Fig. 4: Kinetic observation of dissociation and association of the clusters of the Syt-1F349A clamping mutant in real-time.
A Representative TIRF image of clusters formed with Syt-1 mutant F349A mutant. B The copy number of Syt-1 F349A mutant in each cluster can be determined by step-bleaching, and from the initial intensity. The distribution of copy numbers of Syt-1 F349A clusters displays a single-peak with small cluster size (n = 573). The blue dashed line represents the distribution of wildtype Syt-1 clusters, which serves as a reference. C Determination of unit koff for dissociation of Syt-1 F349A clusters. The initial dissociation slopes were plotted as function of initial sizes, N(0), and displayed a proportional relationship (n = 15). D Averaged growth curves of Syt-1 F349A cluster and comparison with theoretical predictions. All the curves were averaged into a single one (filled red diamonds; n = 12). Growth predictions (black solid line) from Eq. 11 of the averaged growth curves over the whole process, using the kon from (E) and the koff from dissociation data in (C), displayed good matches with the averaged experimental curves. E The initial slope of the averaged growth curve of Syt-1 F349A clusters allows the determination of kon for the formation of Syt-1 F349A clusters (same as Fig. 3E) (n = 12). F Summary of kinetic and thermodynamic parameters of the oligomerization of wild-type (WT) Syt-1 and the F349A mutant (n = 4 for surface densities).
