technical report Rapid and efficient clathrin mediated endocytosis revealed in genome edited mammalian cells Jeffrey B Doyon1 3 Bryan Zeitler2 3 Jackie Cheng1 3 Aaron T Cheng1 3 Jennifer M Cherone2 Yolanda Santiago2 Andrew H Lee2 Thuy D Vo2 Yannick Doyon2 Jeffrey C Miller2 David E Paschon2 Lei Zhang2 Edward J Rebar2 Philip D Gregory2 Fyodor D Urnov2 and David G Drubin1 4 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 Clathrin mediated endocytosis CME is characterized by recruitment of clathrin triskelia composed of heavy and light chains to the plasma membrane and their assembly into polygonal cages that mediate membrane invagination During late stages of this process the GTPase dynamin is recruited to the necks of these invaginations1 where it functions in vesicle release through membrane scission and the subsequent internalization of plasma membrane molecules extracellular fluid and specific ligands from the environment 2 3 Three decades of evidence directly connect perturbation of CME to a broad range of pathophysiological outcomes including atherosclerosis4 disorders of the peripheral and central nervous system5 and infection by the hepatitis C virus6 The study of CME has been greatly advanced through the use of fluorescent fusion proteins7 In yeast direct genomic tagging of pairs of genes with different fluorescent tags has allowed researchers to define a very regular series of spatiotemporal events for CME in living cells8 Conversely in mammalian cells these events have been described as being much more heterogeneous and inefficient 9 12 but the inability to do precise genome editing has forced researchers to rely heavily on overexpression of fusion proteins1 9 14 Furthermore these fusion proteins are often derived from a different cell type or species from the cell type being studied and encode non native splice variants An accurate description of endocytic dynamics is the foundation for understanding the mechanism and regulation of this crucial process Because evidence from fields ranging from developmental and cell biology to plant physiology has shown that overexpression can result in protein mislocalization aggregation and altered signalling 15 20 we sought to re examine the highly dynamic process of CME using fluorescent fusion proteins expressed from their native loci RESULTS ZFN mediated editing as an efficient and accurate method for generating stable cell lines expressing fluorescent protein fusions from native loci To engineer precise gene fusions at endogenous loci we used zinc finger nucleases ZFNs 21 designed to cleave their target genes near the stop codons The resulting double strand breaks were mended by homologydirected repair using an exogenously supplied DNA donor that encodes a fluorescent tag Fig 1a In all cases ZFNs and donor constructs were cotransfected into the cells The high editing efficiency and accuracy of the open reading frame ORF addition process coupled with the expression of proteins bearing fluorescent markers allowed us to obtain the desired cells simply by using fluorescence activated cell sorting FACS without drug selection Supplementary Fig S1a and Table S1 top Single cellderived clones were then generated by limiting dilution We found that ZFN treatment had a negligible impact on cell survival Supplementary Department of Molecular and Cell Biology University of California Berkeley Berkeley California 94720 USA 2Sangamo BioSciences Incorporated Richmond California 94804 USA 3These authors contributed equally to this work 4 Correspondence should be addressed to D G D e mail drubin berkeley edu 1 Received 27 September 2010 accepted 21 December 2010 published online 06 February 2011 DOI 10 1038 ncb2175 nature cell biology advance online publication 1 TGA ATG mkCLTAEN 55 Exon 7 ZFN target TA X TA X TA E CL CL rC L BSC 1 Control mk b Mr K 70 mk CLTA Co c D ntr ol N t e c h n i c a l r e p o rt GFP CLTA CLTA RFP ZFN CLTA R 7 7 7 7 7 7 7 6 4 3 9 77 7 7 77 7 7 7 Left HA ZFN CLTA L RFP 35 Right HA CLTA Donor RFP Actin d f Time min 30 Control e 45 40 mkCLTAX 35 mkCLTAX Mean lifetime s 50 mkCLTAEN Control 15 30 25 20 mkCLTAEN mkCLTAX rCLTAX Figure 1 Editing of CLTA using ZFNs in BSC 1 cells a Schematic representation of the strategy for integration of RFP at the CLTA locus White boxes exons of CLTA HA donor plasmid region of homology to CLTA sequence Blue letters stop codon The grey box indicates the region of CLTA exon 7 surrounding the translation stop codon the underlined sequences indicate the recognition stretches of the individual zinc finger nucleases ZFN CLTA L and ZFN CLTA R respectively b Out out PCR showing targeted integration of RFP Control parental cell line mkCLTAEN singleallele CLTA RFP tagged genome edited line c Western blot analysis of cell lysates immunoblotted for CLTA RFP and actin Note RFP antibody crossreactivity with GFP mkCLTAX stable CLTA RFP overexpression line rCLTAX stable GFP CLTA rat brain derived overexpression line d Epifluorescence image of mkCLTAEN cells expressing endogenous CLTA RFP Scale bar 10 m e CCP dynamics were assessed in the indicated BSC 1 cell lines by quantifying the lifetime of fluorescently tagged CLTA proteins at the plasma membrane Data are means s e m Tracks 30 734 50 250 n 5 11 and 15 cells for the mkCLTAX mkCLTAEN and
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