Molecular Mechanisms Underlying The Protection Of SNARE Complex Assembly By Munc18-1 And Munc13-1

The information transfer between neurons depends on the release of neurotransmitters.Synaptic vesicle exocytosis is an essential process in neurotransmitter release.The core molecular machinery mediating synaptic exocytosis is the soluble N-ethylmaleimidesensitive factor(NSF)attachment protein(SNAP)receptor(SNARE)complex,which is assembled by SNAREs Syntaxin-1,SNAP-25,and Synaptobrevin-2.The zippering of Synaptobrevin-2 on synaptic vesicles together with Syntaxin-1 and SNAP-25 on the plasma membrane brings synaptic vesicles and the plasma membrane into close proximity,leading to SNARE complex formation that catalyzes membrane fusion.After membrane fusion,NSF and α-SNAP disassemble the SNARE complex to recycle the SNAREs for another round of fusion.However,NSF and α-SNAP can also disassemble the SNARE complex before fusion,leading to the abrogation of membrane fusion.Recent studies have shown that Munc18-1and Munc13-1,regulators of SNARE complex assembly,can protect SNARE complex assembly in the presence of NSF and α-SNAP.Although an increasing number of studies have reported the molecular mechanisms by which Munc18-1 and Munc13-1 regulate SNARE complex assembly,the molecular mechanisms underlying their protection of SNARE complex assembly from disassembly by NSF and α-SNAP during membrane fusion remain unclear.Using several approaches such as F(?)rster resonance energy transfer(FRET)experiments,fluorescence anisotropy assays,dynamic light scattering,confocal microscopy,and sedimentation-based assays,the underlying mechanisms of Munc18-1 and Munc13-1protective roles in SNARE complex assembly were investigated,and the results are as follows:(1)The protective roles of Munc18-1 and Munc13-1 in SNARE complex assembly were verified by FRET experiments,revealing that the protective roles of Munc18-1 and Munc13-1 are strictly relying on the Mun18-1–Munc13-1-regulated pathway of SNARE complex assembly,rather than simply binding to the assembled SNARE complex;(2)Separate studies on the protective roles of Munc18-1 and Munc13-1 revealed that Munc18-1 and Munc13-1 each,independently,have protective effects during SNARE complex assembly,and revealed that Munc18-1 and Munc13-1 can jointly protect SNARE complex assembly to enhance protection efficiency;(3)Elucidated that the protective effect of Munc18-1 relies on the interaction with the Cterminal part of Synaptobrevin-2 during SNARE complex assembly,and found that the protective effect of Munc13-1 is attributed to its ability in templating the assembling SNAREs;(4)Fluorescence anisotropy assays were performed to investigate the interaction between α-SNAP and the SNARE complex,revealing the inability of α-SNAP to bind properly to the SNARE complex assembled through the Munc18-Munc13-regulated pathway,thus elucidating the potential molecular mechanism by which this assembly pathway resists NSF and α-SNAP;(5)Confocal microscopy was applied to screen the conditions that induce liquid-liquid phase separation of SNAREs and the SNARE complex,revealing that Munc18-1 can be recruited into the condensed droplets formed by liquid-liquid phase separation of the SNARE complex and protects liquid-liquid phase separation of the SNARE complex from inhibition by α-SNAP.In conclusion,this thesis shed new light on understanding the protective mechanisms of Munc18-1 and Munc13-1 in SNARE complex-mediated synaptic exocytosis.