title: Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.
abstract: Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we 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. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.
authors.affiliation: 1] Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94720, USA.
authors.forename: Jeffrey B
authors.initials: JB
authors.lastname: Doyon
authors.forename: Bryan
authors.initials: B
authors.lastname: Zeitler
authors.forename: Jackie
authors.initials: J
authors.lastname: Cheng
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Cheng
authors.forename: Jennifer M
authors.initials: JM
authors.lastname: Cherone
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Santiago
authors.forename: Andrew H
authors.initials: AH
authors.lastname: Lee
authors.forename: Thuy D
authors.initials: TD
authors.lastname: Vo
authors.forename: Yannick
authors.initials: Y
authors.lastname: Doyon
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: David E
authors.initials: DE
authors.lastname: Paschon
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Rebar
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.forename: David G
authors.initials: DG
authors.lastname: Drubin
mesh_terms: D000818:Animals; D001483:Base Sequence; D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D016678:Genome; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
chemical_list: D002966:Clathrin; D049452:Green Fluorescent Proteins; D034285:Dynamin II