Fig. 2: Kinetic observation of dissociation of Syt-1 clusters in real-time.

A Schematic illustration of dissociation of wildtype Syt-1 clusters after flowing in Ca²⁺-free buffer containing 50 mM HEPES, 140 mM KCl, 10% glycerol, and 1 mM TCEP. B Representative TIRF images of the Syt-1 cluster at different time during the process. Dissociation starts at t = 160 s upon the flow of buffer. C Representative curves of the fluorescent intensity of Syt-1 clusters versus time showing the dissociation of clusters of different sizes, “small” clusters are in the left three panels (in red) and “large” clusters in the right two panels (green). The blue dashed lines in the left two panels indicate the step-dissociation of small Syt-1 clusters. N(0) indicates the estimated initial size of the cluster. D Schematic illustration of the two pathways that Syt-1 may utilize for dissociation: the free end disassembly model in which dissociation can only occur at the two ends, and the isotropic internal disassembly model in which every Syt-1 molecule can dissociate from the cluster with equal probability. The initial disassembly slope allows discriminating between the two models. E Determination of the slopes of the initial dissociation of the 5 representative clusters shown in (C). The intensity, I(t), versus t curves were converted into the copy number, N(t), versus t curves. (F) Determination of unit k_off of dissociation of WT Syt-1 clusters. The initial slopes were plotted as function of initial sizes, N(0), and the data were fitted with the free end disassembly model (purple dashed line with R² = 0) and the isotropic disassembly model (black solid line with R² = 0.98), respectively. The R² values suggests the experimental data is best described by the isotropic internal dissociation process (n = 27).