Miriam B. Goodman

All Publications

• C. elegans touch receptor neurons direct mechanosensory complex organization via repurposing conserved basal lamina proteins. Current biology : CB Das, A., Franco, J. A., Mulcahy, B., Wang, L., Chapman, D., Jaisinghani, C., Pruitt, B. L., Zhen, M., Goodman, M. B. 2024

  Abstract

  The sense of touch is conferred by the conjoint function of somatosensory neurons and skin cells. These cells meet across a gap filled by a basal lamina, an ancient structure found in metazoans. Using Caenorhabditis elegans, we investigate the composition and ultrastructure of the extracellular matrix at the epidermis and touch receptor neuron (TRN) interface. We show that membrane-matrix complexes containing laminin, nidogen, and the MEC-4 mechano-electrical transduction channel reside at this interface and are central to proper touch sensation. Interestingly, the dimensions and spacing of these complexes correspond with the discontinuous beam-like extracellular matrix structures observed in serial-section transmission electron micrographs. These complexes fail to coalesce in touch-insensitive extracellular matrix mutants and in dissociated neurons. Loss of nidogen reduces the density of mechanoreceptor complexes and the amplitude of the touch-evoked currents they carry. Thus, neuron-epithelium cell interfaces are instrumental in mechanosensory complex assembly and function. Unlike the basal lamina ensheathing the pharynx and body wall muscle, nidogen recruitment to the puncta along TRNs is not dependent upon laminin binding. MEC-4, but not laminin or nidogen, is destabilized by point mutations in the C-terminal Kunitz domain of the extracellular matrix component, MEC-1. These findings imply that somatosensory neurons secrete proteins that actively repurpose the basal lamina to generate special-purpose mechanosensory complexes responsible for vibrotactile sensing.

  View details for DOI 10.1016/j.cub.2024.06.013

  View details for PubMedID 38964319

• A high-throughput behavioral screening platform for measuring chemotaxis by C. elegans. PLoS biology Fryer, E., Guha, S., Rogel-Hernandez, L. E., Logan-Garbisch, T., Farah, H., Rezaei, E., Mollhoff, I. N., Nekimken, A. L., Xu, A., Seyahi, L. S., Fechner, S., Druckmann, S., Clandinin, T. R., Rhee, S. Y., Goodman, M. B. 2024; 22 (6): e3002672

  Abstract

  Throughout history, humans have relied on plants as a source of medication, flavoring, and food. Plants synthesize large chemical libraries and release many of these compounds into the rhizosphere and atmosphere where they affect animal and microbe behavior. To survive, nematodes must have evolved the sensory capacity to distinguish plant-made small molecules (SMs) that are harmful and must be avoided from those that are beneficial and should be sought. This ability to classify chemical cues as a function of their value is fundamental to olfaction and represents a capacity shared by many animals, including humans. Here, we present an efficient platform based on multiwell plates, liquid handling instrumentation, inexpensive optical scanners, and bespoke software that can efficiently determine the valence (attraction or repulsion) of single SMs in the model nematode, Caenorhabditis elegans. Using this integrated hardware-wetware-software platform, we screened 90 plant SMs and identified 37 that attracted or repelled wild-type animals but had no effect on mutants defective in chemosensory transduction. Genetic dissection indicates that for at least 10 of these SMs, response valence emerges from the integration of opposing signals, arguing that olfactory valence is often determined by integrating chemosensory signals over multiple lines of information. This study establishes that C. elegans is an effective discovery engine for determining chemotaxis valence and for identifying natural products detected by the chemosensory nervous system.

  View details for DOI 10.1371/journal.pbio.3002672

  View details for PubMedID 38935621

• Mechanosensitive membrane proteins: Usual and unusual suspects in mediating mechanotransduction. The Journal of general physiology Goodman, M. B., Haswell, E. S., Vásquez, V. 2023; 155 (3)

  Abstract

  This Viewpoint, which accompanies a Special Issue focusing on membrane mechanosensors, discusses unifying and unique features of both established and emerging mechanosensitive (MS) membrane proteins, their distribution across protein families and phyla, and current and future challenges in the study of these important proteins and their partners. MS membrane proteins are essential for tissue development, cellular motion, osmotic homeostasis, and sensing external and self-generated mechanical cues like those responsible for touch and proprioception. Though researchers' attention and this Viewpoint focus on a few famous ion channels that are considered the usual suspects as MS mechanosensors, we also discuss some of the more unusual suspects, such as G-protein coupled receptors. As the field continues to grow, so too will the list of proteins suspected to function as mechanosensors and the diversity of known MS membrane proteins.

  View details for DOI 10.1085/jgp.202213248

  View details for PubMedID 36696153

• Visualizing Neurons Under Tension In Vivo with Optogenetic Molecular Force Sensors. Methods in molecular biology (Clifton, N.J.) Sanfeliu-Cerdán, N., Lin, L. C., Dunn, A. R., Goodman, M. B., Krieg, M. 2023; 2600: 239-266

  Abstract

  The visualization of mechanical stress distribution in specific molecular networks within a living and physiologically active cell or animal remains a formidable challenge in mechanobiology. The advent of fluorescence-resonance energy transfer (FRET)-based molecular tension sensors overcame a significant hurdle that now enables us to address previously technically limited questions. Here, we describe a method that uses genetically encoded FRET tension sensors to visualize the mechanics of cytoskeletal networks in neurons of living animals with sensitized emission FRET and confocal scanning light microscopy. This method uses noninvasive immobilization of living animals to image neuronal β-spectrin cytoskeleton at the diffraction limit, and leverages multiple imaging controls to verify and underline the quality of the measurements. In combination with a semiautomated machine-vision algorithm to identify and trace individual neurites, our analysis performs simultaneous calculation of FRET efficiencies and visualizes statistical uncertainty on a pixel by pixel basis. Our approach is not limited to genetically encoded spectrin tension sensors, but can also be used for any kind of ratiometric imaging in neuronal cells both in vivo and in vitro.

  View details for DOI 10.1007/978-1-0716-2851-5_16

  View details for PubMedID 36587102

• Image-based axon model highlights heterogeneity in initiation of damage. Biophysical journal Wang, L. M., Goodman, M. B., Kuhl, E. 2022

  Abstract

  Head injury simulations predict the occurrence of traumatic brain injury by placing a threshold on the calculated strains for axon tracts within the brain. However, a current roadblock to accurate injury prediction is the selection of an appropriate axon damage threshold. While several computational studies have used models of the axon cytoskeleton to investigate damage initiation, these models all employ an idealized, homogeneous axonal geometry. This homogeneous geometry with regularly spaced microtubules, evenly distributed throughout the model, overestimates axon strength because in reality, the axon cytoskeleton is heterogeneous. In the heterogeneous cytoskeleton, the weakest cross section determines the initiation of failure, but these weak spots are not present in a homogeneous model. Addressing one source of heterogeneity in the axon cytoskeleton, we present a new semi-automated image analysis pipeline for using serial section transmission electron micrographs (ssTEM) to reconstruct the microtubule geometry of an axon. The image analysis procedure locates microtubules within the images, traces them throughout the image stack, and reconstructs the microtubule structure as a finite element mesh. We demonstrate the image analysis approach using a C. elegans touch receptor neuron due to the availability of high-quality ssTEM datasets. The results of the analysis highlight the heterogeneity of the microtubule structure in the spatial variation of both microtubule number and length. Simulations comparing this image-based geometry to homogeneous geometries show that structural heterogeneity in the image-based model creates significant spatial variation in deformation. The homogeneous geometries, on the other hand, deform more uniformly. Since no single homogeneous model can replicate the mechanical behavior of the image-based model, our results argue that heterogeneity in axon microtubule geometry should be considered in determining accurate axon failure thresholds.

  View details for DOI 10.1016/j.bpj.2022.11.2946

  View details for PubMedID 36461640

• Engineering Bright and Mechanosensitive Alkaline-Earth Rare-Earth Upconverting Nanoparticles. The journal of physical chemistry letters McLellan, C. A., Siefe, C., Casar, J. R., Peng, C. S., Fischer, S., Lay, A., Parakh, A., Ke, F., Gu, X. W., Mao, W., Chu, S., Goodman, M. B., Dionne, J. A. 2022: 1547-1553

  Abstract

  Upconverting nanoparticles (UCNPs) are an emerging platform for mechanical force sensing at the nanometer scale. An outstanding challenge in realizing nanometer-scale mechano-sensitive UCNPs is maintaining a high mechanical force responsivity in conjunction with bright optical emission. This Letter reports mechano-sensing UCNPs based on the lanthanide dopants Yb3+ and Er3+, which exhibit a strong ratiometric change in emission spectra and bright emission under applied pressure. We synthesize and analyze the pressure response of five different types of nanoparticles, including cubic NaYF4 host nanoparticles and alkaline-earth host materials CaLuF, SrLuF, SrYbF, and BaLuF, all with lengths of 15 nm or less. By combining optical spectroscopy in a diamond anvil cell with single-particle brightness, we determine the noise equivalent sensitivity (GPa/√Hz) of these particles. The SrYb0.72Er0.28F@SrLuF particles exhibit an optimum noise equivalent sensitivity of 0.26 ± 0.04 GPa/√Hz. These particles present the possibility of robust nanometer-scale mechano-sensing.

  View details for DOI 10.1021/acs.jpclett.1c03841

  View details for PubMedID 35133831

• Reciprocal Interactions Between TGF-beta Signaling and Collagens - Insights from C. elegans. Developmental dynamics : an official publication of the American Association of Anatomists Goodman, M. B., Savage-Dunn, C. 2021

  Abstract

  Studies in genetically tractable organisms such as the nematode Caenorhabditis elegans have led to pioneering insights into conserved developmental regulatory mechanisms. For example, Smad signal transducers for the TGF-beta superfamily were first identified in C. elegans and in the fruit fly Drosophila. Recent studies of TGF-beta signaling and the extracellular matrix (ECM) in C. elegans have forged unexpected links between signaling and the ECM, yielding novel insights into the reciprocal interactions that occur across tissues and spatial scales, and potentially providing new opportunities for the study of biomechanical regulation of gene expression. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/dvdy.423

  View details for PubMedID 34537996

• DEG/ENaC/ASIC channels vary in their sensitivity to anti-hypertensive and non-steroidal anti-inflammatory drugs. The Journal of general physiology Fechner, S., D'Alessandro, I., Wang, L., Tower, C., Tao, L., Goodman, M. B. 2021; 153 (4)

  Abstract

  The degenerin channels, epithelial sodium channels, and acid-sensing ion channels (DEG/ENaC/ASICs) play important roles in sensing mechanical stimuli, regulating salt homeostasis, and responding to acidification in the nervous system. They have two transmembrane domains separated by a large extracellular domain and are believed to assemble as homomeric or heteromeric trimers. Based on studies of selected family members, these channels are assumed to form nonvoltage-gated and sodium-selective channels sensitive to the anti-hypertensive drug amiloride. They are also emerging as a target of nonsteroidal anti-inflammatory drugs (NSAIDs). Caenorhabditis elegans has more than two dozen genes encoding DEG/ENaC/ASIC subunits, providing an excellent opportunity to examine variations in drug sensitivity. Here, we analyze a subset of the C. elegans DEG/ENaC/ASIC proteins to test the hypothesis that individual family members vary not only in their ability to form homomeric channels but also in their drug sensitivity. We selected a panel of C. elegans DEG/ENaC/ASICs that are coexpressed in mechanosensory neurons and expressed gain-of-function or d mutants in Xenopus laevis oocytes. We found that only DEGT‑1d, UNC‑8d, and MEC‑4d formed homomeric channels and that, unlike MEC‑4d and UNC‑8d, DEGT‑1d channels were insensitive to amiloride and its analogues. As reported for rat ASIC1a, NSAIDs inhibit DEGT‑1d and UNC‑8d channels. Unexpectedly, MEC‑4d was strongly potentiated by NSAIDs, an effect that was decreased by mutations in the putative NSAID-binding site in the extracellular domain. Collectively, these findings reveal that not all DEG/ENaC/ASIC channels are amiloride-sensitive and that NSAIDs can both inhibit and potentiate these channels.

  View details for DOI 10.1085/jgp.202012655

  View details for PubMedID 33656557

• Nanoscale Structure and Mechanics of Skin in a C. elegans Model of Touch Sensation Rezaeil, E., Savage-Dunn, C., Goodman, M. B. CELL PRESS. 2021: 234A–235A

  View details for Web of Science ID 000629601401391

• Expansion microscopy of C. elegans. eLife Yu, C. J., Barry, N. C., Wassie, A. T., Sinha, A., Bhattacharya, A., Asano, S., Zhang, C., Chen, F., Hobert, O., Goodman, M. B., Haspel, G., Boyden, E. S. 2020; 9

  Abstract

  We recently developed expansion microscopy (ExM), which achieves nanoscale-precise imaging of specimens at ~70 nm resolution (with ~4.5x linear expansion) by isotropic swelling of chemically processed, hydrogel-embedded tissue. ExM of C. elegans is challenged by its cuticle, which is stiff and impermeable to antibodies. Here we present a strategy, expansion of C. elegans (ExCel), to expand fixed, intact C. elegans. ExCel enables simultaneous readout of fluorescent proteins, RNA, DNA location, and anatomical structures at resolutions of ~65-75 nm (3.3-3.8x linear expansion). We also developed epitope-preserving ExCel, which enables imaging of endogenous proteins stained by antibodies, and iterative ExCel, which enables imaging of fluorescent proteins after 20x linear expansion. We demonstrate the utility of the ExCel toolbox for mapping synaptic proteins, for identifying previously unreported proteins at cell junctions, and for gene expression analysis in multiple individual neurons of the same animal.

  View details for DOI 10.7554/eLife.46249

  View details for PubMedID 32356725

• The plant terpenoid carvone is a chemotaxis repellent for C. elegans. microPublication. Biology Ellington, C., Hayden, A., LaGrange, Z., Luccioni, M., Osman, M., Ramlan, L., Vogt, M., Guha, S., Goodman, M., O'Connell, L. 2020; 2020

  View details for DOI 10.17912/micropub.biology.000231

  View details for PubMedID 32550506

• Touch-induced Mechanical Strain in Somatosensory Neurons is Independent of Extracellular Matrix Mutations in C. elegans. Molecular biology of the cell Nekimken, A. L., Pruitt, B. L., Goodman, M. B. 2020: mbcE20010049

  Abstract

  Cutaneous mechanosensory neurons are activated by mechanical loads applied to the skin, and these stimuli are proposed to generate mechanical strain within sensory neurons. Using a microfluidic device to deliver controlled stimuli to intact animals and large, immobile, and fluorescent protein-tagged mitochondria as fiducial markers in the touch receptor neurons (TRNs), we visualized and measured touch-induced mechanical strain in C. elegans worms. At steady-state, touch stimuli sufficient to activate TRNs induce an average strain of 3.1% at the center of the actuator and this strain decays to near zero at the edges of the actuator. We also measured strain in animals carrying mutations affecting links between the extracellular matrix (ECM) and the TRNs but could not detect any differences in touch-induced mechanical strain between wild-type and mutant animals. Collectively, these results demonstrate that touching the skin induces local mechanical strain in intact animals and suggest that a fully intact ECM is not essential for transmitting mechanical strain from the skin to cutaneous mechanosensory neurons.

  View details for DOI 10.1091/mbc.E20-01-0049

  View details for PubMedID 32579427

• Alkaline-earth Rare-earth Upconverting Nanoparticles as Bio-compatible Mechanical Force Sensors McLellan, C. A., Siefe, C. P., Fischer, S., Casar, J. R., Swearer, D. F., Goodman, M. B., Dionne, J. A., IEEE IEEE. 2020

  View details for Web of Science ID 000612090003343

• Opportunities and challenges in achieving co-management in marine protected areas in East Africa: a comparative case study Journal of the Indian Ocean Region O'Leary, J. K., Goodman, M., Tuda, A., Machumu, M., West, L. 2020

  View details for DOI 10.1080/19480881.2020.1825201

• Parallel Processing of Two Mechanosensory Modalities by a Single Neuron in C.elegans. Developmental cell Tao, L., Porto, D., Li, Z., Fechner, S., Lee, S. A., Goodman, M. B., Xu, X. Z., Lu, H., Shen, K. 2019

  Abstract

  Neurons convert synaptic or sensory inputs into cellular outputs. It is not well understood how a single neuron senses, processes multiple stimuli, and generates distinct neuronal outcomes. Here, we describe the mechanism by which the C.elegans PVD neurons sense two mechanical stimuli: external touch and proprioceptive body movement. These two stimuli are detected by distinct mechanosensitive DEG/ENaC/ASIC channels, which trigger distinct cellular outputs linked to mechanonociception and proprioception. Mechanonociception depends on DEGT-1 and activates PVD's downstream command interneurons through its axon, while proprioception depends on DEL-1, UNC-8, and MEC-10 to induce local dendritic Ca2+ increase and dendritic release of a neuropeptide NLP-12. NLP-12 directly modulates neuromuscular junction activity through the cholecystokinin receptor homolog on motor axons, setting muscle tone and movement vigor. Thus, the same neuron simultaneously uses both its axon and dendrites as output apparatus to drive distinct sensorimotor outcomes.

  View details for DOI 10.1016/j.devcel.2019.10.008

  View details for PubMedID 31735664

• Progressive recruitment of distal MEC-4 channels determines touch response strength in C. elegans. The Journal of general physiology Katta, S., Sanzeni, A., Das, A., Vergassola, M., Goodman, M. B. 2019

  Abstract

  Touch deforms, or strains, the skin beyond the immediate point of contact. The spatiotemporal nature of the touch-induced strain fields depend on the mechanical properties of the skin and the tissues below. Somatosensory neurons that sense touch branch out within the skin and rely on a set of mechano-electrical transduction channels distributed within their dendrites to detect mechanical stimuli. Here, we sought to understand how tissue mechanics shape touch-induced mechanical strain across the skin over time and how individual channels located in different regions of the strain field contribute to the overall touch response. We leveraged Caenorhabditis elegans' touch receptor neurons as a simple model amenable to in vivo whole-cell patch-clamp recording and an integrated experimental-computational approach to dissect the mechanisms underlying the spatial and temporal dynamics we observed. Consistent with the idea that strain is produced at a distance, we show that delivering strong stimuli outside the anatomical extent of the neuron is sufficient to evoke MRCs. The amplitude and kinetics of the MRCs depended on both stimulus displacement and speed. Finally, we found that the main factor responsible for touch sensitivity is the recruitment of progressively more distant channels by stronger stimuli, rather than modulation of channel open probability. This principle may generalize to somatosensory neurons with more complex morphologies.

  View details for DOI 10.1085/jgp.201912374

  View details for PubMedID 31533952

• Somatosensory neurons integrate the geometry of skin deformation and mechanotransduction channels to shape touch sensing. eLife Sanzeni, A., Katta, S., Petzold, B., Pruitt, B. L., Goodman, M. B., Vergassola, M. 2019; 8

  Abstract

  Touch sensation hinges on force transfer across the skin and activation of mechanosensitive ion channels along the somatosensory neurons that invade the skin. This skin-nerve sensory system demands a quantitative model that spans the application of mechanical loads to channel activation. Unlike prior models of the dynamic responses of touch receptor neurons in Caenorhabditis elegans (Eastwood et al., 2015), which substituted a single effective channel for the ensemble along the TRNs, this study integrates body mechanics and the spatial recruitment of the various channels. We demonstrate that this model captures mechanical properties of the worm's body and accurately reproduces neural responses to simple stimuli. It also captures responses to complex stimuli featuring non-trivial spatial patterns, like extended or multiple contacts that could not be addressed otherwise. We illustrate the importance of these effects with new experiments revealing that skin-neuron composites respond to pre-indentation with increased currents rather than adapting to persistent stimulation.

  View details for DOI 10.7554/eLife.43226

  View details for PubMedID 31407662

• Optically Robust and Biocompatible Mechanosensitive Upconverting Nanoparticles ACS CENTRAL SCIENCE Lay, A., Sheppard, O. H., Siefe, C., McLellan, C. A., Mehlenbacher, R. D., Fischer, S., Goodman, M. B., Dionne, J. A. 2019; 5 (7): 1211–22

  View details for DOI 10.1021/acscentsci.9b00300

  View details for Web of Science ID 000476928300014

• Optically Robust and Biocompatible Mechanosensitive Upconverting Nanoparticles. ACS central science Lay, A., Sheppard, O. H., Siefe, C., McLellan, C. A., Mehlenbacher, R. D., Fischer, S., Goodman, M. B., Dionne, J. A. 2019; 5 (7): 1211-1222

  Abstract

  Upconverting nanoparticles (UCNPs) are promising tools for background-free imaging and sensing. However, their usefulness for in vivo applications depends on their biocompatibility, which we define by their optical performance in biological environments and their toxicity in living organisms. For UCNPs with a ratiometric color response to mechanical stress, consistent emission intensity and color are desired for the particles under nonmechanical stimuli. Here, we test the biocompatibility and mechanosensitivity of α-NaYF4:Yb,Er@NaLuF4 nanoparticles. First, we ligand-strip these particles to render them dispersible in aqueous media. Then, we characterize their mechanosensitivity (∼30% in the red-to-green spectral ratio per GPa), which is nearly 3-fold greater than those coated in oleic acid. We next design a suite of ex vivo and in vivo tests to investigate their structural and optical properties under several biorelevant conditions: over time in various buffers types, as a function of pH, and in vivo along the digestive tract of Caenorhabditis elegans worms. Finally, to ensure that the particles do not perturb biological function in C. elegans, we assess the chronic toxicity of nanoparticle ingestion using a reproductive brood assay. In these ways, we determine that mechanosensitive UCNPs are biocompatible, i.e., optically robust and nontoxic, for use as in vivo sensors to study animal digestion.

  View details for DOI 10.1021/acscentsci.9b00300

  View details for PubMedID 31403071

  View details for PubMedCentralID PMC6661856

• How Caenorhabditis elegans Senses Mechanical Stress, Temperature, and Other Physical Stimuli. Genetics Goodman, M. B., Sengupta, P. 2019; 212 (1): 25–51

  Abstract

  Caenorhabditis elegans lives in a complex habitat in which they routinely experience large fluctuations in temperature, and encounter physical obstacles that vary in size and composition. Their habitat is shared by other nematodes, by beneficial and harmful bacteria, and nematode-trapping fungi. Not surprisingly, these nematodes can detect and discriminate among diverse environmental cues, and exhibit sensory-evoked behaviors that are readily quantifiable in the laboratory at high resolution. Their ability to perform these behaviors depends on <100 sensory neurons, and this compact sensory nervous system together with powerful molecular genetic tools has allowed individual neuron types to be linked to specific sensory responses. Here, we describe the sensory neurons and molecules that enable C. elegans to sense and respond to physical stimuli. We focus primarily on the pathways that allow sensation of mechanical and thermal stimuli, and briefly consider this animal's ability to sense magnetic and electrical fields, light, and relative humidity. As the study of sensory transduction is critically dependent upon the techniques for stimulus delivery, we also include a section on appropriate laboratory methods for such studies. This chapter summarizes current knowledge about the sensitivity and response dynamics of individual classes of C. elegans mechano- and thermosensory neurons from in vivo calcium imaging and whole-cell patch-clamp electrophysiology studies. We also describe the roles of conserved molecules and signaling pathways in mediating the remarkably sensitive responses of these nematodes to mechanical and thermal cues. These studies have shown that the protein partners that form mechanotransduction channels are drawn from multiple superfamilies of ion channel proteins, and that signal transduction pathways responsible for temperature sensing in C. elegans share many features with those responsible for phototransduction in vertebrates.

  View details for PubMedID 31053616

• How Caenorhabditis elegans Senses Mechanical Stress, Temperature, and Other Physical Stimuli GENETICS Goodman, M. B., Sengupta, P. 2019; 212 (1): 25–51

  View details for DOI 10.1534/genetics.118.300241

  View details for Web of Science ID 000466802000003

• Funders should evaluate projects, not people LANCET Raymond, J. L., Goodman, M. B. 2019; 393 (10171): 494–95

  View details for Web of Science ID 000458184300013

• Funders should evaluate projects, not people. Lancet (London, England) Raymond, J. L., Goodman, M. B. 2019; 393 (10171): 494–95

  View details for PubMedID 30739667

• Synaptic Communication upon Gentle Touch. Neuron Fechner, S., Goodman, M. B. 2018; 100 (6): 1272–74

  Abstract

  Gentle touch sensation in mammals depends on synaptic transmission from primary sensory cells (Merkel cells) to secondary sensory neurons. Hoffman etal. (2018) identify norepinephrine and beta2-adrendergic receptors as the neurotransmitter-receptor pair responsible for sustained touch responses. The findings may deepen understanding of how drugs affect touch and pain sensation.

  View details for DOI 10.1016/j.neuron.2018.12.001

  View details for PubMedID 30571937

• Mechanosensitive upconverting nanoparticles for visualizing mechanical forces in vivo Lay, A., Siefe, C., Fischer, S., Mehlenbacher, R., Das, A., Nekimken, A., Ke, F., Mao, W., Pruitt, B., Cohen, B., Alivisatos, P., Goodman, M., Dionne, J. AMER CHEMICAL SOC. 2018

  View details for Web of Science ID 000447600003857

• The tactile receptive fields of freely moving Caenorhabditis elegans nematodes INTEGRATIVE BIOLOGY Mazzochette, E. A., Nekimken, A. L., Loizeau, F., Whitworth, J., Huynh, B., Goodman, M. B., Pruitt, B. L. 2018; 10 (8): 450–63

  View details for DOI 10.1039/c8ib00045j

  View details for Web of Science ID 000441400700002

• The tactile receptive fields of freely moving Caenorhabditis elegans nematodes. Integrative biology : quantitative biosciences from nano to macro Mazzochette, E. A., Nekimken, A. L., Loizeau, F., Whitworth, J., Huynh, B., Goodman, M. B., Pruitt, B. L. 2018

  Abstract

  Sensory neurons embedded in skin are responsible for the sense of touch. In humans and other mammals, touch sensation depends on thousands of diverse somatosensory neurons. By contrast, Caenorhabditis elegans nematodes have six gentle touch receptor neurons linked to simple behaviors. The classical touch assay uses an eyebrow hair to stimulate freely moving C. elegans, evoking evasive behavioral responses. This assay has led to the discovery of genes required for touch sensation, but does not provide control over stimulus strength or position. Here, we present an integrated system for performing automated, quantitative touch assays that circumvents these limitations and incorporates automated measurements of behavioral responses. The Highly Automated Worm Kicker (HAWK) unites a microfabricated silicon force sensor holding a glass bead forming the contact surface and video analysis with real-time force and position control. Using this system, we stimulated animals along the anterior-posterior axis and compared responses in wild-type and spc-1(dn) transgenic animals, which have a touch defect due to expression of a dominant-negative alpha-spectrin protein fragment. As expected from prior studies, delivering large stimuli anterior and posterior to the mid-point of the body evoked a reversal and a speed-up, respectively. The probability of evoking a response of either kind depended on stimulus strength and location; once initiated, the magnitude and quality of both reversal and speed-up behavioral responses were uncorrelated with stimulus location, strength, or the absence or presence of the spc-1(dn) transgene. Wild-type animals failed to respond when the stimulus was applied near the mid-point. These results show that stimulus strength and location govern the activation of a characteristic motor program and that the C. elegans body surface consists of two receptive fields separated by a gap.

  View details for PubMedID 30027970

• Immunofluorescence reveals neuron-specific promoter activity in non-neuronal cells. microPublication. Biology Lear, S. K., Das, A., Goodman, M. B. 2018; 2018

  View details for DOI 10.17912/8FDA-CK77

  View details for PubMedID 32550391

  View details for PubMedCentralID PMC7282515

• Loss of CaMKI function disrupts salt aversive learning in C. elegans. The Journal of neuroscience : the official journal of the Society for Neuroscience Lim, J. P., Fehlauer, H., Das, A., Saro, G., Glauser, D. A., Brunet, A., Goodman, M. B. 2018

  Abstract

  The ability to adapt behavior to environmental fluctuations is critical for survival of organisms ranging from invertebrates to mammals. Caenorhabditis elegans can learn to avoid sodium chloride when it is paired with starvation. This behavior may help animals avoid areas without food. While some genes have been implicated in this salt aversive learning behavior, critical genetic components, and the neural circuit in which they act, remain elusive. Here, we show that the sole worm ortholog of mammalian CaMKI/IV, CMK-1, is essential for salt aversive learning behavior in C. elegans hermaphrodites. We find that CMK-1 acts in the primary salt-sensing ASE neurons to regulate this behavior. By characterizing the intracellular calcium dynamics in ASE neurons using microfluidics, we find that loss of cmk-1 has subtle effects on sensory-evoked calcium responses in ASE axons and their modulation by salt conditioning. Our study implicates the expression of the conserved CaMKI/CMK-1 in chemosensory neurons as a regulator of behavioral plasticity to environmental salt in C. elegansSIGNIFICANCE STATEMENTLike other animals, the nematode Caenorhabditis elegans depends on salt for survival and navigates toward high concentrations of this essential mineral. Besides its role as an essential nutrient, salt also causes osmotic stress at high concentrations. A growing body of evidence indicates that C. elegans balances the requirement for salt with the danger it presents through a process called salt aversive learning. We show that this behavior depends on expression of a calcium/calmodulin-dependent kinase, CMK-1, in the ASE salt sensing neurons. Our study identifies CMK-1 and salt-sensitive chemosensory neurons as key factors in this form of behavioral plasticity.

  View details for PubMedID 29875264

• The extraordinary AFD thermosensor of C-elegans PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY Goodman, M. B., Sengupta, P. 2018; 470 (5): 839–49

  Abstract

  The nematode C. elegans exhibits complex thermal experience-dependent navigation behaviors in response to environmental temperature changes of as little as 0.01°C over a > 10°C temperature range. The remarkable thermosensory abilities of this animal are mediated primarily via the single pair of AFD sensory neurons in its head. In this review, we describe the contributions of AFD to thermosensory behaviors and temperature-dependent regulation of organismal physiology. We also discuss the mechanisms that enable this neuron type to adapt to recent temperature experience and to exhibit extraordinary thermosensitivity over a wide dynamic range.

  View details for PubMedID 29218454

  View details for PubMedCentralID PMC5945307

• Ultrasound Elicits Behavioral Responses through Mechanical Effects on Neurons and Ion Channels in a Simple Nervous System JOURNAL OF NEUROSCIENCE Kubanek, J., Shukla, P., Das, A., Baccus, S. A., Goodman, M. B. 2018; 38 (12): 3081–91

  Abstract

  Focused ultrasound has been shown to stimulate excitable cells, but the biophysical mechanisms behind this phenomenon remain poorly understood. To provide additional insight, we devised a behavioral-genetic assay applied to the well-characterized nervous system of Caenorhabditis elegans nematodes. We found that pulsed ultrasound elicits robust reversal behavior in wild-type animals in a pressure-, duration-, and pulse protocol-dependent manner. Responses were preserved in mutants unable to sense thermal fluctuations and absent in mutants lacking neurons required for mechanosensation. Additionally, we found that the worm's response to ultrasound pulses rests on the expression of MEC-4, a DEG/ENaC/ASIC ion channel required for touch sensation. Consistent with prior studies of MEC-4-dependent currents in vivo, the worm's response was optimal for pulses repeated 300-1000 times per second. Based on these findings, we conclude that mechanical, rather than thermal, stimulation accounts for behavioral responses. Further, we propose that acoustic radiation force governs the response to ultrasound in a manner that depends on the touch receptor neurons and MEC-4-dependent ion channels. Our findings illuminate a complete pathway of ultrasound action, from the forces generated by propagating ultrasound to an activation of a specific ion channel. The findings further highlight the importance of optimizing ultrasound pulsing protocols when stimulating neurons via ion channels with mechanosensitive properties.SIGNIFICANCE STATEMENT How ultrasound influences neurons and other excitable cells has remained a mystery for decades. Although it is widely understood that ultrasound can heat tissues and induce mechanical strain, whether or not neuronal activation depends on heat, mechanical force, or both physical factors is not known. We harnessed Caenorhabditis elegans nematodes and their extraordinary sensitivity to thermal and mechanical stimuli to address this question. Whereas thermosensory mutants respond to ultrasound similar to wild-type animals, mechanosensory mutants were insensitive to ultrasound stimulation. Additionally, stimulus parameters that accentuate mechanical effects were more effective than those producing more heat. These findings highlight a mechanical nature of the effect of ultrasound on neurons and suggest specific ways to optimize stimulation protocols in specific tissues.

  View details for DOI 10.1523/JNEUROSCI.1458-17.2018

  View details for Web of Science ID 000428156900016

  View details for PubMedID 29463641

  View details for PubMedCentralID PMC5864152

• Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Fehlauer, H., Nekimken, A. L., Kim, A. A., Pruitt, B. L., Goodman, M. B., Krieg, M. 2018

  Abstract

  One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

  View details for PubMedID 29553526

• Bright, Mechanosensitive Upconversion with Cubic-Phase Heteroepitaxial Core-Shell Nanoparticles. Nano letters Lay, A. n., Siefe, C. n., Fischer, S. n., Mehlenbacher, R. D., Ke, F. n., Mao, W. L., Alivisatos, A. P., Goodman, M. B., Dionne, J. A. 2018

  Abstract

  Lanthanide-doped nanoparticles are an emerging class of optical sensors, exhibiting sharp emission peaks, high signal-to-noise ratio, photostability, and a ratiometric color response to stress. The same centrosymmetric crystal field environment that allows for high mechanosensitivity in the cubic-phase (α), however, contributes to low upconversion quantum yield (UCQY). In this work, we engineer brighter mechanosensitive upconverters using a core-shell geometry. Sub-25 nm α-NaYF4:Yb,Er cores are shelled with an optically inert surface passivation layer of ∼4.5 nm thickness. Using different shell materials, including NaGdF4, NaYF4, and NaLuF4, we study how compressive to tensile strain influences the nanoparticles' imaging and sensing properties. All core-shell nanoparticles exhibit enhanced UCQY, up to 0.14% at 150 W/cm2, which rivals the efficiency of unshelled hexagonal-phase (β) nanoparticles. Additionally, strain at the core-shell interface can tune mechanosensitivity. In particular, the compressive Gd shell results in the largest color response from yellow-green to orange or, quantitatively, a change in the red to green ratio of 12.2 ± 1.2% per GPa. For all samples, the ratiometric readouts are consistent over three pressure cycles from ambient to 5 GPa. Therefore, heteroepitaxial shelling significantly improves signal brightness without compromising the core's mechano-sensing capabilities and further, promotes core-shell cubic-phase nanoparticles as upcoming in vivo and in situ optical sensors.

  View details for PubMedID 29927609

• Forces applied during classical touch assays for Caenorhabditis elegans PLOS ONE Nekimken, A. L., Mazzochette, E. A., Goodman, M. B., Pruitt, B. L. 2017; 12 (5)

  Abstract

  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 Nekimken, A. L., Fehlauer, H., Kim, A. A., Manosalvas-Kjono, S. N., Ladpli, P., Memon, F., Gopisetty, D., Sanchez, V., Goodman, M. B., Pruitt, B. L., Krieg, M. 2017

  Abstract

  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 Krieg, M., Stuehmer, J., Cueva, J. G., Fetter, R., Spilker, K., Cremers, D., Shen, K., Dunn, A. R., Goodman, M. B. 2017; 6

  View details for DOI 10.7554/eLife.20172

  View details for Web of Science ID 000394255800001

• Molecules empowering animals to sense and respond to temperature in changing environments CURRENT OPINION IN NEUROBIOLOGY Glauser, D. A., Goodman, M. B. 2016; 41: 92-98

  Abstract

  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 Lockhead, D., Schwarz, E. M., O'Hagan, R., Bellotti, S., Krieg, M., Barr, M. M., Dunn, A. R., Sternberg, P. W., Goodman, M. B. 2016; 27 (23): 3717-3728

  View details for DOI 10.1091/mbc.E16-06-0473

  View details for Web of Science ID 000389601600004

• Grabbing brain activity on the go. Proceedings of the National Academy of Sciences of the United States of America Clandinin, T. R., Goodman, M. B. 2016; 113 (8): 1965-7

  View details for DOI 10.1073/pnas.1524219113

  View details for PubMedID 26842834

  View details for PubMedCentralID PMC4776519

• Tissue mechanics govern the rapidly adapting and symmetrical response to touch PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Eastwood, A. L., Sanzeni, A., Petzold, B. C., Park, S., Vergassola, M., Pruitt, B. L., Goodman, M. B. 2015; 112 (50): E6955-E6963

  Abstract

  Interactions with the physical world are deeply rooted in our sense of touch and depend on ensembles of somatosensory neurons that invade and innervate the skin. Somatosensory neurons convert the mechanical energy delivered in each touch into excitatory membrane currents carried by mechanoelectrical transduction (MeT) channels. Pacinian corpuscles in mammals and touch receptor neurons (TRNs) in Caenorhabditis elegans nematodes are embedded in distinctive specialized accessory structures, have low thresholds for activation, and adapt rapidly to the application and removal of mechanical loads. Recently, many of the protein partners that form native MeT channels in these and other somatosensory neurons have been identified. However, the biophysical mechanism of symmetric responses to the onset and offset of mechanical stimulation has eluded understanding for decades. Moreover, it is not known whether applied force or the resulting indentation activate MeT channels. Here, we introduce a system for simultaneously recording membrane current, applied force, and the resulting indentation in living C. elegans (Feedback-controlled Application of mechanical Loads Combined with in vivo Neurophysiology, FALCON) and use it, together with modeling, to study these questions. We show that current amplitude increases with indentation, not force, and that fast stimuli evoke larger currents than slower stimuli producing the same or smaller indentation. A model linking body indentation to MeT channel activation through an embedded viscoelastic element reproduces the experimental findings, predicts that the TRNs function as a band-pass mechanical filter, and provides a general mechanism for symmetrical and rapidly adapting MeT channel activation relevant to somatosensory neurons across phyla and submodalities.

  View details for DOI 10.1073/pnas.1514138112

  View details for Web of Science ID 000366404200018

  View details for PubMedID 26627717

  View details for PubMedCentralID PMC4687575

• Feeling force: physical and physiological principles enabling sensory mechanotransduction. Annual review of cell and developmental biology Katta, S., Krieg, M., Goodman, M. B. 2015; 31: 347-71

  Abstract

  Organisms as diverse as microbes, roundworms, insects, and mammals detect and respond to applied force. In animals, this ability depends on ionotropic force receptors, known as mechanoelectrical transduction (MeT) channels, that are expressed by specialized mechanoreceptor cells embedded in diverse tissues and distributed throughout the body. These cells mediate hearing, touch, and proprioception and play a crucial role in regulating organ function. Here, we attempt to integrate knowledge about the architecture of mechanoreceptor cells and their sensory organs with principles of cell mechanics, and we consider how engulfing tissues contribute to mechanical filtering. We address progress in the quest to identify the proteins that form MeT channels and to understand how these channels are gated. For clarity and convenience, we focus on sensory mechanobiology in nematodes, fruit flies, and mice. These themes are emphasized: asymmetric responses to applied forces, which may reflect anisotropy of the structure and mechanics of sensory mechanoreceptor cells, and proteins that function as MeT channels, which appear to have emerged many times through evolution.

  View details for DOI 10.1146/annurev-cellbio-100913-013426

  View details for PubMedID 26566115

• Mechanical systems biology of C. elegans touch sensation. BioEssays Krieg, M., Dunn, A. R., Goodman, M. B. 2015; 37 (3): 335-344

  Abstract

  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. eLife Kelley, M., Yochem, J., Krieg, M., Calixto, A., Heiman, M. G., Kuzmanov, A., Meli, V., Chalfie, M., Goodman, M. B., Shaham, S., Frand, A., Fay, D. S. 2015; 4

  Abstract

  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

  View details for PubMedCentralID PMC4395870

• Feeling Force: Physical and Physiological Principles Enabling Sensory Mechanotransduction ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY, VOL 31 Katta, S., Krieg, M., Goodman, M. B. 2015; 31: 347-371

  View details for DOI 10.1146/annurev-cellbio-100913-013426

  View details for Web of Science ID 000367292400016

• CaMKI-Dependent Regulation of Sensory Gene Expression Mediates Experience-Dependent Plasticity in the Operating Range of a Thermosensory Neuron NEURON Yu, Y. V., Bell, H. W., Glauser, D. A., Van Hooser, S. D., Goodman, M. B., Sengupta, P. 2014; 84 (5): 919-926

  Abstract

  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

  View details for PubMedCentralID PMC4258139

• The Balance between Cytoplasmic and Nuclear CaM Kinase-1 Signaling Controls the Operating Range of Noxious Heat Avoidance NEURON Schild, L. C., Zbinden, L., Bell, H. W., Yu, Y. V., Sengupta, P., Goodman, M. B., Glauser, D. A. 2014; 84 (5): 983-996

  Abstract

  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. Current biology Vásquez, V., Scherrer, G., Goodman, M. B. 2014; 24 (12): R566-9

  Abstract

  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 Krieg, M., Dunn, A. R., Goodman, M. B. 2014; 16 (3): 224-233

  Abstract

  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 Krieg, M., Dunn, A. R., Goodman, M. B. 2014; 16 (3): 224-233

  Abstract

  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

• 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 Luo, L., Cook, N., Venkatachalam, V., Martinez-Velazquez, L. A., Zhang, X., Calvo, A. C., Hawk, J., MacInnis, B. L., Frank, M., Ng, J. H., Klein, M., Gershow, M., Hammarlund, M., Goodman, M. B., Colon-Ramos, D. A., Zhang, Y., Samuel, A. D. 2014; 111 (7): 2776-2781

  Abstract

  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. Cell reports Vásquez, V., Krieg, M., Lockhead, D., Goodman, M. B. 2014; 6 (1): 70-80

  Abstract

  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

• PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in Caenorhabditis elegans neurons. eLife Richardson, C. E., Spilker, K. A., Cueva, J. G., Perrino, J., Goodman, M. B., Shen, K. 2014; 3

  Abstract

  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

  View details for PubMedCentralID PMC3932522

• PTRN-1, a microtubule minus end-binding CAMSAP homolog, promotes microtubule function in Caenorhabditis elegans neurons. eLife Richardson, C. E., Spilker, K. A., Cueva, J. G., Perrino, J., Goodman, M. B., Shen, K. 2014; 3: e01498

  Abstract

  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

  View details for PubMedCentralID PMC3932522

• Thermotaxis navigation behavior. WormBook : the online review of C. elegans biology Goodman, M. B., Klein, M., Lasse, S., Luo, L., Mori, I., Samuel, A., Sengupta, P., Wang, D. 2014: 1-10

  Abstract

  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

• Assaying mechanosensation. WormBook : the online review of C. elegans biology Chalfie, M. 2014: 1-13

  Abstract

  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

• 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 Wang, D., O'Halloran, D., Goodman, M. B. 2013; 142 (4): 437-449

  Abstract

  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

  View details for PubMedCentralID PMC3787776

• Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis PLOS GENETICS Frederic, M. Y., Lundin, V. F., Whiteside, M. D., Cueva, J. G., Tu, D. K., Kang, S. Y., Singh, H., Baillie, D. L., Hutter, H., Goodman, M. B., Brinkman, F. S., Leroux, M. R. 2013; 9 (10)

  Abstract

  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

• MEMS-based force-clamp analysis of the role of body stiffness in C. elegans touch sensation. Integrative biology Petzold, B. C., Park, S., Mazzochette, E. A., Goodman, M. B., Pruitt, B. L. 2013; 5 (6): 853-864

  Abstract

  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 Bellanger, J., Cueva, J. G., Baran, R., Tang, G., Goodman, M. B., Debant, A. 2012; 125 (22): 5417-5427

  Abstract

  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 PHYSIOLOGY Eastwood, A. L., Goodman, M. B. 2012; 27 (5): 282-290

  Abstract

  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 CURRENT BIOLOGY Cueva, J. G., Hsin, J., Huang, K. C., Goodman, M. B. 2012; 22 (12): 1066-1074

  Abstract

  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

  View details for PubMedCentralID PMC3670109

• How We Feel: Ion Channel Partnerships that Detect Mechanical Inputs and Give Rise to Touch and Pain Perception NEURON Geffeney, S. L., Goodman, M. B. 2012; 74 (4): 609-619

  Abstract

  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

  View details for PubMedCentralID PMC3371091

• Electrophysiological Methods for Caenorhabditis elegans Neurobiology CAENORHABDITIS ELEGANS: CELL BIOLOGY AND PHYSIOLOGY, SECOND EDITION Goodman, M. B., Lindsay, T. H., Lockery, S. R., Richmond, J. E. 2012; 107: 409-436

  Abstract

  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 Glauser, D. A., Johnson, B. E., Aldrich, R. W., Goodman, M. B. 2011; 108 (51): 20790-20795

  Abstract

  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

  View details for PubMedCentralID PMC3251113

• Alternatively spliced domains interact to regulate BK potassium channel gating PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johnson, B. E., Glauser, D. A., Dan-Glauser, E. S., Halling, D. B., Aldrich, R. W., Goodman, M. B. 2011; 108 (51): 20784-20789

  Abstract

  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

  View details for PubMedCentralID PMC3251094

• DEG/ENaC but Not TRP Channels Are the Major Mechanoelectrical Transduction Channels in a C. elegans Nociceptor NEURON Geffeney, S. L., Cueva, J. G., Glauser, D. A., Doll, J. C., Lee, T. H., Montoya, M., Karania, S., Garakani, A. M., Pruitt, B. L., Goodman, M. B. 2011; 71 (5): 845-857

  Abstract

  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

  View details for PubMedCentralID PMC3170654

• The DEG/ENaC Protein MEC-10 Regulates the Transduction Channel Complex in Caenorhabditis elegans Touch Receptor Neurons JOURNAL OF NEUROSCIENCE Arnadottir, J., O'Hagan, R., Chen, Y., Goodman, M. B., Chalfie, M. 2011; 31 (35): 12695-12704

  Abstract

  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 GENETICS Glauser, D. A., Chen, W. C., Agin, R., MacInnis, B. L., Hellman, A. B., Garrity, P. A., Tan, M., Goodman, M. B. 2011; 188 (1): 91-U150

  Abstract

  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

  View details for PubMedCentralID PMC3120139

• Caenorhabditis elegans Body Mechanics Are Regulated by Body Wall Muscle Tone BIOPHYSICAL JOURNAL Petzold, B. C., Park, S., Ponce, P., Roozeboom, C., Powell, C., Goodman, M. B., Pruitt, B. L. 2011; 100 (8): 1977-1985

  Abstract

  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 Park, S., Petzold, B. C., Goodman, M. B., Pruitt, B. L. 2011; 82 (4)

  Abstract

  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 Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B., Nachury, M. V. 2010; 107 (50): 21517-21522

  Abstract

  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

  View details for PubMedCentralID PMC3003046

• Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis in C. elegans and Drosophila GENES & DEVELOPMENT Garrity, P. A., Goodman, M. B., Samuel, A. D., Sengupta, P. 2010; 24 (21): 2365-2382

  Abstract

  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 NEURON Klassen, M. P., Wu, Y. E., Maeder, C. I., Nakae, I., Cueva, J. G., Lehrman, E. K., Tada, M., Gengyo-Ando, K., Wang, G. J., Goodman, M., Mitani, S., Kontani, K., Katada, T., Shen, K. 2010; 66 (5): 710-723

  Abstract

  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 Glauser, D. A., Goodman, M. B. 2010; 13 (5): 528-529

  View details for DOI 10.1038/nn0510-528

  View details for Web of Science ID 000277065700005

  View details for PubMedID 20421897

• The Dystrophin Complex Controls BK Channel Localization and Muscle Activity in Caenorhabditis elegans PLOS GENETICS Kim, H., Pierce-Shimomura, J. T., Oh, H. J., Johnson, B. E., Goodman, M. B., McIntire, S. L. 2009; 5 (12)

  Abstract

  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

  View details for PubMedCentralID PMC2788698

• SU-8 force sensing pillar arrays for biological measurements LAB ON A CHIP Doll, J. C., Harjee, N., Klejwa, N., Kwon, R., Coulthard, S. M., Petzold, B., Goodman, M. B., Pruitt, B. L. 2009; 9 (10): 1449-1454

  Abstract

  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

  View details for PubMedCentralID PMC2818990

• The quest for action potentials in C. elegans neurons hits a plateau NATURE NEUROSCIENCE Lockery, S. R., Goodman, M. B. 2009; 12 (4): 377-378

  Abstract

  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) Park, S., Petzold, B., Goodman, M. B., Pruitt, B. L. 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 Ramot, D., MacInnis, B. L., Lee, H., Goodman, M. B. 2008; 28 (47): 12546-12557

  Abstract

  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 Hueston, J. L., Herren, G. P., Cueva, J. G., Buechner, M., Lundquist, E. A., Goodman, M. B., Suprenant, K. A. 2008; 8

  Abstract

  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 NATURE NEUROSCIENCE Ramot, D., MacInnis, B. L., Goodman, M. B. 2008; 11 (8): 908-915

  Abstract

  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

  View details for PubMedCentralID PMC2587641

• Artificial dirt: Microfluidic substrates for nematode neurobiology and behavior JOURNAL OF NEUROPHYSIOLOGY Lockery, S. R., Lawton, K. J., Doll, J. C., Faumont, S., COULTHARD, S. M., Thiele, T. R., Chronis, N., McCormick, K. E., Goodman, M. B., Pruitt, B. L. 2008; 99 (6): 3136-3143

  Abstract

  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 Brown, A. L., Liao, Z., Goodman, M. B. 2008; 131 (6): 605-616

  Abstract

  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

  View details for PubMedCentralID PMC2391253

• The Parallel Worm Tracker: A Platform for Measuring Average Speed and Drug-Induced Paralysis in Nematodes PLOS ONE Ramot, D., Johnson, B. E., Berry, T. L., Carnell, L., Goodman, M. B. 2008; 3 (5)

  Abstract

  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

  View details for PubMedCentralID PMC2373883

• Patch clamp recording of ion channels expressed in Xenopus oocytes. Journal of visualized experiments : JoVE Brown, A. L., Johnson, B. E., Goodman, M. B. 2008

  Abstract

  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

  View details for PubMedCentralID PMC3234036

• Making patch-pipettes and sharp electrodes with a programmable puller. Journal of visualized experiments : JoVE Brown, A. L., Johnson, B. E., Goodman, M. B. 2008

  Abstract

  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

  View details for PubMedCentralID PMC3233862

• Pressure-polishing pipettes for improved patch-clamp recording. Journal of visualized experiments : JoVE Johnson, B. E., Brown, A. L., Goodman, M. B. 2008

  Abstract

  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

  View details for PubMedCentralID PMC3234038

• Nanoscale organization of the MEC-4 DEG/ENaC sensory mechanotransduction channel in Caenorhabditis elegans touch receptor neurons JOURNAL OF NEUROSCIENCE Cueva, J. G., Mulholland, A., Goodman, M. B. 2007; 27 (51): 14089-14098

  Abstract

  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 NATURE Chalasani, S. H., Chronis, N., Tsunozaki, M., Gray, J. M., Ramot, D., Goodman, M. B., Bargmann, C. I. 2007; 450 (7166): 63-?

  Abstract

  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 Park, S., Goodman, M. B., Pruitt, B. L. 2007; 104 (44): 17376-17381

  Abstract

  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 Brown, A. L., Fernandez-Illescas, S. M., Liao, Z., Goodman, M. B. 2007; 129 (2): 161-173

  Abstract

  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

  View details for PubMedCentralID PMC2154353

• Mechanosensation. WormBook : the online review of C. elegans biology Goodman, M. B. 2006: 1-14

  Abstract

  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 NATURE NEUROSCIENCE O'Hagan, R., Chalfie, M., Goodman, M. B. 2005; 8 (1): 43-50

  Abstract

  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 Park, S. J., Goodman, M. B., Pruitt, B. L. 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 Goodman, M. B., Lumpkin, E. A., Ricci, A., Tracey, W. D., Kernan, M., Nicolson, T. SOC NEUROSCIENCE. 2004: 9220–22

  View details for DOI 10.1523/JNEUROSCI.3342-04.2004

  View details for PubMedID 15496654

• Deconstructing C-elegans sensory mechanotransduction SCIENCE Goodman, M. B. 2004; 306 (5695): 427-428

  View details for Web of Science ID 000224626500036

• Eppendorf essay winner. Deconstructing C. elegans sensory mechanotransduction. Science (New York, N.Y.) Goodman, M. B. 2004; 306 (5695): 427-8

  View details for DOI 10.1126/science.1105624

  View details for PubMedID 15486284

• Sensation is painless TRENDS IN NEUROSCIENCES Goodman, M. B. 2003; 26 (12): 643-645

  Abstract

  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 Goodman, M. B., Schwarz, E. M. 2003; 65: 429-452

  Abstract

  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 NATURE Chelur, D. S., Ernstrom, G. G., Goodman, M. B., Yao, C. A., Chen, L., O'Hagan, R., Chalfie, M. 2002; 420 (6916): 669-673

  Abstract

  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 NATURE Goodman, M. B., Ernstrom, G. G., Chelur, D. S., O'Hagan, R., Yao, C. A., Chalfie, M. 2002; 415 (6875): 1039-1042

  Abstract

  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 Goodman, M. B., Lockery, S. R. 2000; 100 (1-2): 13-15

  Abstract

  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 NEURON Goodman, M. B., Hall, D. H., Avery, L., Lockery, S. R. 1998; 20 (4): 763-772

  Abstract

  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 Lockery, S. R., Goodman, M. B. 1998; 293: 201-217

  View details for Web of Science ID 000075762300013

  View details for PubMedID 9711611

• Variations in the ensemble of potassium currents underlying resonance in turtle hair cells JOURNAL OF PHYSIOLOGY-LONDON Goodman, M. B., Art, J. J. 1996; 497 (2): 395-412

  Abstract

  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 BIOPHYSICAL JOURNAL Goodman, M. B., Art, J. J. 1996; 71 (1): 430-442

  Abstract

  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 Art, J. J., Goodman, M. B. NEW YORK ACAD SCIENCES. 1996: 103–122

  View details for Web of Science ID A1996BF92G00009

  View details for PubMedID 8694408

• A KINETIC DESCRIPTION OF THE CALCIUM-ACTIVATED POTASSIUM CHANNEL AND ITS APPLICATION TO ELECTRICAL TUNING OF HAIR-CELLS PROGRESS IN BIOPHYSICS & MOLECULAR BIOLOGY Wu, Y. C., Art, J. J., Goodman, M. B., Fettiplace, R. 1995; 63 (2): 131-158

  View details for Web of Science ID A1995RC14300002

  View details for PubMedID 7624477

• RAPID-SCANNING CONFOCAL MICROSCOPY METHODS IN CELL BIOLOGY, VOL 38 Art, J. J., Goodman, M. B. 1993; 38: 47-77

  View details for Web of Science ID A1993BZ58T00002

  View details for PubMedID 8246787

• ACTIVATION OF THE INOSITOL TRISPHOSPHATE 2ND MESSENGER SYSTEM BY CAMP IN A MOUSE FIBROBLAST CELL-LINE MOLECULAR AND CELLULAR BIOCHEMISTRY HORN, V. J., Sheehy, P. A., Goodman, M. B., Ambudkar, I. S. 1991; 101 (1): 43-49

  Abstract

  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 Jones, S. V., Barker, J. L., Goodman, M. B., Brann, M. R. 1990; 421: 499-519

  Abstract

  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 ENDOCRINOLOGY Lewis, D. L., Goodman, M. B., STJOHN, P. A., Barker, J. L. 1988; 123 (1): 611-621

  Abstract

  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