Fig. 3: Kinetic observation of association of Syt-1 clusters in real-time.
A Schematic illustration of association of wildtype Syt-1 clusters after flowing in Syt-1 solution. B Representative TIRF images of the Syt-1 cluster at different time points during the process. Association starts at t = 390 s upon the flow of the protein solution. C Representative curves of the fluorescent intensity of Syt-1 versus time showing the association of Syt-1 that grew into clusters of different sizes. The growth of small clusters was marked in red (left two panels), while large clusters in green (right two panels). The blue dashed lines in the left two panels indicate the step-wise association of individual Syt-1 molecules. D Averaged growth curves of wildtype Syt-1 cluster and comparison with theoretical predictions. According the bimodal size distribution, all growth curves were divided into two classes with a cutoff between 9 and 10 copies of Syt-1: a slow-growth class that result in small clusters, and a fast-growth class that result in large clusters. In each class, all the curves were averaged into a single one (filled red diamonds and filled green circles; n = 10 for small clusters and n = 9 for large clusters). Growth predictions (black solid lines) from Eq. 11 of the two averaged growth curves over the whole process, using the two kon from (E) and the single koff from dissociation data in Fig. 2F, displayed good matches with the averaged experimental curves. E Determination of kon of association of WT Syt-1 clusters on averaged growth curves of the clusters. In each class, kon were obtained from the initial growth of the mean cluster size (filled red diamonds and filled green circles, respectively), <N(t)>, versus t curves, which generated a characteristic size, <N > , for each class (n = 10 for small clusters and n = 9 for large clusters).
