Miriam B. Goodman - US grants

The ability to detect mechanical energy by activation of specialized ion channels, termed mechano-gated ion channels, is shared by all cellular organisms and underlies the mechanical senses of touch, hearing, and balance. The molecules responsible for these events are not known in any eukaryote. Twelve candidates have been identified by genetic analysis of the response to gentle touch in Caenorhabditis elegans, including three genes (mec-4, mec-6 and mec-10) postulated to encode subunits of the mechano-gated ion channel. Genetic analyses have fostered a detailed model for channel activation which I propose to test using whole-cell electrical recording. Mechano-gated ion currents will be measured in wild-type C. elegans in order to characterize their mechanical sensitivity, ion permeability, and pharmacology. The importance of individual gene products will be studied by measuring mechano-gated ion currents n animals that carry null alleles of the putative ion channel subunits. the effects of defects in genes encoding components of the proposed subcellular scaffold will also be examined. Finally, experiments involving in vitro mutagenesis of putative ion channel subunits and analysis of functional effects by in vivo expression are proposed to identify proteins contributing to the ion pore. The implications of this work lie in the possibility of delineating the molecular components of eukaryotic mechanosensory apparatus and of analyzing their function biophysically in a genetically tractable organism, the nematode C. elegans.

DESCRIPTION (provided by applicant): The long-term objective of the proposed work is to understand mechanisms of mechanotransduction. The ability to detect mechanical energy is essential for our senses of touch, hearing, and balance as well as for on-going regulation of posture, osmotic balance, and blood pressure. In recent years, classical and forward genetic approaches in nematodes, fruit flies, zebra fish, and mice have yielded a small, but expanding list of proteins needed for touch and hearing. However, it is not clear whether or not all of these proteins function in sensory mechanotransduction or, if so, how they might act in concert to convert mechanical energy into electrical signals in neurons. We propose to investigate the molecular physiology of touch-sensitive neurons in the nematode Caenorhabditis elegans. For several reasons, C. elegans is a nearly ideal animal for this research. Now, extensive collections of touch-insensitive mutants, powerful molecular-genetic tools, and an unparalleled description of nervous system anatomy, co-exist with the ability to record electrical signals from single, identified mechanosensory neurons. Newly discovered parallels in the physiology of C. elegans vertebrate touch-sensitive neurons significantly increase the value of C. elegans as a model system. In this proposal, we focus on two classes of mechanosensory neurons (nonciliated PLM neurons and ciliated ASH neurons). Electrophysiological recordings will be made from PLM and ASH in normal animals to determine how mechanotransduction differs in these distinct classes of mechanosensory cells (Aims #1A, 3A). We will determine the cellular function of proteins predicted to form sensory transduction channels and voltage-gated K channels by comparing sensory responses in wild type and mutant cells (AIMS 1A, 3B, & 3C). To investigate how cellular architecture contributes to force transfer, we will also record from mutant animals with defects in extracellular and intracellular structures (Aim 1B). Finally, we will express channels needed for PLM function in heterologous cells and determine how known accessory proteins regulate single channel activity (Aim 2). What is learned from these studies will clarify basic mechanisms of mechanotransduction and could improve the diagnosis and treatment of sensory neuropathies associated with both inherited and acquired diseases.

Body temperature has profound influences on all aspects of animal biology. All animals, including humans, have the ability to regulate their body temperature by moving to favorable areas in the environment. This behavioral thermoregulation is thought to involve thermosensory neurons in the skin, an internal set point and complex feedback. A major challenge in understanding behavioral thermoregulation is to identify animals amenable to mechanistic analysis. With only 302 nerve cells (neurons), the roundworm C. elegans is an almost perfect organism for such studies. It is the only animal in which the shape of every neuron and its connections to other neurons is known (the wiring diagram). A sub-circuit of 11 bilaterallysymmetric neurons mediate initial responses to temperature. All of these neurons can be identified in living animals, including a pair of neurons that sense cooling and warming (the AFD cells) and are critical for thermotaxis.

In C. elegans, but not in mammals, it is possible to directly measure how these neurons respond to temperature. The proposed research leverages these advantages of C. elegans and takes an integrated approach, analyzing temperature sensation at the molecular and cellular level. Experiments are proposed to deconstruct the molecular networks that make temperature sensation possible and to discover genes responsible for the development or function of neurons that link sensation to behavior. Because the wiring diagram is known, future experiments can investigate the neural circuit linking temperature sensation by AFD to motor output. In C. elegans, the AFD neurons are critical for successful thermotaxis. Other nematodes, which are parasites of agriculturally important plants and animals, are thought to use thermotaxis as part of a host-finding strategy. Some of these parasitic nematodes are known to have neurons similar to AFD, suggesting that the proposed research could have broader implications. In particular, what is learned about the molecular networks responsible for temperature could provide entry points for new research into methods for controlling nematode species that threaten agriculture.

The intellectual merit of this work lies in the establishment of a new animal model for mechanistic analysis of temperature sensation and its behavioral consequences. It has the potential to offer insight into universal molecular, cellular and network-level mechanisms by which sensory information provides essential, real-time feedback for movement. The work derives broad impact from its multidisciplinary approach, combining the experimental and conceptual tools of genetics, cell physiology, and behavioral studies. Since its inception, high school students and undergraduates have contributed to the work, including women and minority participants in the Stanford Summer Research Program. Research opportunities for undergraduates will continue to be an integral part of the proposed work. Additionally, the PI encourages lab members to participate in community outreach activities and provides release time for this important work.

[unreadable] DESCRIPTION (provided by applicant): Body temperature has profound influences on all aspects of animal biology. Mammals have the ability to control body temperature by adjusting their physiology (e.g. non-shivering thermogenesis, perspiration) and by behavioral measures (e.g. huddling, locating areas of more favorable temperatures or thermotaxis). This process is thought to involve an internal set point and complex feedback loops that rely on thermoreceptor neurons in the skin and in the hypothalamus. Our understanding of the connections between such thermoreceptor neurons and motor centers is incomplete at best. Even less is known about the mechanisms of sensorimotor integration responsible for behavioral thermoregulation and its modification during short-term and long-term acclimation. In the nematode C. elegans, by contrast, a single pair of thermoreceptor neurons is linked to changes in behavioral responses to thermal gradients (thermotaxis). Their connections with interneurons and, ultimately, with motor neurons are known. Thus, in C. elegans, it is possible to understand how thermoreceptor neuron signaling affects behavior at a level of detail that is not possible in mammals. In this exploratory proposal, we seek to expand understanding of the link between temperature sensation and behavior by investigating the mechanism by which mutations can disrupt this link and by developing new tools for altering neuronal function. We focus on a mutant isolated in our laboratory that exhibits an intriguing defect in sensorimotor integration. We seek to extend this work by screening for genes responsible for the development and/or function of neurons that link thermosensation to motor output and to develop new tools for analyzing behavioral responses to neuron activation. The general strategy is to combine classical genetics with quantitative behavioral analysis and in vivo whole-cell patch-clamp recording in order to elucidate the relationship between temperature sensation and its behavioral consequences. The long-term objective of the proposed is to establish C. elegans as a new model for the study of behavioral thermoregulation and sensorimotor integration. The relevance of this project to human health lies in its potential to uncover universal motifs in sensorimotor integration and to provide insight into thermal dysregulation. [unreadable] [unreadable] All animals, including humans, have the ability to regulate their body temperature by moving to favorable areas in the environment. This ability relies on nerve cells that sense temperature and communicate with the nervous system to produce the needed movements. This complex process is only poorly understood. To improve understanding, we will study a simple roundworm that has only 302 neurons. Two of these neurons sense temperature and appear to aid a form of thermoregulation. What is learned from this study will clarify basic mechanisms of temperature sensation as well as learning and could improve understanding of thermoregulation and its dysfunction in disease and in response to recreational drugs such as ecstasy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The gentlest breeze, the roughest sandpaper, and the sharpest pin are detected by our somatosensory system, which is composed of thousands of touch-sensitive mechanoreceptor neurons embedded in the skin. It is widely understood that physical force is transduced into neural signals through activation of specialized ion channels (called 'mechano-electrical transduction' or MeT channels), but understanding of the molecular and physical basis of this process remains rudimentary. All animals have the ability to sense touch and recent work has shown that nematode and mammalian somatosensory neurons have similar response dynamics (reviewed in Geffeney and Goodman, Neuron 74:609, 2012). This functional conservation is seen in neurons that rely on either DEG/ENaC or TRP channel proteins to form MeT channels, a finding which implies that response properties are conferred primarily by the cellular environment and not intrinsic to the ion channel proteins. The long-term goal of the proposed research program is to understand how the cell membrane and cytoskeleton regulate the delivery of physical force to MeT channels expressed in touch receptor neurons (TRNs) and discover the structural rearrangements associated with touch-evoked MeT channel gating. The general approach will be to combine in vivo recording of MeT channel gating in identified TRNs with genetic perturbations in C. elegans nematodes that 1) alter biosynthesis of polyunsaturated fatty acids and cell membrane function, 2) disrupt the microtubule and spectrin cytoskeleton and 3) disrupt selected domains in the MEC-4 proteins crucial to the formation of the sodium-selective native MeT channels,. This work will be paired with optogenetics studies designed to identify factors that affect MeT channels, but not downstream signaling events and with biophysical analysis of MeT channels expressed in heterologous cells. The proposed research combines expertise in sensory biophysics, in vivo electrical recording from identified C. elegans neurons, genetic analysis, to derive a profound understanding of the sense of touch. What is learned from these studies has the potential to improve understanding of touch sensation and its dysfunction in disease, during chemotherapy and as a consequence of normal aging.

DESCRIPTION (provided by applicant): The long-term goal of this research is to discover the force transmission and force transduction pathways responsible for touch and proprioception. These senses are essential for social communication and every aspect of daily life from sitting, to standing, to running; however, their function is disrupted in both inherited and acquired diseases, including HIV-AIDS and diabetes. Even partial loss of sensory function as in diabetic peripheral neuropathy (DPN) has devastating consequences; DPN affects an estimated 15 million Americans and is the dominant risk factor in lower limb amputations. Thus, the loss of touch and proprioception is common, associated not only with discomfort and pain, but also with a decrease in the quality of life. Despite this, diagnostic tools and treatments for the dysfunction of touch and proprioception remain poorly developed, principally because little is known about how these senses work. This knowledge gap reflects a lack of adequate devices for delivering controlled mechanical stimuli and of animal models amenable to analysis of the mechanobiology of touch sensation. The objective of the proposed research is to bridge this gap by developing new devices for controlled force delivery, improved animal models for dissecting force transmission and transduction pathways, and new analytical methods for fundamental study of the relevant mechanics of this basic life process. The proposed research uses the simple roundworm, Caenorhabditis elegans, because more is understood about its sense of touch than that of any other animal. Research using C. elegans has successfully revealed mechanistic aspects of several fundamental and conserved biological processes, including touch sensation. It was in C. elegans, for instance, that the first ion channel proteins required for touch sensation were identified ~20 years ago. Because analogous proteins are expressed in mammalian touch receptor neurons, they may also contribute to touch sensation. At present, C. elegans is the only animal in which we know which proteins form the mechano-electrical transduction channels responsible for detecting force in touch receptor neurons. This knowledge enables a level of analysis that is not currently available in mammalian models. The central hypothesis we are testing is that both force-sensitivity and response dynamics are determined by the interplay of skin mechanics, neuron position, and intracellular, cytoskeletal structures. To test this hypothesis, we will develop new metrics for quantitative assessment of touch sensitivity; new microfabricated tools suitable for delivering pN-5N forces, new in vitro models of touch receptor neurons, and build new models of force transmission and force transduction. The specific aims are: 1) Test the hypothesis that skin mechanics, neuron position, and the neuronal cytoskeleton regulate touch sensitivity in vivo; 2) Assess the impact of body wall muscle tone and internal hydrostatic pressure on touch sensation in vivo; 3) Identify mechanisms of mechano- electrical transduction channel activation and adaptation.

DESCRIPTION (provided by applicant): Our senses provide us with the information that we need to perform all the essential functions of life: navigate the world, avoid danger, find food, choose mates and nurture our offspring. Each sensory modality is tuned to a specific set of stimuli; this tuning sets the limits of our sensory experience. Thus understanding how sensory cells detect and transduce sensory stimuli is critical to understanding how the nervous system responds to the environment to generate appropriate behaviors and perceptions. Moreover, disruption of sensory signaling in diseases ranging from blindness to deafness to insensitivity to pain can be devastating for humans. Despite ample evidence of shared molecular components and shared principles for transforming sensory input to neural signals across the senses, researchers investigating distinct senses rarely have opportunities to interact directly. With new technologies emerging at an ever- increasing rate, the timely sharing of information is needed to greatly accelerate research in sensory biology. The goal of this conference proposal is to assemble leading researchers across all sensory modalities (vision, smell, taste, touch, temperature, pain) and create an event designed to spark new collaborations among established and emerging investigators as well as graduate students and postdoctoral fellows. The conference, entitled Sensory Transduction, will be the 68th Annual Symposium of the Society of General Physiologists (SGP), a conference that has long been recognized as a pioneering and high- impact meeting for physiologists, cell biologists, and biophysicists. The venue, the campus of the Marine Biological Laboratory in Woods Hole, MA, is a magical place that affords participants with an intimate environment and ample opportunities to share scientific insight in both formal and informal settings.

? DESCRIPTION (provided by applicant): The development and function of the nervous system is regulated not only by electrical and biochemical signals, but also by mechanical inputs. For instance, most if not all neurons generate mechanical stress and experience strain during migration, axon outgrowth, and dendritic spine remodeling. With a few notable exceptions, however, the study of cell mechanics has been neglected in neuroscience. We seek to fill this knowledge gap by investigating the genetic and physical basis of how neurons withstand mechanical stress focusing on C. elegans touch receptor neurons as a model. Key entry points for this investigation are the findings that loss of unc-70 ß spectrin function makes C. elegans neurons vulnerable to damage induced by normal movement and the well-known function for actin-spectrin networks in protecting red blood cells from the mechanical strains generated as they transit through tiny capillaries. In new work, we establish a simple visible assay for loss of spectrin function in the ventral touch receptor neurons and create transgenic animals expressing a genetically-encoded strain sensor that enables us to visualize how body bending and touch sensation affects stress in neural actin-spectrin networks. The proposed research combines genetic dissection, high-speed quantitative confocal microscopy, and in vitro biochemistry to investigate the role of spectrin networks in mechanical neuroprotection and sensory mechanoelectrical transduction. This work has the potential to transform understanding of neuronal cell mechanics and the contribution of actin-spectrin networks in this process. The new knowledge we seek to acquire could provide insight into the genetic basis of mechanical neuroprotection and potential risk factors related to traumatic brain injury.

? DESCRIPTION (provided by applicant): The development and function of the nervous system is regulated not only by electrical and biochemical signals, but also by mechanical inputs. For instance, most if not all neurons generate mechanical stress and experience strain during migration, axon outgrowth, and dendritic spine remodeling. With a few notable exceptions, however, the study of cell mechanics has been neglected in neuroscience. We seek to fill this knowledge gap by investigating the genetic and physical basis of how neurons withstand mechanical stress focusing on C. elegans touch receptor neurons as a model. Key entry points for this investigation are the findings that loss of unc-70 ß spectrin function makes C. elegans neurons vulnerable to damage induced by normal movement and the well-known function for actin-spectrin networks in protecting red blood cells from the mechanical strains generated as they transit through tiny capillaries. In new work, we establish a simple visible assay for loss of spectrin function in the ventral touch receptor neurons and create transgenic animals expressing a genetically-encoded strain sensor that enables us to visualize how body bending and touch sensation affects stress in neural actin-spectrin networks. The proposed research combines genetic dissection, high-speed quantitative confocal microscopy, and in vitro biochemistry to investigate the role of spectrin networks in mechanical neuroprotection and sensory mechanoelectrical transduction. This work has the potential to transform understanding of neuronal cell mechanics and the contribution of actin-spectrin networks in this process. The new knowledge we seek to acquire could provide insight into the genetic basis of mechanical neuroprotection and potential risk factors related to traumatic brain injury.

ABSTRACT Cells push and pull on their surroundings, generating and sensing mechanical forces. We experience these forces each time our heart beats, our ears hear, or a wound heals. Mechanical forces affect other processes like stem-cell proliferation and differentiation, ion-channel gating, synaptic plasticity, and the development and maintenance of cell organization, multicellular tissues, organs, and animals. Despite its widespread importance in biology, the influence of mechanical stress on cell and tissue function remains poorly understood. The few tools that exist for measuring forces today are either direct, but unsuitable for use in living cells and tissues or indirect, relying on biochemical or molecular changes or simplified mechanical models to infer mechanical stresses. The proposed research unites materials science, photophysics, and fundamental biology to develop biocompatible optical reporters of mechanical force designed to monitor and quantify forces within and between cells and those generated by organs. The technology we seek to develop is based on state-of-the-art inorganic upconverting nanoparticles (NP) that are tiny (<10nm), biocompatible, and emit light in a manner that depends on mechanical compression or extension. We will synthesize and optimize NP reporters of compressive, tensile and shear forces, determining the influence of surface chemistry on mechanosensitivity and biocompatibility, and demonstrate a first-in-animals pilot study using first-generation NP mechanoreporters to determine forces generated by food consumption in C. elegans nematodes, an invertebrate model used to investigate many fundamental biological principles.

Skin, muscle, joints, and internal organs encapsulate specialized sensory neurons that detect mechanical cues in the form of touch and movement. The ability to perform most, if not all of the essential activities of daily living depends on information from these somatosensory, proprioceptive, and visceral sensory neurons. Thus, a better understanding of their function and sensitivity to mechanical and chemical stress is of vital importance for health. This research program focuses on the skin-neuron composite tissues responsible for touch and seeks to decipher how mechanical force is translated from the skin surface to embedded sensory neurons and converted into electrical signals that give rise to tactile perceptions. The work combines genetic dissection in a simple invertebrate (C. elegans nematodes) with electron microscopy, high-performance tools (self-sensing cantilevers) for delivering mechanical stimuli under feedback control and for optically monitoring tissue deformation and neuronal activation with electrophysiology and calcium imaging. The research team includes biologists, engineers and physicists and integrates experimental work with theory and simulation. In addition to seeking a comprehensive understanding of mechanosensation by skin-neuron composites, the research program will also address the outstanding question of how neurons bend without breaking. Based on preliminary work, we also plan to leverage our knowledge of touch sensation and its molecular basis to investigate how chemical stressors linked to diabetes (glucose) and chemotherapy (paclitaxel) affect the function and morphology of skin-neuron composites. The knowledge we seek to acquire is relevant to all animals, including humans that rely on skin-neuron composites for touch sensation.