Figure 6: Syntaxin1a and munc18-1 interaction mode switches dynamically post exocytosis.

(a) Left panel—FCS autocorrelation data show identical diffusion rates of syntaxin1a and munc18-1 in synapses before and during maximal stimulation. Right panel—Syntaxin1a and munc18-1 molecular diffusion models differ before and during maximal stimulation, with a 100% enrichment of molecular complexes exhibiting caged motion and low diffusion rates during stimulation (lower left quandrant of graph). (b) Destabilizing the N-peptide interaction results in a dissociation of syntaxin1a and munc18-1 specifically in synapses but a rapid interaction mode switch occurs during maximal stimulation. Left panel—FCS autocorrelation data deliver significantly different diffusion rates for munc18-1 and syntaxin1a in resting synapse, that converge during depolarizations. Right panel—EGFP-syntaxin1a^S14ECR and munc18-1 molecular diffusion during maximal stimulation: munc18-1 molecules significantly slow during stimulation. Inset: expanded axes scales showing munc18-1 and EGFP-syntaxin1a^S14ECR diffusion rates and modes in resting synapses. Symbols are as for a. Notably, the slow, caged population of molecules is absent during stimulations, in contrast to the wild-type syntaxin1a system. (c) Representative FLIM analyses of FRET between wt EGFP-syntaxin1a (donor) and mCherry-munc18-1 (acceptor); molecular interaction was reported as a mean donor fluorescence lifetime shorter than the mean non-FRET control across neurons (blue line); this threshold (solid vertical line) did not alter during maximal stimulation. Reduced FRET was detected between EGFP-syntaxin1a^S14ECR and mCherry-munc18-1 before stimulation (red line) but this increased significantly after maximal stimulation (dashed grey line). This induction of interaction was abolished in the presence of NEM to inhibit SNARE disassembly post exocytosis (grey line). (d) FLIM maps illustrating spatial restriction of syntaxin1a^S14ECR–munc18-1 interactions to varicosities after maximal stimulation are dependent on SNARE disassembly. Colour bar indicates donor fluorescence lifetime—shorter (blue) values indicate FRET and direct protein–protein interaction. Scale 1 ns (blue)—2 ns (red). Scale bar, 5 μm. (e) Cartoon incorporating our data into a refined model of the syntaxin1a–munc18-1 interaction pathway.