Jessica E. Tanis, Ph.D. - US grants

DESCRIPTION (provided by applicant): Type II taste cells, which respond to sweet, bitter and umami tastants lack morphologically defined synapses. Instead, these taste cells utilize depolarization-evoked non-vesicular ATP release to communicate with afferent gustatory nerves. The overarching goal of the proposed studies is to determine how CALHM1 and CALHM3 contribute to a hetero-oligomeric ATP release channel in Type II taste cells. CALHM1, the pore forming subunit of an ion channel regulated by both voltage and extracellular Ca2+, is expressed in Type II taste cells and the brain. Calhm1 knockout mice cannot perceive sweet, bitter and umami tastants and exhibit defects in tastant-evoked ATP release from Type II taste cells. This suggests that CALHM1 plays an important physiological role in taste perception. However, there are differences in the kinetic properties of native CALHM1-dependent currents in Type II taste cells compared to CALHM1 currents in heterologous systems. It is important to identify the reasons for these inconsistencies to further our understanding of ATP release from Type II taste cells and taste perception. There are five additional Calhm family members of unknown function in humans. CALHM2 and CALHM3 are also expressed in Type II taste cells, but on their own, cannot form functional ion channels. Strikingly, co- expression of CALHM3 with CALHM1 in heterologous systems gave rise to a novel conductance with fast activation kinetics similar to those of the CALHM1-dependent currents observed in Type II taste cells. This led to the hypothesis that CALHM1 and CALHM3 may form a hetero-oligomeric ATP release channel in vivo. To determine the molecular basis for voltage-dependent gating of CALHM channels and define the role of CALHM3 in taste perception, a combination of electrophysiological and cell biological approaches will be utilized. CALHM proteins do not exhibit conserved architecture with canonical voltage-gated ion channels. To gain insight into the molecular determinants for the voltage-dependent gating of CALHM1, electrophysiology studies of chimeric channels and point mutants will be performed. This will lead to the identification of the activation gate and channel pore as well as specific residues required for voltage-dependent gating and ion selectivity properties. Knowledge gained from defining the molecular basis for CALHM1 gating will be critical for interpretation of similar electrophysiological studies that will be conducted o determine how CALHM3 contributes to the novel gating properties of the putative CALHM1 / CALHM3 hetero-oligomeric channel. To define the role of CALHM3 in taste perception as well as ATP release and CALHM1-dependent currents in Type II taste cells, behavioral, cell biological and electrophysiological analyses of Calhm3 knockout mice will be performed. This study will lead to an understanding of the molecular mechanism for CALHM channel gating and the role of CALHM3 in the perception of sweet, bitter and umami taste compounds.

Project Summary Extracellular vesicles (EVs) are membrane-wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication. Roles for EVs in physiological processes as well as pathological conditions including neurodegenerative diseases and cancer have been established. Given the presence of EVs in diverse body fluids, there is also great interest in using these vesicles as biomarkers for disease detection and engineering EVs for therapeutics. Investigation of the release of EVs containing fluorescently-tagged cargo from identified cells in the model system C. elegans can provide insight into unresolved questions concerning conserved mechanisms of EV biogenesis and cargo selection in vivo. We discovered that the calcium homeostasis modulator ion channel CLHM-1 is cargo in EVs released from cilia of male-specific sensory neurons. Remarkably, when we coexpressed tdTomato- tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in distinct EV subpopulations. We have found that the PKD-2 and CLHM-1 containing EVs do not utilize the same biogenesis and release mechanisms, are discharged in different quantities, and do not have the same physiological function. Our overarching goal is to draw upon the strengths of our genetic system and cutting edge imaging and mass spectrometry approaches to define mechanisms underlying formation of EV subpopulations and the physiological significance of EV heterogeneity. Our proposed research will utilize our unique transgenic animals that express fluorescently tagged EV cargoes at endogenous levels. Advanced imaging techniques including confocal microscopy with Airyscan detection and immunogold labeling for transmission electron microscopy will enable us to characterize the size, morphology, and ciliary release site(s) of EVs as well as the impact of lateral lipid asymmetry in the ciliary membrane on cargo sorting. Through a candidate approach, we will define the role of flippases, floppases and scramblases, which control transbilayer lipid asymmetry, in the biogenesis of the EV subsets. We will then explore how cellular stress conditions that disrupt plasma membrane phospholipid homeostasis impact EV cargo sorting and release. To identify other cargoes in the CLHM-1 EV subset, we will perform mass spectrometry on GFP-tagged CLHM-1 vesicles isolated by flow cytometry. Finally, we will identify the hermaphrodite-derived stimulus that induces an increase in formation of CLHM-1 containing EVs from male ciliated neurons as well as the importance of EV release for animal communication and ciliary function. This work will lead to an understanding of how an individual cell generates heterogeneous EV populations with different physiological functions, impacting broadly on our comprehension of basic biogenesis and cargo sorting mechanisms utilized in vivo.

Project Summary At the neuromuscular junction (NMJ), postsynaptic nicotinic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. Defects in cholinergic signaling are the primary cause of severe muscle weakness observed in individuals with congenital myasthenic syndromes and the autoimmune syndrome myasthenia gravis. In addition, clinical features of some congenital myopathies and muscular dystrophies suggest underlying cholinergic signaling defects. Together, this highlights the importance of determining how signaling through AChRs is regulated at the NMJ. While mechanisms that lead to the clustering of postsynaptic AChRs have been well studied, little is known about how receptor insertion and endocytosis is controlled to maintain synaptic efficacy. The body wall muscles in the model organism C. elegans are functionally comparable to vertebrate skeletal muscles. Sinusoidal locomotion occurs as a result of activation of postsynaptic AChRs on one side of the animal, which causes muscle contraction, while simultaneous stimulation of GABAA receptors on the opposite side of the animal triggers muscle relaxation. To identify novel factors that regulate postsynaptic cholinergic signaling we performed a genome wide RNAi screen for gene knockdowns that altered C. elegans sensitivity to the AChR agonist levamisole. One knockdown that caused levamisole hypersensitivity was epn-1, the homolog of mammalian Epsin, which functions to recruit specific cargoes and induce membrane curvature during endocytosis. We discovered that loss of epn-1 resulted in an increase in AChRs, but surprisingly, a decrease in GABAA receptors on the plasma membrane. This led us to hypothesize that EPN-1 as well as some of the other screen isolates regulate trafficking of postsynaptic receptors to maintain appropriate neuromuscular transmission. Our overarching goal is to define the mechanisms that control postsynaptic receptor abundance and localization at the NMJ by characterizing genes identified in our screen. We will use an integrated approach, performing innovative genetic, imaging, biomechanical profiling, and optogenetic experiments. Our study will enable us to develop a broad understanding of mechanisms underlying postsynaptic receptor trafficking at the NMJ, as well as identify novel gene targets for future studies and therapeutic design. I will build upon my strong foundation in genetics, neuroscience, physiology, and C. elegans research to develop a comprehensive and meaningful research program under the mentorship of Dr. Velia Fowler and Dr. Robert Akins who have expertise in skeletal muscle contraction and NMJ development in children with muscle diseases, respectively. This research plan will be carried out in the Department of Biological Sciences and excellent core facilities at the University of Delaware. The Delaware Center for Musculoskeletal Research will provide access to strong mentors, career development resources, and a collaborative interdisciplinary community of scientists.