Dissertation Defense: Tong Shu, Yale University, “Molecular Mechanisms of Regulated SNARE Assembly Revealed by Single-Molecule Force Spectroscopy”

Neurons in the brain communicate with each other by release of neurotransmitters at synaptic junctions. Neurotransmitter release is mediated by three membrane-anchored SNARE proteins (soluble N-ethylmaleimide sensitive factor attachment protein receptors) and various regulatory proteins, including Munc13-1, Munc18-1, Synaptotagmin, complexin, NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein). By forming proper SNARE complex, SNARE proteins couple their folding and assembly to membrane fusion in a regulatory protein-dependent manner. To achieve sub-millisecond synaptic vesicle fusion, SNARE assembly pathway needs to be highly regulated in several aspects, promoting fast and accurate initial SNARE assembly, clamping half-zippered SNARE complex before Ca2+ influx and accomplishing full SNARE zippering for membrane fusion within sub-millisecond upon Ca2+ influx. However, the exact molecular mechanisms of these processes are poorly understood despite decades of researches. Here, we addressed one of the processes using high-resolution optical tweezer. We found that Munc13-1, Munc18-1, and two SNAREs – Syntaxin-1 and VAMP2 associate into a weak tetrameric complex. The third SNARE protein SNAP-25B rapidly binds the two SNAREs in the complex to form a proper ternary SNARE complex, which likely displaces the two regulatory proteins from the resultant four-helix bundle. Therefore, Munc13-1 and Munc18-1 cooperatively chaperone SNARE assembly, a process required for neurotransmitter release. In summary, the findings demonstrate new evidences in understanding the molecular mechanism of sub-millisecond fast Ca2+-triggered synaptic vesicle exocytosis.
Thesis Advisor: James Rothman/Yongli Zhang