Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.
J Cell Biol. 2013 May 27;201(5):673-80. doi: 10.1083/jcb.201212103. Epub 2013 May 20.
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.
连接蛋白在膜运输中起着普遍的作用，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，连接蛋白相对于囊泡融合的时空动力学特征还没有很好地描述。研究最广泛的连接复合物是外泌体，它将囊泡定位到质膜上的特定位置。通过使用哺乳动物遗传替代策略，我们能够将荧光标记的 Sec8 组装到外泌体复合物中，通过生化、运输和形态学标准证明其具有功能。超灵敏的活细胞成像显示，Sec8-TagRFP 移动到来自内体再循环隔室的囊泡的细胞皮质上。令人惊讶的是，Sec8 一直与囊泡在一起，直到融合孔完全扩张，这支持了与 SNARE 融合机制的潜在偶联。细胞突起处 Sec8 的光漂白恢复荧光分析表明，相当一部分是不可移动的。此外，Sec8 动态地重新定位到膜扩展的部位，这表明它可能在早期细胞极化过程中对局部线索做出反应。
