Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion.
Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion.
复制标题
DOI：
10.1083/jcb.201212103
复制
发表时间：
2013-05-27
期刊：
影响因子：
--
通讯作者：
Toomre D
中科院分区：
文献类型：
--
作者：
Rivera-Molina F;Toomre D
活细胞成像的外囊亚基Sec8揭示了蛋白质的时空动态如何与其在囊泡融合中的作用相关。系绳在膜转运过程中起着普遍的作用，并影响囊泡附着的特异性。与可溶性N-乙基马来酰亚胺敏感的融合附着蛋白受体(SNARES)不同，与囊泡融合相关的系链的时空动力学特征很差。研究最广泛的系链复合体是外囊，它在空间上将囊泡定位于质膜上的位置。通过使用哺乳动物基因替换策略，我们能够将荧光标记的Sec8组装到外囊复合体中，通过生化、运输和形态标准证明它具有功能。超灵敏的活细胞成像显示，Sec8-TagRFP以小泡的形式移动到细胞皮质，小泡优先起源于内吞循环室。令人惊讶的是，Sec8仍然带有囊泡，直到融合孔完全扩张，支持与SNARE融合机制的潜在耦合。光漂白后细胞突起处Sec8的荧光恢复分析表明，很大一部分是静止的。此外，Sec8动态地重新定位到膜扩张的位置，这表明它可能在细胞早期极化过程中对局部信号做出反应。
Live-cell imaging of the exocyst subunit Sec8 reveals how the protein’s spatiotemporal dynamics correlate with its roles in vesicle fusion. Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.
登录
查看更多内容
影响因子：
6.9
作者：
Pecenková T;Hála M;Kulich I;Kocourková D;Drdová E;Fendrych M;Toupalová H;Zársky V
通讯作者：
Zársky V
影响因子：
64.5
作者：
NOVICK, P;FIELD, C;SCHEKMAN, R
通讯作者：
SCHEKMAN, R
影响因子：
16.2
作者：
Hsu, SC;Hazuka, CD;Scheller, RH
通讯作者：
Scheller, RH
影响因子：
64.5
作者：
Jaiswal JK;Rivera VM;Simon SM
通讯作者：
Simon SM
DOI：
10.1111/j.1600-0854.2012.01353.x
发表时间：
2012-07
期刊：
Traffic (Copenhagen, Denmark)
影响因子：
--
作者：
Heider MR;Munson M
通讯作者：
Munson M