Merrifield Christien J, Perrais David, Zenisek David
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom.
Laboratoire de Physiologie Cellulaire de la Synapse, CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1 rue Camille Saint Saëns, 33077 Bordeaux Cedex, France.
Cell. 2005 May 20;121(4):593-606. doi: 10.1016/j.cell.2005.03.015.
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.
在网格蛋白介导的内吞作用过程中，膜分裂标志着一个载有货物的网格蛋白包被小窝（CCP）与细胞外部分离。在这里，我们使用对pH敏感的货物进行活细胞成像，以秒级的时间分辨率可视化单个CCP处网格蛋白包被囊泡（CCV）的形成。我们表明，CCP具有高度动态性，并且可以连续产生多个囊泡。使用交替的倏逝场和落射荧光照明，我们表明CCP内陷和分裂紧密耦合，分裂与CCP从质膜的最大位移以及动力蛋白和F-肌动蛋白结合蛋白cortactin-DsRed的峰值募集同时发生。最后，用拉特肌动蛋白-B干扰肌动蛋白聚合会大大降低膜分裂的效率，并影响CCP动力学的许多方面。我们提出，CCP内陷、肌动蛋白聚合和CCV形成高度协调以实现高效的内吞作用。
