Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Ca2+ triggered exocytosis of synaptic vesicle releases neurotransmitter to synaptic cleft, which is critical for the ability of neurons to communicate with one another and essential for brain functions. The neuronal SNAREs syntaxin 1A, SNAP-25, and VAMP2 play a central role in fusion of synaptic vesicles with plasma membrane through assembly of SNARE complex in chasm of two membranes that bridges the synaptic vesicles and plasma membrane closely together even through fusion. However, to gain a precise temporal control of the release which is essential for healthy brain activities, other regulatory proteins such as synaptogamins, complexins, Munc18-1, and Munc13, are also required. Defining how each proteins interact and function together to orchestrate fast and synchronous synaptic vesicles fusion is essential for understanding the mechanism of neurotransmitter release and neuron communication. Though structural and functional studies have yielded extensive knowledge about physiological role and molecular mechanism of these proteins, a clear and comprehensive picture of the whole process from vesicles docking to Ca2+ triggered fast fusion is still elusive.
In this work, we used recently developed total internal reflection (TIR) microscopy based single molecular assays to study the regulatory mechanism of synaptotagmin 1 (Syt1) and Munc18-1, which is significant and innovative that the new assays are capable of resolving SNARE assembly, vesicles docking, lipid mixing, hemifusion, and fusion pore opening steps, thereby facilitate to delineate the function of regulatory proteins onto individual fusion steps. Combining with site-directed spin labeling (SDSL) EPR and other biochemical and biophysical assays, our results showed that the linker region of Syt1 between its transmembrane domain and cytoplasm C2 domains, which contains the basic amino acid-rich N-terminal region and the acidic amino acid-rich C-terminal region, is essential for its two signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. We also found that the polybasic region of Syt1, which is known important for SNARE-binding, may not be as essential for fusion pore opening as the Ca2+ binding and membrane penetration regions of Syt1. These results delineate multiple functions of Syt1 along the pathway of Ca2+-triggered exocytosis in unprecedented detail. Moreover, we found that though Munc18-1 could promote SNARE assembly and lipid mixing in the absence of Syt1, the enhancement is abolished in the presence of Syt1 as well as that Munc18-1 didn’t affect fusion pore opening mediated by SNAREs and Syt1/Ca2+. This work shed some light on a puzzle for a long time that whether Munc18-1 is part of the central fusion machinery in neuronal system.
Lou, Xiaochu, "Mechanistic insights into the regulation of neuronal SNAREs mediate membrane fusion: interplay between core fusion machinery and regulatory proteins" (2015). Graduate Theses and Dissertations. 14612.