Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells.
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.
Merrifield
Christien J
CJ
MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, United Kingdom. Electronic address: cjm@mrc-lmb.cam.ac.uk.
Perrais
David
D
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.
Zenisek
David
D
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06520.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000187:drug effects / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism*; D051356:Cortactin; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008840:Microfilament Proteins / Q000187:drug effects / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018715:Microscopy, Video; D011108:Polymers; D041701:Swiss 3T3 Cells; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D051356:Cortactin; C495869:Cttn protein, mouse; D008840:Microfilament Proteins; D011108:Polymers; D013844:Thiazoles; D053778:Thiazolidines; C037068:latrunculin B