| My laboratory's research is focused on understanding the molecular mechanisms that orchestrate the development, structural organization and secretory signaling functions of the presynaptic nerve terminal. I study the nematode C. elegans (Figure 1) because experimentally it can be manipulated with ease both molecularly and genetically, yet it still retains many features common to vertebrate systems. C. elegans uses all of the major transmitters, possesses a complex transmitter receptor and ion channel repertoire, and shares most molecular components with the vertebrate presynaptic apparatus. |
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| I find synapses (Figure 2) fascinating because they are highly organized subcellular structures with an intricate architecture that is designed to facilitate rapid and reliable cell-cell signaling. In particular, my lab has concentrated on studying the presynaptic nerve terminal that secretes neurotransmitter via vesicular fusion of synaptic vesicles specifically at release sites called active zones. Synaptic vesicles are used repetitively via cycles of exocytosis and endocytosis. This process is often referred to as the synaptic vesicle cycle (Figure 3). |
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| Currently, three major areas of research are under investigation in my lab. First, the lab is dissecting the role of two vesicle-associated rab GTPase proteins in regulating both synaptic transmission in the neurons and secretion of peptides in the intestine. Second, we are delineating molecular mechanisms which orchestrate the assembly and function of the presynaptic density (or active zone) of the synapse. Third, we are taking several approaches to identify signals that mediate the decision to assemble and maintain synaptic connections. To address these questions we use a combination of genetics, pharmacology, physiology, molecular biology and cell biology. |
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Rab proteins regulating exocytosis First, the lab is dissecting the role of synaptic proteins in regulating exocytosis. Our general approach has been to characterize the defects associated with disrupting individual components of the nerve terminal. Recently, we have been concentrating on the role of two rab proteins in regulating synaptic transmission (Figure 4). Rab proteins are a large family of small membrane-associated GTPases that associate with distinct membrane compartments. Recent findings from my lab have revealed that both RAB-3 and RAB-27 function to regulate synaptic transmission. These two rab proteins are coordinately regulated by a single nucleotide exchange factor called AEX-3. Disrupting AEX-3 leads to a 4-fold reduction in the efficiency of neuromuscular synaptic transmission. One unique phenotype of both AEX-3 and RAB-27 mutants is that they also disrupt the defecation motor program of C. elegans. A variety of genetic evidence indicates that this behavioral pathway is critically dependent on secretion of a peptide. Thus, the two rab proteins may define distinct populations of vesicles at synapses with distinct transmitter content. We have recently identified two additional gene products required for the defecation motor program and are exploring their relationship to the RAB3/27 pathway. Our current goal is elucidating the mechanisms by which these rab proteins modulate the secretory system. Specifically, we are attempting to identify novel components of the pathway using genetic approaches and positioning these components in the pathway by defining genetic, biochemical, and cell biological interactions among the identified components. |
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Active Zone Assembly Vesicle fusion occurs in the vicinity of the presynaptic density in a domain of the synapse called the active zone. This electron dense specialization contains numerous proteins that play critical roles in the regulation of calcium-regulated exocytosis. We are specifically interested in understanding how this structure is assembled and how proteins are selectively targeted to this subcellular domain. C. elegans is particularly suited for examining this question because at the ultrastructural level the presynaptic density is robust and well-defined at most C. elegans synapses. Proteins that localize to the active zone include calcium channels, CAST, and the priming factors UNC-13 and Rim. We've shown that in the worm these proteins target to the active zone independently of one another. We have also identified domains of Rim (Figure 5) and CAST that are required for these proteins to target to the active zone. We are now using genetic, molecular and biochemical approaches to identify the scaffold proteins that we hypothesize act as structural foundations for the assembly of Rim, CAST, and UNC-13. |
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Synapse formation The third project in the lab uses in vivo synaptic tags such as synaptobrevin-GFP, and more recently GFP-rab3 (Figure 6), as tools to identify genes regulating both synapse assembly and growth. We have previously identified a retrograde signal from head muscle to neurons that regulates process sprouting and growth of presynaptic specializations. We are searching for constituents of this signaling process using an RNAi screening approach. A second interest in this area is understanding how specific neurons are able to select their synaptic partners. Having recently developed GFP-rab3 as a new marker for visualizing synapses in live animals that is approximately 20-fold brighter than synaptobrevin-GFP, we are now attempting to isolate mutants that specifically disrupt this recognition process using new incarnations of genetic screens we previously used to isolate the synaptic gene rpm-1. In addition, in collaboration with Rachel Wong's lab, we are attempting to develop GFP-rab3 as a tool for visualizing synapses in zebrafish to explore the molecular mechanisms that underlie the specificity of partner selection during synapse formation. |
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