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

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.

[1]  M. Brown,et al.  The low-density lipoprotein pathway and its relation to atherosclerosis. , 1977, Annual review of biochemistry.

[2]  J. Riordan,et al.  Multiple proteolytic systems, including the proteasome, contribute to CFTR processing , 1995, Cell.

[3]  Satoshi Omura,et al.  Degradation of CFTR by the ubiquitin-proteasome pathway , 1995, Cell.

[4]  H. Shibuya,et al.  A BMP-inducible gene, dlx5, regulates osteoblast differentiation and mesoderm induction. , 1999, Developmental biology.

[5]  M. Matsuo‐Takasaki,et al.  Distinct roles for distal‐less genes Dlx3 and Dlx5 in regulating ectodermal development in Xenopus , 2001, Molecular reproduction and development.

[6]  A. Klug,et al.  A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter , 2001, Nature Biotechnology.

[7]  E. Eisenberg,et al.  Clathrin exchange during clathrin-mediated endocytosis , 2001, The Journal of cell biology.

[8]  F. Brodsky,et al.  Biological basket weaving: formation and function of clathrin-coated vesicles. , 2001, Annual review of cell and developmental biology.

[9]  W. Almers,et al.  Imaging actin and dynamin recruitment during invagination of single clathrin-coated pits , 2002, Nature Cell Biology.

[10]  W. Marshall,et al.  Analysis of Clathrin-mediated Endocytosis of Epidermal Growth Factor Receptor by RNA Interference*[boxs] , 2004, Journal of Biological Chemistry.

[11]  Kartik Chandran,et al.  Endocytosis by Random Initiation and Stabilization of Clathrin-Coated Pits , 2004, Cell.

[12]  W. Almers,et al.  Neural Wiskott Aldrich Syndrome Protein (N-WASP) and the Arp2/3 complex are recruited to sites of clathrin-mediated endocytosis in cultured fibroblasts. , 2004, European journal of cell biology.

[13]  S. Schmid,et al.  SNX9 regulates dynamin assembly and is required for efficient clathrin-mediated endocytosis. , 2005, Molecular biology of the cell.

[14]  Jeffrey C. Miller,et al.  Highly efficient endogenous human gene correction using designed zinc-finger nucleases , 2005, Nature.

[15]  David Zenisek,et al.  Coupling between Clathrin-Coated-Pit Invagination, Cortactin Recruitment, and Membrane Scission Observed in Live Cells , 2005, Cell.

[16]  David G. Drubin,et al.  A Modular Design for the Clathrin- and Actin-Mediated Endocytosis Machinery , 2005, Cell.

[17]  Danqing Zhu,et al.  Mutations in the pleckstrin homology domain of dynamin 2 cause dominant intermediate Charcot-Marie-Tooth disease , 2005, Nature Genetics.

[18]  P. Camilli,et al.  GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission , 2006, Nature.

[19]  S. Simon,et al.  Dynamics of clathrin and adaptor proteins during endocytosis. , 2006, American journal of physiology. Cell physiology.

[20]  Å. Engqvist-Goldstein,et al.  A Hip1R–cortactin complex negatively regulates actin assembly associated with endocytosis , 2007, The EMBO journal.

[21]  M. Matsui,et al.  LC3, an Autophagosome Marker, Can be Incorporated into Protein Aggregates Independent of Autophagy: Caution in the Interpretation of LC3 Localization , 2007, Autophagy.

[22]  Darius Moradpour,et al.  Replication of hepatitis C virus , 2007, Nature Reviews Microbiology.

[23]  J. Renauld,et al.  JAK kinases overexpression promotes in vitro cell transformation , 2008, Oncogene.

[24]  S. Schmid,et al.  Real-Time Visualization of Dynamin-Catalyzed Membrane Fission and Vesicle Release , 2008, Cell.

[25]  S. Schmid,et al.  Isoform and splice-variant specific functions of dynamin-2 revealed by analysis of conditional knock-out cells. , 2008, Molecular biology of the cell.

[26]  Michael Z. Lin,et al.  Improving the photostability of bright monomeric orange and red fluorescent proteins , 2008, Nature Methods.

[27]  Emanuele Cocucci,et al.  Distinct Dynamics of Endocytic Clathrin-Coated Pits and Coated Plaques , 2009, PLoS biology.

[28]  T. Kirchhausen,et al.  Imaging endocytic clathrin structures in living cells. , 2009, Trends in cell biology.

[29]  R. Jaenisch,et al.  Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases , 2009, Nature Biotechnology.

[30]  D. Loerke,et al.  Endocytic accessory proteins are functionally distinguished by their differential effects on the maturation of clathrin-coated pits. , 2009, Molecular biology of the cell.

[31]  Sandra L Schmid,et al.  Cargo and Dynamin Regulate Clathrin-Coated Pit Maturation , 2009, PLoS biology.

[32]  Marcel Mettlen,et al.  Dissecting dynamin's role in clathrin-mediated endocytosis. , 2009, Biochemical Society transactions.

[33]  Gaudenz Danuser,et al.  Cargo- and adaptor-specific mechanisms regulate clathrin-mediated endocytosis , 2010, The Journal of cell biology.

[34]  Thuy D. Vo,et al.  Transient cold shock enhances zinc-finger nuclease–mediated gene disruption , 2010, Nature Methods.

[35]  Lei Zhang,et al.  Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. , 2010, Genome research.

[36]  E. Rebar,et al.  Genome editing with engineered zinc finger nucleases , 2010, Nature Reviews Genetics.

[37]  Thuy D Vo,et al.  Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures , 2011, Nature Methods.