Tsuboi Takashi, Fukuda Mitsunori
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Genes Cells. 2007 Apr;12(4):511-9. doi: 10.1111/j.1365-2443.2007.01070.x.
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.
在我们之前的研究中，我们发现稳定表达突触结合蛋白（Syt）VII的PC12细胞系比原始PC12细胞具有更强的Ca(2+)依赖性激素释放能力。然而，Syt VII增强激素分泌的确切分子机制尚未阐明。在本研究中，我们建立了一个稳定表达Syt VII-绿色荧光蛋白（Syt VII-GFP）或其Ca(2+)结合位点缺陷突变体（D172N/D303N替换；Syt VII-DN-GFP）的PC12细胞系，并通过全内反射荧光（TIRF）显微镜检查了Syt VII-GFP表达对致密核心囊泡胞吐动力学的影响。Syt VII-GFP和Syt VII-DN-GFP均与致密核心囊泡标记物、单体红色荧光蛋白（mRFP）标记的神经肽Y（NPY-mRFP）和青色荧光蛋白（CFP）标记的组织纤溶酶原激活剂（tPA-CFP）共定位良好。Syt VII-GFP的表达增加了致密核心囊泡胞吐事件的数量，而Syt VII-DN-GFP的表达或用特异性小干扰RNA（siRNA）敲低Syt VII-GFP则减少了胞吐事件的数量。监测单个tPA-CFP释放事件发现，在表达Syt VII-GFP的细胞中“完全释放”事件增加，但在表达Syt VII-DN-GFP或Syt VII沉默的细胞中则没有增加。我们的数据表明，Syt VII可能通过调节融合孔开放来调节神经内分泌PC12细胞中Ca(2+)依赖性致密核心囊泡胞吐的动力学。
