Science ID A1996VY21300008
View details for PubMedID 8961183
Positive feedback by a potassium-selective inward rectifier enhances tuning in vertebrate hair cells
1996; 71 (1): 430-442
Electrical resonance in vertebrate hair cells shapes receptor potentials and tunes each cell to a narrow band of frequencies. We have investigated the contribution of a potassium-selective inward rectifier (IR) to electrical resonance, isolating outward current carried by IR from other ionic currents active in the physiological voltage range (-75 to -30 mV) using a combination of potassium and calcium channel antagonists. IR expression is tightly regulated in the turtle's auditory epithelium, as revealed by the observation that its size declines systematically with resonant frequency. A critical feature of IR is the rapid inhibition produced by depolarization, which results in a negative slope in the steady-state current-voltage relation in the vicinity of the resting potential (-50 mV). The increasing block of outward current produced by depolarization is functionally equivalent to activating an inward current, suggesting that IR provides positive feedback and, in hair cells, serves an electrical function ordinarily reserved for voltage-dependent sodium and calcium currents. Additional support for this idea comes from the observation that superfusion with cesium selectively reduces IR and eliminates resonance in cells tuned to low frequencies and degrades resonant quality in cells tuned to more than 50 Hz.
View details for Web of Science ID A1996UV90800046
View details for PubMedID 8804626
- Ionic conductances and hair cell tuning in the turtle cochlea Conference on New Directions in Vestibular Research NEW YORK ACAD SCIENCES. 1996: 103–122
- A KINETIC DESCRIPTION OF THE CALCIUM-ACTIVATED POTASSIUM CHANNEL AND ITS APPLICATION TO ELECTRICAL TUNING OF HAIR-CELLS PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY 1995; 63 (2): 131-158
- RAPID-SCANNING CONFOCAL MICROSCOPY METHODS IN CELL BIOLOGY, VOL 38 1993; 38: 47-77
ACTIVATION OF THE INOSITOL TRISPHOSPHATE 2ND MESSENGER SYSTEM BY CAMP IN A MOUSE FIBROBLAST CELL-LINE
MOLECULAR AND CELLULAR BIOCHEMISTRY
1991; 101 (1): 43-49
Intracellular Ca2+ mobilization events were assessed in mouse L cells, which contain native prostaglandin E1 receptors and transfected human beta 2 adrenergic receptors. Both Fura2 (single cell measurements) and Quin 2, (cuvette assays) were used to determine [Ca2+]i levels. Our results demonstrate that in the transfected cells there is a dose-dependent increase in [Ca2+]i in response to isoproterenol (0.1 nM-100 nM), which is inhibited by the beta-adrenergic antagonist, propranolol, and is a result of intracellular Ca2+ release. [Ca2+]i in these cells was also increased by prostaglandin E1, 8 bromo cyclic AMP, and aluminum fluoride. Both 8 bromo cAMP and isoproterenol induced a rapid increase in the levels of IP1, IP2, and IP3. The data presented demonstrate that the elevation of intracellular cyclic AMP induces an increase in IP3 production which leads to an elevation in [Ca2+]i. We propose that this cyclic AMP dependent activation of the IP3 generating system occurs at a post-receptor site.
View details for Web of Science ID A1991EY40400005
View details for PubMedID 1849229
INOSITOL TRISPHOSPHATE MEDIATES CLONED MUSCARINIC RECEPTOR-ACTIVATED CONDUCTANCES IN TRANSFECTED MOUSE FIBROBLAST A9 L CELLS
JOURNAL OF PHYSIOLOGY-LONDON
1990; 421: 499-519
1. The mechanism by which cloned m1 and m3 muscarinic receptor subtypes activate Ca2+-dependent channels was investigated with whole-cell and cell-attached patch-clamp recording techniques and with Fura-2 Ca2+ indicator dye measurements in cultured A9 L cells transfected with rat m1 and m3 cDNAs. 2. The Ca2+-dependent K+ and Cl- currents induced by muscarinic receptor stimulation were dependent on GTP. Responses were reduced when GTP was excluded from the intracellular recording solution or when GDP-beta-S was added. Intracellular GTP-gamma-S activated spontaneous fluctuations and permitted only one acetylcholine-(ACh) induced current response. These results implicate GTP-binding proteins (G protein) in the signal transduction pathway. This G protein is probably not pertussis toxin-sensitive as the ACh-induced electrical response was not abolished by pertussis toxin treatment. 3. Cell-attached single-channel recordings revealed activation of ion channels within the patch during application of ACh outside the patch, implying that second messengers might be involved in the ACh-induced response. Two types of K+ channel were activated, a discrete channel of 36 pS and channel activity calculated to be about 5 pS. 4. Application of 8-bromo cyclic AMP or 1-oleoyl-1,2-acetylglycerol (OAG) produced no electrical response and did not affect the ACh-induced responses. Phorbol myristic acetate (PMA) evoked no electrical response, but reduced the ACh-induced responses. 5. Inclusion of inositol 1,4,5-trisphosphate (IP3) in the intracellular pipette solution activated outward currents at -50 mV associated with an increase in conductance. The IP3-induced current response reversed polarity at -65 mV and showed a dependence on K+. Increasing the intracellular free Ca2+ concentration ([Ca2+]i) from 20 nM to 1 microM also induced an outward current response associated with an increase in conductance. Inclusion of inositol 1,3,4,5-tetrakisphosphate (IP4) in the intracellular solution had no effect on the A9 L cells. 6. Fura-2 measurements revealed ACh-induced increases in Cai2+. The Ca2+ responses were abolished by atropine showing that they were muscarinic in nature. Removal of extracellular Ca2+ did not affect the initial ACh-induced increase in Cai2+ but subsequent Cai2+ responses to ACh were depressed, suggesting depletion of Ca2+ intracellular stores. Residual though small responses continued to be elicited by ACh. Barium (5 mM) had little effect and cobalt slightly reduced the ACh-induced Ca2+ response. 7. The ACh-induced currents recorded at -50 mV were unaffected by removal of extracellular Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
View details for Web of Science ID A1990CN26000029
View details for PubMedID 1693402
CALCIUM CURRENTS AND FURA-2 SIGNALS IN FLUORESCENCE-ACTIVATED CELL SORTED LACTOTROPHS AND SOMATOTROPHS OF RAT ANTERIOR-PITUITARY
1988; 123 (1): 611-621
Optical and electrical recording techniques were applied to single primary pituitary cells to characterize the types of voltage-dependent calcium currents (ICa) and levels of intracellular calcium ([Ca2+]i). GH-containing somatotrophs and PRL-containing lactotrophs were isolated from adult female rats using fluorescence-activated cell-sorting techniques and were maintained in culture for 1-4 days. Whole cell patch-clamp recordings were made to analyze the ICa, and [Ca2+]i was measured with fura-2. Cell type was verified after each recording by indirect immunocytochemistry. GH and PRL cells could be divided into two groups: silent and spontaneously active. Silent cells had stable membrane potentials and stable levels of [Ca2+]i. Spontaneously active cells exhibited spontaneous action potentials and large fluctuations in [Ca2+]i. Two types of ICa were found: a low threshold, transient current which was insensitive to the dihydropyridine -Bay 5417 (the negative isomer of Bay K 8644), and a high threshold, sustained current which was enhanced by -Bay 5417. Both types of ICa were present in PRL and GH cells, but each cell type differed quantitatively in the proportion of each current type. While the GH cells had a more prominent, low threshold, transient ICa, the PRL cells had a more prominent, high threshold, sustained ICa. The enhancement of ICa by -Bay 5417 was greater in the PRL cells, which have a larger dihydropyridine-sensitive ICa. Parallel fura-2 measurements showed an increase in [Ca2+]i in response to 50 mM KCl and -Bay 5417 for both lactotrophs and somatotrophs.
View details for Web of Science ID A1988P042000078
View details for PubMedID 2454814