Cysteine -string protein and Hsc70: A chaperone complex regulating neurotransmitter release

The functionality of neural networks is predominantly derived from chemical synapses, which facilitate information processing by transmitting and modulating neuronal signals through pre- and postsynaptic mechanisms. This study examined the function of Cysteine-string Protein (CSP) and Heat Shock Cognate Protein (Hsc70) at nerve terminals. CSP is associated with synaptic and numerous other secretory vesicles and has been previously implicated in promoting presynaptic calcium entry. My studies of transmitter release at Drosophila csp null-mutant neuromuscular junctions showed, however, that a defect in calcium entry cannot cause the loss of neurotransmitter release in the mutants. Imaging of presynaptic calcium levels combined with electrophysiological recordings suggested that CSP might mediate a calcium-sensitive step of vesicle fusion downstream of calcium entry, inhibit calcium entry, and protect calcium homeostasis at nerve terminals against thermal stress. Previous in vitro studies suggest that CSP acts as a co-factor of Hsc70, but an exocytotic role of Hsc70 was not known—only the uncoating of clathrin-coated vesicles during vesicle recycling. My genetic studies of Drosophila Hsc70 supported a novel exocytotic function for Hsc70 in evoked release. In particular, I identified defects in hsc70 mutants similar to those of csp mutants suggesting that Hsc70 might interact with CSP as part of a chaperone system. Surprisingly, loss of Hsc70 protein did not significantly affect vesicle recycling, suggesting a more redundant role of Hsc70 in recycling than previously assumed. The final project in this thesis describes an in vivo structure/function analysis of CSP, which has three highly conserved domains: an N-terminal J domain assumed to interact with Hsc70, a linker domain, and a lipidated cysteine-string domain. I found that the J domain is essential for CSP\u27s calcium homeostasis function. Moreover, the J domain is apparently important but not essential for CSP\u27s role in vesicle fusion. Surprisingly, the highly conserved linker domain is not essential for any known function of CSP while the least conserved region, the C-terminus, is essential for CSP\u27s role in regulated neurotransmitter release. My studies support the hypothesis that a CSP/Hsc70 complex facilitates G protein mediated inhibition of calcium channels and promotes a late step of vesicle fusion