Munc18's role in regulating Syntaxin's spatial organization, SNARE complex assembly, and vesicle fusion 4 views

The majority of neuronal communication occurs via chemical synapses, in which neurotransmitters are released from presynaptic neurons into the synaptic cleft where they are sensed by neurotransmitter receptors on postsynaptic neurons. Neurotransmitters and hormones are released from secretory cells by SNARE-mediated exocytosis, which involves fusion between secretory vesicles and the plasma membranes of the secreting cells. In neurons, the core protein machinery necessary for membrane fusion consists of three SNARE proteins: Syntaxin-1a and SNAP25 on the plasma membrane and synaptobrevin-2 on the vesicle membrane. These proteins assemble into a four-helix bundle called SNARE complex that allows the membranes to overcome energetic barriers for fusion. SNARE complex assembly is highly orchestrated in cells, where the secretory vesicles first need to be docked and primed in a fusion-ready state at the plasma membrane. From this state, fusion can be triggered by the influx of calcium into the cell. Key regulatory proteins in SNARE assembly include Munc18 and Munc13 that facilitate docking and priming of secretory vesicles, and Synaptotagmin and Complexin that ensure that fusion is coupled to and triggered by calcium influx. Despite extensive studies of the fusion machinery, many mechanistic details and the molecular sequence of events during SNARE complex assembly are still poorly understood. In this thesis, I focus on Munc18, a cytosolic protein essential for neurotransmitter release. Munc18’s role in SNARE complex assembly is multifaceted and complex. It primarily involves Munc18’s interactions with Syntaxin on the plasma membrane. Munc18 aids vesicle docking by organizing Syntaxin into nanoscale clusters and has been shown to both inhibit and stimulate SNARE complex assembly in vitro. Initial Munc18-Syntaxin interaction induces Syntaxin to adopt a closed conformation and is proposed to serve as a checkpoint during the assembly preventing Syntaxin multimerization and premature interactions with other SNARE proteins. Munc18’s inhibition of premature SNARE (self-)assembly is released by other regulatory proteins and Munc18 transitions to stimulate functional SNARE assembly by promoting Syntaxin-SNAP25 and Syntaxin-Synaptobrevin interactions. While the Munc18-Syntaxin interaction has been extensively studied in solution, it is less clear how these two proteins interact in biological and model membranes where Syntaxin is known to oligomerize. To fill these gaps, I reconstituted SNARE proteins in model membranes and performed an array of biochemical and biophysical ensemble and single particle assays to characterize the effects of Munc18 binding on the SNAREs’ distribution and conformation. I show that the membrane lipid-dependent oligomerization of Syntaxin affects the binding of Munc18 to Syntaxin and Syntaxin/SNAP25 complexes. I describe that the different modes of Munc18-Syntaxin and Munc18-Syntaxin/SNAP25 interaction have consequences on Munc18’s effect on Syntaxin’s oligomerization and orientation relative to the membrane surface, as well as on docking and fusion of purified insulin granules. My experiments also indicate the existence of Munc18/Syntaxin/SNAP25 acceptor complex that can mediate membrane fusion. Using cryoEM, we were able to obtain low-resolution structures of Munc18/Syntaxin and Munc18/Syntaxin/SNAP25 complexes in model membranes, showcasing that the architectures of these two complexes are not the same. The existence of a functional Munc18/Syntaxin/SNAP25 acceptor complex is further supported by experiments, in which Syntaxin and SNAP25 were co-reconstituted in model membranes at different ratios and in which I utilized Munc18 mutations to show their effects on SNARE complex assembly.

PHD (Doctor of Philosophy)

SNARE proteins; membrane fusion; Syntaxin-1a; Munc18; exocytosis; membrane lipids

English

2026-07-21