Abstract

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. We study this process by live-cell microscopy in yeast and mammalian cells. The yeast studies have revealed a regular sequence of events necessary for endocytic vesicle formation involving some 60 proteins, which induce a highly choreographed series of changes in membrane geometry, ultimately resulting in scission and vesicle release. To analyze endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we previously targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus (1). The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells. The high regularity of CME dynamics in these genome-edited cells has now been exploited to interrogate by RNAi and chemical inhibition how endocytic proteins and the actin cytoskeleton contribute to endocytic vesicle formation and to the regulation and dynamics of the process. These studies demonstrate the importance of actin assembly for robust endocytic dynamics in mammalian cells.

1. Doyon JB, Zeitler B, Cheng J, Cheng AT, Cherone JM, Santiago Y, Lee AH, Vo TD, Doyon Y, Miller JC, Paschon DE, Zhang L, Rebar EJ, Gregory PD, Urnov FD, Drubin DG. (2011) Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat Cell Biol. 13(3):331-7.