Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells

被引：179

作者：

Doyon, Jeffrey B. ^{[1
]}

Zeitler, Bryan ^{[2
]}

Cheng, Jackie ^{[1
]}

Cheng, Aaron T. ^{[1
]}

Cherone, Jennifer M. ^{[2
]}

Santiago, Yolanda ^{[2
]}

Lee, Andrew H. ^{[2
]}

Vo, Thuy D. ^{[2
]}

Doyon, Yannick ^{[2
]}

Miller, Jeffrey C. ^{[2
]}

Paschon, David E. ^{[2
]}

Zhang, Lei ^{[2
]}

Rebar, Edward J. ^{[2
]}

Gregory, Philip D. ^{[2
]}

Urnov, Fyodor D. ^{[2
]}

Drubin, David G. ^{[1
]}

机构：

[1] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA

[2] Sangamo BioSci Inc, Richmond, CA 94804 USA

来源：

NATURE CELL BIOLOGY | 2011年 / 13卷 / 03期

关键词：

ZINC; GENE; PROTEASOME; PROTEINS; COMPLEX; CFTR;

D O I：

10.1038/ncb2175

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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.

引用

页码：331 / U327

页数：20

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