The long term goal of this research is to elucidate molecular mechanisms involved in neurotransmitter release at mature chemical synapses.
The specific aims of the proposed research are to study, using electrophysiological, novel cell biological and traditional molecular genetic methods, mutations in four Drosophila genes which probably affect synaptic vesicle recycling, a little-studied process vital for presynaptic function. There are four major aims: 1) To characterize genetically four genes, chc encoding a clathirin heavy chain, stoned, e(shi)A and e(shi)B, which have been identified in the investigator's laboratory, and are likely involved in synaptic vesicle recycling. 2) To investigate the involvement of clathirin heavy chain and other novel clathrin associated proteins in presynaptic function. 3) To determine the molecular and physiological role of the e(shi)A gene in synaptic vesicle recycling. 4) To develop new assays for analyzing different intermediate steps or pathways in synaptic vesicle recycling. The experiments will use electrophysiological and quantitative cell biological assays to study potential defects in synaptic vesicle fusion and recycling in identified mutant synapses. Morphological methods will be used to examine the disposition of synaptic vesicle membrane in mutant nerve terminals. Recombinant DNA techniques will be used to identify molecules affected by the mutations, in order to examine their phylogenetic conservation, their subcellular localization and their intracellular traffic during the cycling of synaptic vesicle membrane. If successful, these experiments will constitute the first in vivo analysis of molecular mechanisms in synaptic vesicle recycling.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS034889-03
Application #
2883689
Study Section
Neurology C Study Section (NEUC)
Program Officer
Leblanc, Gabrielle G
Project Start
1997-03-01
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
3
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Arizona
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Sanyal, Subhabrata; Jennings, Tricia; Dowse, Harold et al. (2006) Conditional mutations in SERCA, the Sarco-endoplasmic reticulum Ca2+-ATPase, alter heart rate and rhythmicity in Drosophila. J Comp Physiol B 176:253-63
Narayanan, Radhakrishnan; Leonard, Marilyn; Song, Byeong Doo et al. (2005) An internal GAP domain negatively regulates presynaptic dynamin in vivo: a two-step model for dynamin function. J Cell Biol 169:117-26
Sanyal, S; Consoulas, C; Kuromi, H et al. (2005) Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability. Genetics 169:737-50
Rikhy, Richa; Ramaswami, Mani; Krishnan, K S (2003) A temperature-sensitive allele of Drosophila sesB reveals acute functions for the mitochondrial adenine nucleotide translocase in synaptic transmission and dynamin regulation. Genetics 165:1243-53
Estes, Patricia S; Jackson, Taryn C; Stimson, Daniel T et al. (2003) Functional dissection of a eukaryotic dicistronic gene: transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants. Genetics 165:185-96
Narayanan, Radhakrishnan; Ramaswami, Mani (2003) Regulation of dynamin by nucleoside diphosphate kinase. J Bioenerg Biomembr 35:49-55
Chen, Mai-Lei; Green, David; Liu, Lei et al. (2002) Unique biochemical and behavioral alterations in Drosophila shibire(ts1) mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling. J Neurobiol 53:319-29
Krishnan, K S; Rikhy, R; Rao, S et al. (2001) Nucleoside diphosphate kinase, a source of GTP, is required for dynamin-dependent synaptic vesicle recycling. Neuron 30:197-210
Stimson, D T; Estes, P S; Rao, S et al. (2001) Drosophila stoned proteins regulate the rate and fidelity of synaptic vesicle internalization. J Neurosci 21:3034-44
Sanyal, S; Tolar, L A; Pallanck, L et al. (2001) Genetic interaction between shibire and comatose mutations in Drosophila suggest a role for snap-receptor complex assembly and disassembly for maintenance of synaptic vesicle cycling. Neurosci Lett 311:21-4

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