Rapid and Efficient Clathrin-Mediated Endocytosis Revealed in Genome-Edited Mammalian Cells

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 The study of CME has been greatly advanced through the use of pathway by which cells selectively internalize molecules from fluorescent fusion proteins7. In yeast, direct genomic tagging of pairs of the plasma membrane and surrounding environment. Previous genes with different fluorescent tags has allowed researchers to define live-cell imaging studies using ectopically overexpressed a very regular series of spatiotemporal events for CME in living cells8. fluorescent fusions of endocytic proteins indicated that Conversely, in mammalian cells these events have been described as mammalian CME is a highly dynamic but inefficient and being much more heterogeneous and inefficient9–12, but the inability heterogeneous process. In contrast, studies of endocytosis in to do precise genome editing has forced researchers to rely heavily on budding yeast using fluorescent protein fusions expressed at overexpression of fusion proteins1,9–14 . Furthermore, these fusion pro- physiological levels from native genomic loci have revealed a teins are often derived from a different cell type or species from the cell process that is very regular and efficient. To analyse endocytic type being studied, and encode non-native splice variants. An accurate dynamics in mammalian cells in which endogenous protein description of endocytic dynamics is the foundation for understanding stoichiometry is preserved, we targeted zinc finger nucleases the mechanism and regulation of this crucial process. Because evidence (ZFNs) to the clathrin light chain A and dynamin-2 genomic from fields ranging from developmental and cell biology to plant physi- loci and generated cell lines expressing fluorescent protein ology has shown that overexpression can result in protein mislocaliza- fusions from each locus. The genome-edited cells exhibited tion, aggregation and altered signalling15–20, we sought to re-examine enhanced endocytic function, dynamics and efficiency when the highly dynamic process of CME using fluorescent fusion proteins compared with previously studied cells, indicating that CME expressed from their native loci. is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a RESULTS robust tool for expressing protein fusions at endogenous levels ZFN-mediated editing as an efficient and accurate method to faithfully report subcellular localization and dynamics. for generating stable cell lines expressing fluorescent protein fusions from native loci Clathrin-mediated endocytosis (CME) is characterized by recruitment To engineer precise gene fusions at endogenous loci, we used zinc finger of clathrin triskelia, composed of heavy and light chains, to the plasma nucleases (ZFNs)21 designed to cleave their target genes near the stop membrane and their assembly into polygonal cages that mediate mem- codons. The resulting double-strand breaks were mended by homology- brane invagination. During late stages of this process the GTPase directed repair using an exogenously supplied DNA donor that encodes a dynamin is recruited to the necks of these invaginations1, where it fluorescent tag (Fig. 1a). In all cases, ZFNs and donor constructs were co- functions in vesicle release through membrane scission and the sub- transfected into the cells. The high-editing efficiency and accuracy of the sequent internalization of plasma-membrane molecules, extracellular open reading frame (ORF) addition process, coupled with the expression fluid and specific ligands from the environment2,3. Three decades of of proteins bearing fluorescent markers, allowed us to obtain the desired evidence directly connect perturbation of CME to a broad range of cells simply by using fluorescence-activated cell sorting (FACS) without pathophysiological outcomes, including atherosclerosis4, disorders drug selection (Supplementary Fig. S1a and Table S1, top). Single-cell- of the peripheral and central nervous system5 and infection by the derived clones were then generated by limiting dilution. We found that hepatitis C virus6. ZFN treatment had a negligible impact on cell survival (Supplementary 1Department 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. 4Correspondence should be addressed to D.G.D. (e-mail: [email protected]) Received 27 September 2010; accepted 21 December 2010; published online 06 February 2011; DOI: 10.1038/ncb2175 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1 TECHNICAL REPORT EN X c X D CLTA Mr(K) Control mkCLTA mkCLTA rCLTA b BSC-1 70- EN ATG TGA Control mkCLTA GFP–CLTA 55- CLTA–RFP Exon 7 ZFN target ZFN-CLTA-R 7&&&7&$$*&$**&&&&*&7**7*&$&7*$$*$*&&$&&&7*7**$ 6/.4$3/9+ $***$*77&*7&&****&*$&&$&*7*$&77&7&**7***$&$&&7 Left HA Right HA 35- ZFN-CLTA-L RFP CLTA Donor RFP d Actin f Time (min) Control 15 30 Control e EN *** X 50 * mkCLTA 45 mkCLTA 40 * 35 X 30 Mean lifetime (s) 25 mkCLTA 20 mkCLTAEN mkCLTAX rCLTAX Figure 1 Editing of CLTA using ZFNs in BSC-1 cells. (a) Schematic stable GFP–CLTA (rat brain-derived) overexpression line. (d) Epifluorescence representation of the strategy for integration of RFP at the CLTA locus. image of mkCLTAEN cells expressing endogenous CLTA–RFP. Scale bar, White boxes, exons of CLTA; HA, donor plasmid region of homology to CLTA 10 μm. (e) CCP dynamics were assessed in the indicated BSC-1 cell lines sequence; Blue letters, stop codon. The grey box indicates the region of CLTA by quantifying the lifetime of fluorescently tagged CLTA proteins at the exon 7 surrounding the translation stop codon; the underlined sequences plasma membrane. Data are means ± s.e.m. Tracks, 30,734–50,250; n = 5, indicate the recognition stretches of the individual zinc finger nucleases 11 and 15 cells for the mkCLTAX, mkCLTAEN and rCLTAX lines, respectively. (ZFN-CLTA-L and ZFN-CLTA-R, respectively). (b) Out-out PCR showing Triple asterisk indicates P < 0.0001 and single asterisk indicates P < 0.05. targeted integration of RFP. Control, parental cell line; mkCLTAEN, single- (f) Time course of Alexa Fluor 488-conjugated human transferrin uptake in allele CLTA–RFP tagged genome-edited line. (c) Western blot analysis of cell the indicated BSC-1 cell lines. Areas enclosed by yellow boxes are enlarged lysates immunoblotted for CLTA, RFP and actin. Note RFP antibody cross- (right) for better visualization. Scale bar, 10 μm. Uncropped images of gels reactivity with GFP. mkCLTAX, stable CLTA–RFP overexpression line; rCLTAX, and blots are shown in Supplementary Fig. S2a. Fig. S1b), suggesting that the editing strategy is an ideal method for indicating that the endogenously tagged CLTA is correctly incorporated generating stable cell lines. The complete panel of cells engineered in into clathrin-coated pits (CCPs). this study is described in Table 1. Genome-edited CLTA–RFP monkey cells exhibit increased CME ZFN-mediated insertion of RFP at the CLTA locus in monkey cells dynamics and function relative to overexpression lines We designed ZFNs targeting the 3ʹ-terminus of clathrin light chain Numerous reports have documented striking heterogeneity in the size, A (CLTA; Fig. 1a and Supplementary Table S2) and first used them dynamics and productivity of CCPs10–12, yet the nature of this heteroge- in African green monkey kidney epithelial cells (BSC-1), which have neity and its functional implications have not been established. Because been used for many important studies of endocytic dynamics10,11,22,23. We determining the time required to form coated pits and vesicles is critical generated a cell line, mkCLTAEN, in which one CLTA allele was tagged for gaining a mechanistic and regulatory understanding of CME11,22–24, with red fluorescent protein (RFP; Table 1 and Supplementary Fig. S1c we sought to quantitatively analyse clathrin dynamics in mkCLTAEN cells and Table S1, bottom). Genotyping and western blot analysis con- using total internal reflection fluorescence (TIRF) microscopy and semi- firmed the accurate addition of the tag to the CLTA locus (Fig. 1b, c and automated particle tracking. Analysis of a well-studied BSC-1-derived Supplementary Fig. S2a). As expected, mkCLTAEN cells demonstrated cell line (rCLTAX, Table 1), which overexpresses rat brain CLTA fused to punctate plasma membrane and perinuclear CLTA–RFP distribution green fluorescent protein (GFP) from a randomly integrated transgene12, (Fig. 1d and Supplementary Fig. S3a), and complete co-localization of yielded an overall mean lifetime for the fusion protein at the plasma CLTA–RFP with clathrin heavy chain (CHC; Supplementary Fig. S3b), membrane of 46 ± 2.2 s (Fig. 1e and Supplementary Fig. S4a, Table S3 2 NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION TECHNICAL REPORT Table 1 Cell lines used in this study Cell lines with marker ORFs added to the endogenous locus Designation Cell line Species Gene Marker mkCLTAEN BSC-1 Cercopithecus aethiops monkey CLTA (clathrin light chain A) RFP hCLTAEN SK-MEL-2 Homo sapiens human CLTA (clathrin light chain A) RFP hDNM2EN SK-MEL-2 Homo sapiens human DNM2 (dynamin 2) GFP hCLTAEN/DNM2EN SK-MEL-2 Homo sapiens human CLTA, DNM2 RFP/GFP Cell lines carrying overexpressed, randomly integrated marker ORFs Designation Cell line Species Gene Marker rCLTAX BSC-1 Cercopithecus aethiops rat brain CLTA (clathrin light chain A) GFP mkCLTAX BSC-1 Cercopithecus aethiops monkey CLTA (clathrin light chain A) RFP hCLTAX SK-MEL-2 Homo sapiens human CLTA (clathrin light chain A) RFP hDNM2X SK-MEL-2 Homo sapiens human DNM2 (dynamin 2) GFP and Movie S1), similar to previously reported values of 39 and 46 s (refs of internalization (Fig. 1f). These results indicate that overexpression 11, 12).

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