In our previous study, we showed that PC12 cell lines stably expressing synaptotagmin (Syt) VII have greater ability to release hormones Ca2+-dependently than the original PC12 cells. However, the precise molecular mechanism of the enhancement of hormone secretion by Syt VII has never been elucidated. In this study, we established a PC12 cell line that stably expresses Syt VII-green fluorescent protein (Syt VII-GFP) or its Ca2+-binding-site-deficient mutant (D172N/D303N substitutions; Syt VII-DN-GFP), and examined the effect of Syt VII-GFP expression on the kinetics of dense-core vesicle exocytosis by total internal reflection fluorescence (TIRF) microscopy. Both Syt VII-GFP and Syt VII-DN-GFP co-localized well with dense-core vesicle markers, monomeric red fluorescent protein (mRFP)-tagged neuropeptide Y (NPY-mRFP) and cyan fluorescent protein (CFP)-tagged tissue plasminogen activator (tPA-CFP). Expression of Syt VII-GFP enhanced the number of dense-core vesicle exocytotic events, whereas expression of Syt VII-DN-GFP or knockdown of Syt VII-GFP with specific small interfering RNA (siRNA) attenuated the number of exocytotic events. Monitoring individual tPA-CFP release events revealed that "full release" events are increased in Syt VII-GFP-expressing cells, but not in Syt VII-DN-GFP-expressing or Syt VII-silenced cells. Our data indicate that Syt VII modulates the kinetics of Ca2+-dependent dense-core vesicle exocytosis in neuroendocrine PC12 cells, possibly by modulating fusion pore opening.
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