Fig. 1: Syt-1 forms clusters on lipid bilayer.
A DOPC:DOPS:DAG:PI(4,5)P2 (71:25:2:2) bilayer is monitored by TIRF microscopy. A When fluorescent wildtype Syt-1 (79–421) is added in solution, protein clusters are observed on the bilayer. A representative image of these clusters is presented here. The buffer is 50 mM HEPES, 140 mM KCl, 10% glycerol, and 1 mM TCEP at pH 7.4. B In absence of Syt-1, no fluorescence is observed. C, D Representative step-bleaching profiles of Syt-1 clusters of different sizes: small cluster (C), where the bleaching steps and corresponding numbers of dyes are indicated, and large cluster (D), where the bleaching profile displays a smooth decay, which can be fitted with an exponential function (red solid line): \(I\left(t\right)={I}_{0}{e}^{-t/{t}_{0}}+B\), where I0 is the initial intensity at the beginning of bleaching, t0 is the time constant of bleaching, and B is the background fluorescence. E The copy number of Syt-1 in each cluster can be determined by step-bleaching, and from the initial intensity (See Materials and Methods). The distribution of Syt-1 copy numbers in clusters displays a double-peak showing there are two types of clusters (n = 437).
