Figure 4.

Synaptotagmin-1 binding to PIP2–syntaxin-1A clusters in artificial stacked supported membranes generated by spin-coating on glass surfaces. (a) Representative confocal images of membranes (69% DOPC, 30% cholesterol, 1% PIP2) containing syntaxin-1A_257–288 labeled with Atto647N (SxTMH; magenta), which were incubated with 100 nM C2AB fragment (labeled with Alexa fluor 488; C2AB-AF488; green) in the absence (purple) or after addition of 200 µM Ca²⁺ (blue) and after chelating previously added Ca²⁺ with EGTA (orange). (b) Fluorescence intensity profiles through the lines marked in a (see Fig. 1). (c,d) Fluorescence (peak counts) between (c) and at (d) the puncta determined from the different images of C2AB-AF488 (± range; 6 positions, 3 independent bilayers; *: P = 0.006 – 0.01, two-sided t-test; see Supp. Fig. 3a for distributions of C2AB binding to the puncta). (e,f) Same as panels a and b in absence of Ca²⁺, but now with fluorescent Top-Fluor PIP2 (0.1 mol% TF-PIP2; green) instead of C2AB. PIP2 was clearly enriched in the SxTMH clusters. (scale bars, 2 µm)