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

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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.

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Figure 1: Editing of CLTA using ZFNs in BSC-1 cells.
Figure 2: Editing of CLTA using ZFNs in SK-MEL-2 cells.
Figure 3: Editing of DNM2 using ZFNs in SK-MEL-2 cells.
Figure 4: Simultaneous editing of both CLTA and DNM2 using ZFNs in SK-MEL-2 cells.
Figure 5: Fluorescence microscopy analysis of the hCLTAEN/DNM2EN cell line.

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Acknowledgements

We thank H. Nolla and A. Valeros for their cell-sorting expertise, A. Fischer and M. Yasukawa for help with cell culture, the Biological Imaging Facility for use of the Imaris software, the Sangamo Production group for technical assistance and members of the Drubin/Barnes lab for critical reading of this manuscript. We also thank T. Kirchhausen, L. Greene, and R. Tsien for providing the BSC-1 GFP–CLTA cell line, human CLTA plasmid, and TagRFP-T plasmid, respectively. J.B.D. and J.C. were supported by postdoctoral fellowships from the Jane Coffin Childs Memorial Fund and The Croucher Foundation, respectively. This work was supported by NIH grant R01 GM65462 to D.G.D.

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J.B.D, B.Z., J.C., A.T.C., T.D.V., Y.D., J.C.M., D.E.P., L.Z., E.J.R., P.D.G., F.D.U. and D.G.D designed the study and experiments. J.B.D, B.Z., J.C., A.T.C., J.M.C., Y.S. and A.H.L. performed the experiments. J.B.D., B.Z., J.C., A.T.C., J.M.C. and F.D.U. analysed the data. J.B.D., B.Z., J.C., A.T.C., F.D.U. and D.G.D. wrote the manuscript.

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Correspondence to David G. Drubin.

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B.Z., J.M.C., Y.S., A.H.L., T.D.V., Y.D., J.C.M., D.E.P., L.Z., E.J.R., P.D.G. and F.D.U are full-time employees of Sangamo BioSciences, Incorporated.

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Doyon, J., Zeitler, B., Cheng, J. et al. Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells. Nat Cell Biol 13, 331–337 (2011). https://doi.org/10.1038/ncb2175

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