Coupling between clathrin-coated-pit invagination, cortactin recruitment, and membrane scission observed in live cells

Cell. 2005 May 20;121(4):593-606. doi: 10.1016/j.cell.2005.03.015.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism*
  • Animals
  • Bridged Bicyclo Compounds, Heterocyclic / pharmacology
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Clathrin-Coated Vesicles / drug effects
  • Clathrin-Coated Vesicles / metabolism*
  • Coated Pits, Cell-Membrane / drug effects
  • Coated Pits, Cell-Membrane / metabolism*
  • Cortactin
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Mice
  • Microfilament Proteins / drug effects
  • Microfilament Proteins / metabolism*
  • Microscopy, Fluorescence
  • Microscopy, Video
  • Polymers
  • Swiss 3T3 Cells
  • Thiazoles / pharmacology
  • Thiazolidines

Substances

  • Actins
  • Bridged Bicyclo Compounds, Heterocyclic
  • Cortactin
  • Cttn protein, mouse
  • Microfilament Proteins
  • Polymers
  • Thiazoles
  • Thiazolidines
  • latrunculin B