Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein
Age-dependent Preferential Dense-Core Vesicle Exocytosis in Neuroendocrine Cells Revealed by Newly Developed Monomeric Fluorescent Timer Protein
复制标题
DOI：
10.1091/mbc.e09-08-0722
复制
发表时间：
2010-01-01
影响因子：
3.3
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
--
作者：
Tsuboi, Takashi;Kitaguchi, Tetsuya;Miyawaki, Atsushi
虽然很明显，神经内分泌细胞中只有少数几个分泌小泡有资格与质膜融合，并将其内容释放到细胞外空间，但调节其胞吐的分子机制尚不清楚。例如，分泌囊泡是根据年龄随机排出还是优先排出，一直存在争议。使用一种新开发的基于蛋白质的荧光计时器，单体Kusabira Green Orange(MK-GO)，它以可预测的时间过程改变颜色，在这里，我们展示了小GTPase Rab27A效应器调节PC12细胞中分泌囊泡的年龄相关的胞吐。当用MK-GO标记的神经肽Y或组织型纤溶酶原激活剂标记小泡时，观察到带有绿色或红色荧光的点状结构。高[K+]刺激可诱导新的(绿色)荧光分泌小泡排出，而不是旧的(红色)小泡排出。正向和负向调节胞吐的RabPhilin和Synaptoagmin-like Protein 4-a(SLP4-a)的过度表达或缺失分别以不同的方式干扰了分泌囊泡的年龄依赖性胞吐。我们的结果表明，Rab27A的两个效应物RabPhilin和SLP4-a的协同作用是调节分泌途径所必需的。
Although it is evident that only a few secretory vesicles accumulating in neuroendocrine cells are qualified to fuse with the plasma membrane and release their contents to the extracellular space, the molecular mechanisms that regulate their exocytosis are poorly understood. For example, it has been controversial whether secretory vesicles are exocytosed randomly or preferentially according to their age. Using a newly developed protein-based fluorescent timer, monomeric Kusabira Green Orange (mK-GO), which changes color with a predictable time course, here we show that small GTPase Rab27A effectors regulate age-dependent exocytosis of secretory vesicles in PC12 cells. When the vesicles were labeled with mK-GO-tagged neuropeptide Y or tissue-type plasminogen activator, punctate structures with green or red fluorescence were observed. Application of high [K+] stimulation induced exocytosis of new (green) fluorescent secretory vesicles but not of old (red) vesicles. Overexpression or depletion of rabphilin and synaptotagmin-like protein4-a (Slp4-a), which regulate exocytosis positively and negatively, respectively, disturbed the age-dependent exocytosis of the secretory vesicles in different manners. Our results suggest that coordinate functions of the two effectors of Rab27A, rabphilin and Slp4-a, are required for regulated secretory pathway.