IN VIVO FUNCTION AND INTERACTION OF SNARES, NSF AND SNAP DURING PRESYNAPTIC VESICLE TRAFFICKING IN DROSOPHILA

Open Access
Author:
Yu, Wenhua
Graduate Program:
Genetics
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
July 02, 2009
Committee Members:
  • Richard W Ordway, Dissertation Advisor
  • Zhi Chun Lai, Committee Chair
  • Richard W Ordway, Committee Member
  • Wendy Hanna Rose, Committee Member
  • Aimin Liu, Committee Member
  • Douglas Cavener, Committee Member
Keywords:
  • synaptic function
  • NSF
  • SNAP
  • SNAREs
  • live imaging
  • FRET
  • Drosophila
Abstract:
Synaptic transmission is a fundamental aspect of nervous system function. At chemical synapses, neurotransmitter-filled synaptic vesicles fuse to the presynaptic plasma membrane at specific sites known as active zones and release neurotransmitters, which act on postsynaptic neurons. SNAREs, NSF and SNAP are core components of the synaptic vesicle fusion apparatus. Gaining insights into their in vivo functions and interactions has been the primary research goal of this dissertation project. Taking advantage of the powerful genetic model system of Drosophila, the project has combined genetic, biochemical and live imaging methods to reveal interesting insights into synaptic protein behavior and interactions at living synapses. This work extends our previous analysis of a temperature-sensitive (TS) paralytic NSF mutant, comatose, and extends previous models describing the spatial organization of SNAREs, NSF and SNAP proteins with respect to presynaptic active zones. Specifically, biochemical analysis demonstrated for the first time that acute disruption of dNSF1 activity in comatose leads to accumulation of protein complexes containing dNSF1, dSNAP and SNAREs, suggesting that mutant dNSF1 is capable of associating in complexes with dSNAP and SNAREs, although its ability to disassemble them is impaired by the missense mutation located in the D1 domain. Moreover, dNSF1, dSNAP and t-SNAREs exhibit activity-dependent redistribution to Peri-Active Zone (PAZ) regions of the plasma membrane in comatose, suggesting the PAZ as the location where NSF-mediated disassembly of SNARE complexes would normally take place. Further, dSNAP exhibits a stable punctuate distribution pattern to the PAZ following its redistribution in comatose but is still mobile and can disassociate from the PAZ, suggesting dSNAP may bind and disassociate from dNSF1 and SNARE complexes independent of SNARE complex disassembly. Moreover, the apparent shift of this equilibrium towards dSNAP binding to the PAZ may reflect accumulation of dNSF1 and SNAREs, which leads to higher capacity of affinity for dSNAP binding at the PAZ. Unlike dSNAP, dNSF1 is fixed at the PAZ following its redistribution in comatose, suggesting that anchorage or immobilization of dNSF1 at the PAZ is mediated by synaptic components or signals other than the classic synaptic vesicle fusion apparatus (SNAREs and SNAP). Live imaging approaches developed and adapted in our laboratory, including in vivo Fluorescence Recovery after Photobleaching (FRAP) and Fluorescence Resonance Energy Transfer (FRET) analysis, have been employed to monitor the behavior and interactions of synaptic proteins at living synapses. Particularly, efforts have been made to establish, optimize and compare two independent FRET methods within the model system. Notably, for the first time in any system, intermolecular FRET was observed between presynaptic proteins and defined activity-dependent protein interactions within living presynaptic boutons. Live imaging analysis has also generated other interesting insights with respect to in vivo orientation of proteins in assembly within complexes and assessments of protein mobility at native synapses. These imaging studies have provided important in vivo evidence from individual living presynaptic boutons which complements and extends previous biochemical analysis in developing molecular models of neurotransmitter release mechanisms.