CYSTEINE STRING PROTEIN IS REQUIRED FOR CALCIUM SECRETION COUPLING OFEVOKED NEUROTRANSMISSION IN DROSOPHILA BUT NOT FOR VESICLE RECYCLING

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Droso phila. CSP has been found to interact in vitro with the clathrin-uncoa ting ATPase HSC70, suggesting a potential role of CSP in vesicle recyc ling. Using FM1-43 imaging, we analyzed whether the ts block of neurot ransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic bout ons of csp mutant neuromuscular junctions fail to destain at 32 degree s C after K+ depolarization, and that FM1-43 dye uptake cannot be evok ed by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and t he calcium ionophore calcimycin stimulate FM1-43 destaining and quanta l release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dep endent calcium signaling pathway must be impaired, either calcium entr y, calcium action, or both. Collectively, our results indicate that th e csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP link s synaptic vesicles to calcium secretion coupling.