Fig. 3.

Disrupting Syt1 oligomerization occludes Ca²⁺ control of exocytosis in PC12 cells. (A) Representative Western blot showing that untransfected F7 cells lack Syt1 expression along with reduced expression of Syt9, a closely related isoform. It further shows that the rescue of Syt1 expression in the F7 cells with Syt1^WT or Syt1^F349A leads to no alteration in the expression of other Syt isoforms and the SNARE proteins (quantitative analysis is provided in SI Appendix, Fig. S2). (B) Representative immunofluorescence analysis using anti-Syt1 antibody shows a similar distribution of the endogenous Syt1 in PC12 cells and the recombinant Syt1^WT or Syt1F^349A expressed in F7 cells. (C) Effect of Syt1^349A mutation on exocytosis assessed by VAMP2-pHluorin TIRFM imaging of single-vesicle fusion events under basal (constitutive) conditions and after KCl-induced depolarization (stimulated with Ca²⁺). Disrupting the Syt1 oligomer dysregulated exocytosis, dramatically increasing (approximately threefold) the typically low levels of constitutive exocytosis and effectively abolishing the Ca²⁺ control of exocytosis. The majority of the exocytotic events with Syt1F^349A rescue are constitutive in nature, as disrupting Ca²⁺ binding (Syt1^3A/F349A) shows little or no effect under either basal or stimulated conditions. ***P < 0.005 Wilcoxon–Mann–Whitney test. n.s., not significant. (D) EM of serial sections was used to characterize the distribution and docking of the dense-core vesicles (DCVs). (Left) Representative micrograph of untransfected (Top) or Syt1^F349A-expressing (Bottom) F7 cells. The DCVs are marked by red arrows. (Right) Cumulative probability plots of DCV distribution relative to the PM. Untransfected F7 cells (red) showed a pronounced right shift in the distribution, with fewer vesicles locating near the PM (P < 0.001 vs. PC12 cells, Kolmogorov–Smirnov test). This impairment in vesicle docking is partially rescued by the expression of either Syt1^WT or Syt1^F349A (blue and green, respectively). Data are from n = 14–32 cells for each condition, with 55 ± 38 vesicles per cell (mean ± SD) from three independent preparations.