Synaptotagmin VII modulates the kinetics of dense-core vesicle exocytosis in PC12 cells.
In our previous study, we showed that PC12 cell lines stably expressing synaptotagmin (Syt) VII have greater ability to release hormones Ca(2+)-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 Ca(2+)-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 Ca(2+)-dependent dense-core vesicle exocytosis in neuroendocrine PC12 cells, possibly by modulating fusion pore opening.
Tsuboi
Takashi
T
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Fukuda
Mitsunori
M
D000818:Animals; D002118:Calcium / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009478:Neuropeptide Y / Q000235:genetics / Q000378:metabolism; D016716:PC12 Cells; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D050857:Synaptotagmins / Q000235:genetics / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000378:metabolism
D008164:Luminescent Proteins; D009478:Neuropeptide Y; D011993:Recombinant Fusion Proteins; D050857:Synaptotagmins; D010959:Tissue Plasminogen Activator; D002118:Calcium