Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells
Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells
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DOI：
10.1016/j.cell.2005.03.015
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发表时间：
2005-05-20
期刊：
影响因子：
64.5
通讯作者：
Zenisek, D
中科院分区：
文献类型：
--
作者：
Merrifield, CJ;Perrais, D;Zenisek, D
在网格蛋白介导的内吞作用过程中，膜分裂标志着载有货物的网格蛋白包被小窝（CCP）从细胞外环境中分离出来。在此，我们利用对pH敏感的货物进行活细胞成像，以秒为时间分辨率观察单个网格蛋白包被小窝处网格蛋白包被囊泡（CCV）的形成。我们发现网格蛋白包被小窝具有高度动态性，能够连续产生多个囊泡。通过交替使用消逝场和落射荧光照明，我们发现网格蛋白包被小窝的内陷和分裂紧密耦合，分裂与网格蛋白包被小窝从质膜的最大位移以及与皮层肌动蛋白 - 红色荧光蛋白（一种发动蛋白和F - 肌动蛋白结合蛋白）的招募峰值同时发生。最后，用松胞菌素 - B干扰肌动蛋白聚合会大幅降低膜分裂的效率，并影响网格蛋白包被小窝动力学的多个方面。我们提出，网格蛋白包被小窝的内陷、肌动蛋白聚合以及网格蛋白包被囊泡的形成是高度协调的，以实现高效的内吞作用。
During clathrin-mediated endocytosis, membrane scission marks the isolation of a cargo-laden clathrin-coated pit (CCP) from the cell exterior. Here we used live-cell imaging of a pH-sensitive cargo to visualize the formation of clathrin-coated vesicles (CCVs) at single CCPs with a time resolution of seconds. We show that CCPs are highly dynamic and can produce multiple vesicles in succession. Using alternating evanescent field and epifluorescence illumination, we show that CCP invagination and scission are tightly coupled, with scission coinciding with maximal displacement of CCPs from the plasma membrane and with peak recruitment of cortactin-DsRed, a dynamin and F-actin binding protein. Finally, perturbing actin polymerization with latrunculin-B drastically reduces the efficiency of membrane scission and affects many aspects of CCP dynamics. We propose that CCP invagination, actin polymerization, and CCV formation are highly coordinated for efficient endocytosis.