Figure 3: Syntaxin1a and Munc18-1 are in an immobile complex in resting synapses.

(a) FCS and FCCS were used to probe syntaxin1a–munc18-1 interaction in synapses on a rapid timescale. The dimensions of the excitation spot (W₀ and Z₀) are determined experimentally using purified fluorescent proteins (Supplementary Fig. 3). FCS provides high spatiotemporal resolution of protein diffusion rate and mode (i) interaction (ii) and reaction kinetics (iii). (b) Representative cortical neuron transfected with munc18-1-EGFP (left panel) showing varicosities (white circles) where measurements were acquired. Scale bar, 5 μm. Example spontaneous mEPSC trace (right panel). (c) Representative autocorrelation fits of unfused EGFP and mCherry molecules in neuronal synapses, autocorrelation trace of the same data (insert). No cross-correlation could be detected. (d) Fit residuals of the data in c. (e) Box plot of EGFP and mCherry diffusion in resting synapses. (f) Representative autocorrelation and cross-correlation fit (blue) result of syntaxin1a (green) and munc18-1 (red), raw autocorrelation trace of the same data (insert). (h) Fit residuals of the data in f. (i) Box plot of syntaxin1a and munc18-1 diffusion data in resting synapses (simplified bar charts showing means and s.e.m. for each treatment are presented in Supplementary Fig. 3). The rates of diffusion were calculated from the derived autocorrelation curves; centre lines represent the median; cross indicates the mean; box limits indicate the 25th and 75th percentiles and whiskers extend to minimum and maximum points. Notches are 95% confidence intervals that two medians differ. There are no statistical differences between groups. (j) Graphical representation of the calculated deviation from Brownian motion of each sample diffusion data. EGFP and mCherry in synapses diffuse with a Brownian motion (indicated by the blue dashed line), indicative of free diffusion in the synaptic cytosol, whereas syntaxin1a and munc18-1 molecules deviate from this behavior, indicative of membrane anchoring and thus interaction (as monomeric munc18-1 is a soluble protein). In this plot (left), the central black bars indicate the median, with error bars indicating s.e.m. Right panel: plotting ‘Deviation from Brownian motion’ against diffusion rate reveals two populations of mCherry-munc18-1 behaviours in synapses; both populations have low diffusion rates but differ in diffusional behavior—one group of molecules diffuse in a directed manner and the other appears caged.