The goal of the proposed research is to identify and understand the function of the molecules required for synaptic function. In recent years we have seen great progress in this field as a number of synaptic proteins have been identified by biochemical means. However, it is not clear how each of these proteins functions in the synapse nor have all of the proteins involved in this fundamental biological process been identified. We have identified synaptic function mutants in the nematode Caenorhabditis elegans. Some of these mutations are in known synaptic proteins. From among these we propose to focus on three proteins which are believed to function in the endocytosis of synaptic vesicles. We will characterize the phenotype of these mutants in detail and thereby test models which have been proposed for the function of these proteins based on biochemical data. Other mutations identified in our screens are in genes encoding novel proteins. We will characterize one of these proteins at a molecular level and determine the function of this protein in synaptic transmission by characterizing the mutant phenotype in detail.
Aim 1. NSF. It has been proposed that NSF may function at a pre-docking step of exocytosis, at the fusion step of exocytosis, or at a post-fusion step preceding, endocytosis. We will test the hypothesis that NSF is required at a post-fusion step; specifically, that it is required for the endocytosis of the vesicle protein synaptobrevin.
Aim 2. u2 subunit of AP2. The clathrin adaptor complex AP2 is believed to be required for endocytosis of synaptic vesicles. However, it is not clear whether synaptic vesicle recycling occurs solely via a clathrin-mediated process. It is also possible that the synapse uses a specialized medium subunit of AP2 rather than the ubiquitous u2 medium subunit. We will test whether the u2 medium subunit of AP2 is required for synaptic vesicle endocytosis.
Aim 3. Synaptojanin. It has been proposed that the inositol phosphatase synaptojanin is required for endocytosis of synaptic vesicles because it is associated with the endocytic machinery. We will determine whether synaptojanin is required for endocytosis of synaptic vesicle components from the plasma membrane. In addition, our preliminary studies indicate that it may be required for the transport of vesicle precursors to the synapse. We will test a model for the mechanism of synaptojanin function in vesicle transport.
Aim 4. RIC-7. Mutations in this gene disrupt cholinergic and GABAergic-mediated behaviors. We will determine the function of this novel synaptic protein using ultrastructural and physiological tools.
| Watanabe, Shigeki; Mamer, Lauren Elizabeth; Raychaudhuri, Sumana et al. (2018) Synaptojanin and Endophilin Mediate Neck Formation during Ultrafast Endocytosis. Neuron 98:1184-1197.e6 |
| Kurshan, Peri T; Merrill, Sean A; Dong, Yongming et al. (2018) ?-Neurexin and Frizzled Mediate Parallel Synapse Assembly Pathways Antagonized by Receptor Endocytosis. Neuron 100:150-166.e4 |
| Topalidou, Irini; Chen, Pin-An; Cooper, Kirsten et al. (2017) The NCA-1 and NCA-2 Ion Channels Function Downstream of Gq and Rho To Regulate Locomotion in Caenorhabditis elegans. Genetics 206:265-282 |
| Chen, Bojun; Liu, Ping; Hujber, Edward J et al. (2017) AIP limits neurotransmitter release by inhibiting calcium bursts from the ryanodine receptor. Nat Commun 8:1380 |
| German, Christopher L; Gudheti, Manasa V; Fleckenstein, Annette E et al. (2017) Brain Slice Staining and Preparation for Three-Dimensional Super-Resolution Microscopy. Methods Mol Biol 1663:153-162 |
| Jang, SoRi; Nelson, Jessica C; Bend, Eric G et al. (2016) Glycolytic Enzymes Localize to Synapses under Energy Stress to Support Synaptic Function. Neuron 90:278-91 |
| Bend, Eric G; Si, Yue; Stevenson, David A et al. (2016) NALCN channelopathies: Distinguishing gain-of-function and loss-of-function mutations. Neurology 87:1131-9 |
| Li, Pengpeng; Merrill, Sean A; Jorgensen, Erik M et al. (2016) Two Clathrin Adaptor Protein Complexes Instruct Axon-Dendrite Polarity. Neuron 90:564-80 |
| Yogev, Shaul; Cooper, Roshni; Fetter, Richard et al. (2016) Microtubule Organization Determines Axonal Transport Dynamics. Neuron 92:449-460 |
| Geisler, Florian; Gerhardus, Harald; Carberry, Katrin et al. (2016) A novel function for the MAP kinase SMA-5 in intestinal tube stability. Mol Biol Cell 27:3855-3868 |
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