Local protein dynamics during microvesicle exocytosis in neuroendocrine cells.
Local protein dynamics during microvesicle exocytosis in neuroendocrine cells.
复制标题
DOI：
10.1091/mbc.e17-12-0716
复制
发表时间：
2018-08-01
影响因子：
3.3
通讯作者：
Taraska JW
中科院分区：
文献类型：
--
作者：
Somasundaram A;Taraska JW
钙触发的胞吐作用是许多生理过程的关键，包括神经递质和神经元和内分泌细胞释放的激素。数十种蛋白质调节胞吐作用，但这些因素在囊泡融合过程中的时间和空间动态仍不清楚。在这里，我们使用全内反射荧光显微镜来可视化活培养的神经内分泌 PC12 细胞中小突触样微泡胞吐作用单个位点的局部蛋白质动态。我们采用双色成像来同时观察膜融合（使用标记有 pHluorin 的囊泡乙酰胆碱 ACh 转运蛋白）和胞吐作用周围时刻相关蛋白的动态。我们的实验表明，许多蛋白质，包括 SNAREs syntaxin1 和 VAMP2、SNARE 调节剂 Tomosyn 和 Rab 蛋白，在融合位点预聚集并在融合时迅速丢失。 ATPase N-乙基马来酰亚胺敏感因子在融合时局部募集。有趣的是，含有 Bin-Amphiphyn-Rvs 结构域的内吞蛋白 amphiphyn1、syndapin2 和内亲素被动态招募到融合位点，并减缓融合位点囊泡膜结合货物的损失。 GTPases dynamin1 和 dynamin2 的过表达对囊泡膜蛋白动力学也有类似的影响。这些结果表明，参与经典网格蛋白介导的内吞作用的蛋白质可以调节突触样微泡的胞吐作用。我们的研究结果提供了对活细胞中微泡融合单个位点的胞吐作用和内吞作用的许多关键因素的动力学、组装和机制作用的见解。
Calcium-triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine ACh transporter tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are preclustered at fusion sites and rapidly lost at fusion. The ATPase N-ethylmaleimide–sensitive factor is locally recruited at fusion. Interestingly, the endocytic Bin-Amphiphysin-Rvs domain–containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the overexpression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.