Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis
2007; Springer Nature; Volume: 26; Issue: 19 Linguagem: Inglês
10.1038/sj.emboj.7601850
ISSN1460-2075
AutoresEiji Morita, Virginie Sandrin, Hyo-Young Chung, Scott G. Morham, Steven P. Gygi, Christopher K. Rodesch, Wesley I. Sundquist,
Tópico(s)Pancreatic function and diabetes
ResumoArticle13 September 2007free access Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis Eiji Morita Eiji Morita Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Virginie Sandrin Virginie Sandrin Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Hyo-Young Chung Hyo-Young Chung Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Scott G Morham Scott G Morham Myriad Genetics Incorporated, Salt Lake City, UT, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Christopher K Rodesch Christopher K Rodesch School of Medicine Fluorescence Microscopy Core Facility, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Wesley I Sundquist Corresponding Author Wesley I Sundquist Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Eiji Morita Eiji Morita Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Virginie Sandrin Virginie Sandrin Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Hyo-Young Chung Hyo-Young Chung Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Scott G Morham Scott G Morham Myriad Genetics Incorporated, Salt Lake City, UT, USA Search for more papers by this author Steven P Gygi Steven P Gygi Department of Cell Biology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Christopher K Rodesch Christopher K Rodesch School of Medicine Fluorescence Microscopy Core Facility, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Wesley I Sundquist Corresponding Author Wesley I Sundquist Department of Biochemistry, University of Utah, Salt Lake City, UT, USA Search for more papers by this author Author Information Eiji Morita1, Virginie Sandrin1, Hyo-Young Chung1, Scott G Morham2, Steven P Gygi3, Christopher K Rodesch4 and Wesley I Sundquist 1 1Department of Biochemistry, University of Utah, Salt Lake City, UT, USA 2Myriad Genetics Incorporated, Salt Lake City, UT, USA 3Department of Cell Biology, Harvard Medical School, Boston, MA, USA 4School of Medicine Fluorescence Microscopy Core Facility, University of Utah, Salt Lake City, UT, USA *Corresponding author. Department of Biochemistry, University of Utah, room 4100, 15N Medical Dr East, Salt Lake City, UT 84132-3201, USA. Tel.: +1 801 585 5402; Fax: +1 801 581 7959; E-mail: [email protected] The EMBO Journal (2007)26:4215-4227https://doi.org/10.1038/sj.emboj.7601850 Correction(s) for this article Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis18 July 2012 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 ‘hinge’ region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis. Introduction TSG101(yeast Vps23p) and ALIX/AIP1(Bro1p) both function at the endosome to help sort membrane proteins into vesicles that bud into the lumen to create multivesicular bodies (MVBs) (Hurley and Emr, 2006; Gill et al, 2007). One important function of this pathway is to target membrane proteins for degradation, which occurs when MVBs fuse with lysosomes and thereby expose the internal vesicles and their contents to the action of lysosomal lipases and hydrolases. TSG101 functions as a subunit of the heterotetrameric ESCRT-I complex (Endosomal Sorting Complex Required for Transport-I), together with three other MVB pathway proteins: VPS28, VPS37, and MVB12 (Chu et al, 2006; Curtiss et al, 2007; Kostelansky et al, 2007; Morita et al, 2007; Oestreich et al, 2007). ALIX also functions in the MVB pathway, where it can interact with several different proteins and complexes, including TSG101/ESCRT-I (Martin-Serrano et al, 2003; Strack et al, 2003; von Schwedler et al, 2003; Odorizzi, 2006). Both TSG101 and ALIX can also bind directly to retroviral Gag proteins, including HIV-1 Gag, and facilitate late stages of virus budding (Demirov and Freed, 2004; Morita and Sundquist, 2004; Bieniasz, 2006). In some contexts, ALIX and TSG101 can substitute for one another in the release of infectious virions, indicating that they can perform similar (or complementary) roles in virus budding (Fisher et al, 2007; Usami et al, 2007). TSG101/ESCRT-I and ALIX both function together with other ESCRT pathway members, including ESCRT-II, ESCRT-III and VPS4. Although mechanistic details are lacking, current models hold that the ESCRT pathway mediates the protein sorting and membrane fission events required for release of cargo-filled vesicles and viruses. Importantly, both MVB vesicles and retroviruses bud away from (rather than into) the cytoplasm, implying that the cytoplasmic ESCRT machinery may help mediate membrane fission events from inside the neck of the budding virus/vesicle. The family of related ESCRT-III proteins appears to play a particularly critical role in this process, by assembling into membrane-bound lattices associated with sites of vesicle formation (Babst et al, 2002; Lin et al, 2005). Once assembled on the membrane, the ESCRT-III subunits are bound and remodeled by the action of the VPS4 ATPases, which redistributes the ESCRT machinery back into the cytoplasm (Babst et al, 1998). Another important cellular process in which a thin membrane tubule must be resolved from within is the final abscission step of cytokinesis (reviewed in McCollum, 2005). During abscission, the thin microtubule-filled midbody that connects the dividing cells is severed to release two discrete daughter cells. Abscission is mediated by proteins of the Flemming body, a dense proteinaceous ring that occupies a central position within the midbody. Many midbody proteins have been identified (Skop et al, 2004), and recent studies have revealed that one such protein, CEP55, performs important roles in organizing the Flemming body and in recruiting a series of late-acting protein required for abscission (Fabbro et al, 2005; Martinez-Garay et al, 2006; Zhao et al, 2006). Cytokinesis proceeds through a series of sequential, but coupled stages that ultimately lead to abscission. Initially, a cleavage furrow is created through constriction of the actomyosin contractile ring. A number of factors help control contractile ring assembly and constriction, including IQGAP1 and Rho-associated kinases such as citron and ROCK1 (reviewed in Machesky, 1998; Matsumura, 2005). The centrosome also appears to play critical roles in helping to regulate the different stages of cytokinesis, and a series of proteins, including centriolin, centrin, and CEP55, concentrate at centrosomes during most of the cell cycle, but then migrate to Flemming bodies during cytokinesis (reviewed in Doxsey et al, 2005). Cleavage furrow ingression eventually creates a thin midbody that connects the dividing cells and the Flemming body forms at the center of the midbody. Some Flemming body components are present throughout furrow ingression, whereas others are delivered late, including cellular membranes, endocytic factors, and secretory vesicles and their associated fusion machinery (McCollum, 2005). Once all of these components are properly assembled and activated, abscission occurs and the two daughters separate completely. As part of our continuing effort to characterize the functions of human TSG101/ESCRT-I and ALIX, we performed a series of proteomic-style experiments aimed at identifying cellular binding partners. These experiments revealed that both ESCRT-I and ALIX bind a series of proteins that localize to centrosomes and midbodies, and function in cytokinesis. When we began this work, there were already several observations linking the ESCRT pathway with centrosomes and cytokinesis, although the potential implications of these links were not widely appreciated. First, Xie et al (1998) reported that TSG101/ESCRT-I can localize to centrosomes and midbodies, and that TSG101 downregulation leads to mitotic abnormalities. Second, Spitzer et al (2006) demonstrated that elc/tsg101 mutants in Arabidopsis exhibited high levels of multinucleate cells, and the authors suggested that this might reflect a cytokinesis defect arising from misregulation of the microtubule cytoskeleton, although this defect was not characterized further. Third, we reported that EAP20/ESCRT-II also concentrates at centrosomes, but did not characterize a centrosomal function for the ESCRT-II complex (Langelier et al, 2006). Fourth, Jin et al (2005) reported that EAP30/ESCRT-II negatively regulates maturation of the meiotic spindle pole body (centrosome) in Schizosaccharomyces pombe, although the authors did not note that EAP30 is a component of ESCRT-II. Finally, Furukawa and co-workers used affinity purification/mass spectrometry methods to show that CEP55 (called ‘C10orf3’) binds both TSG101 and ALIX (called ‘PDCD6IP’), but the role of CEP55 in cytokinesis was not yet known and the authors apparently did not recognize that PDCD6IP corresponds to ALIX (Sakai et al, 2006). Subsequently, while our work was in progress, Carlton and Martin-Serrano (2007) reported that both ESCRT-I and ALIX localize to the Flemming body and are required for abscission in human cells. We have therefore interpreted our observations in the light of their discovery, and proceeded to characterize the roles of ESCRT-I, ALIX, and other ESCRT pathway components in the abscission step of cytokinesis. Results Identification of ALIX and ESCRT-I binding partners involved in cytokinesis Extensive yeast two-hybrid and One-STrEP-tagged affinity purification/mass spectrometry experiments were performed to identify potential binding partners for ESCRT-I and ALIX. Remarkably, these experiments identified more than 10 proteins previously implicated in centrosome and midbody function. Four of these proteins, CEP55, ROCK1, IQGAP1, and CD2AP, were selected for further study because each (1) localizes to the midbody, (2) functions in cytokinesis/abscission, and (3) was identified in at least two independent proteomics screens (summarized in Figure 1A and caption). Our initial surveys indicated that three of these proteins, ROCK1, IQGAP1, and CD2AP, associated with ESCRT-I, and that CEP55 associated with both ESCRT-I and ALIX. Moreover, CD2AP was recently reported as an ALIX binding partner (Usami et al, 2007), indicating that this protein also binds both ESCRT-I and ALIX. Figure 1.Cellular binding partners of ESCRT-I and ALIX. (A) Proteomic identification of ESCRT-I and ALIX binding partners. Upper panel: summarized data from yeast two-hybrid screens of AD prey libraries for DBD-TSG101 and DBD-ALIX binding partners. Binding sites were inferred from the minimal overlapping regions of bait and prey fragments that gave positive interactions. Lower panel: proteins identified as ESCRT-I or ALIX binding partners in affinity purification/mass spectrometric experiments (and absent in control purifications). Bait proteins were expressed as One-STrEP-FLAG (OSF, N-terminal) or FLAG-One-STrEP (FOS, C-terminal) fusions, and OSF-TSG101 was coexpressed with untagged versions of other ESCRT-I subunits (VPS28 and VPS37A-D) in samples labeled ‘TSG101/ESCRT-I’. (B) Cytokinesis proteins co-precipitate with ALIX and ESCRT-I. ‘Bait’ FLAG-ALIX and OSF-TSG101 or empty vector controls were tested for co-precipitation with overexpressed Myc-tagged ‘prey’ proteins or with endogenous IQGAP1 (lower right). Western blots show (1) prey protein levels in soluble lysates (middle panels, Lysate, IB: anti-Myc), (2) bait proteins bound to anti-FLAG (FLAG-ALIX) or StrepTactin (OSF-TSG/ESCRT-I) matrices (lower panels, IP: anti-FLAG or Strep), or (3) prey proteins co-precipitated onto the matrix (upper panels, IB: anti-Myc or anti-IQGAP1). Note that untagged versions of the three other ESCRT-I subunits (VPS37B, MVB12A, and VPS28) were always coexpressed with OSF-TSG101, and that others have also reported TSG101-CEP55, ALIX-CEP55, and ALIX-CD2AP interactions (Sakai et al, 2006; Usami et al, 2007). (C) Yeast two-hybrid mapping of the TSG101 binding sites for IQGAP11463–1547. TSG101-AD fusions (rows 2–6) or control AD constructs (row 1, Empty) were coexpressed together with IQGAP11463–1547-DBD (column 2) or control DBD constructs (column 1, Empty) and tested for positive yeast two-hybrid interactions (left) or co-transformation (control, right). Note that IQGAP11463-1547 shows positive interactions with both the stalk and head regions of TSG101. (D) Yeast two-hybrid mapping of the TSG101 binding sites for ROCK1. The experiment is analogous to that in panel C, except that TSG101 constructs were expressed as DBD fusions and ROCK1 was expressed as an AD fusion. Note that ROCK1 shows positive interactions with both the stalk and core regions of TSG101. (E) Summary of TSG101 interactions with IQGAP1 and ROCK1. Domain abbreviations: CH, Calponin Homology; IR, IQGAP-Specific Repeat; GRD, GAP-Related Domain; UEV, Ubiquitin E2 Variant; PRR, Proline-Rich Region; Coil, predicted coiled-coil region; RB, Rho-Binding region; Zn, Zinc finger; PH, Plexstrin Homology Domain. Download figure Download PowerPoint Five of the six relevant TSG101/ESCRT-I and ALIX interactions were initially verified by demonstrating that Myc-tagged candidate proteins bound immobilized FLAG-ALIX or OSF-TSG101/ESCRT-I but not control matrices (Figure 1B). The sixth interaction, IQGAP1-TSG101/ESCRT-I, could not be verified in this manner because Myc-IQGAP1 exhibited high background binding to the control matrix. In this case, preferential co-immunoprecipitation of endogenous IQGAP1 by OSF-TSG101/ESCRT-I was documented, albeit at levels only modestly above background. Finally, because the two CD2AP interactions were underrepresented in our initial proteomic screens, we further verified that CD2AP-ALIX and CD2AP-ESCRT-I co-immunoprecipitation reactions were robust and specific in both directions (Supplementary Figures S1A and B). Taken together, these experiments demonstrate that TSG101/ESCRT-I and ALIX interact with a series of proteins that function in cytokinesis. ROCK1 and IQGAP1 interact with the TSG101/ESCRT-I PRR/stalk and headpiece ROCK1 and IQGAP1 have both been implicated in contractile ring assembly and activation (Machesky, 1998; Matsumura, 2005), and may therefore belong to a similar functional class of ESCRT-I binding proteins. As shown in Figures 1C and D, both ROCK1 and IQGAP11465–1547 exhibited positive two-hybrid interactions with TSG101, and these assays were used to map their TSG101 binding requirements. TSG101 constructs were designed to span the N-terminal UEV domain (TSG1–145), the central proline-rich (PRR) and ‘stalk’ regions (TSG101146–311), and the C-terminal ‘headpiece’ (TSG101312–390) (Kostelansky et al, 2007), and also to test requirements for the TSG101 PTAP element (ΔPTAP) and PTAP binding activity (M95A) (Pornillos et al, 2002; Kostelansky et al, 2007). ROCK1 and IQGAP11465–1547 associated specifically with both the TSG101 PRR/stalk and headpiece, indicating that both proteins must make multiple contacts with TSG101/ESCRT-I. The TSG101 stalk and headpiece make a complex series of interactions with other ESCRT-I subunits (Kostelansky et al, 2007; Morita et al, 2007), and we therefore did not attempt to map the IQGAP1 and ROCK1 binding sites more precisely. Nevertheless, our experiments show that both IQGAP1 and ROCK1 associate with the core of TSG101/ESCRT-I (Figure 1E). ALIX and TSG101 bind CEP55 We focused our studies on CEP55 because our initial proteomics screens indicated that this protein bound both TSG101 and ALIX, and because CEP55 localizes to Flemming bodies and is required for abscission (Fabbro et al, 2005; Martinez-Garay et al, 2006; Zhao et al, 2006). The specificity and reciprocity of the CEP55–ESCRT-I and CEP55–ALIX interactions were confirmed in a series of co-immunoprecipitation and directed yeast two-hybrid experiments, which established that (1) OSF-CEP55 specifically co-immunoprecipitated endogenous TSG101 and ALIX (Supplementary Figure S2A), (2) OSF-CEP55 co-immunoprecipitated the entire ESCRT-I complex (not just TSG101) (Supplementary Figure S2B), and (3) CEP55 exhibited yeast two-hybrid interactions with several additional TSG101 binding proteins in the MVB pathway proteins, including HRS, VPS37C, and VPS37D (Figure 2A). Figure 2.CEP55 interactions with ESCRT-I and ALIX. (A) Yeast two-hybrid identification of interactions between CEP55 and proteins of the ESCRT pathway. CEP55-AD fusions or control AD constructs (Empty) were coexpressed together with TSG101-, ALIX-, HRS-, VPS37C- or VPS37D-DBD fusions, or control DBD constructs (Empty), and tested for positive yeast two-hybrid interactions (left) or co-transformation (control, right). CEP55 did not interact with other human ESCRT proteins in this assay (not shown). The different CEP55 constructs are summarized on the right. (B) Mapping of the CEP55 binding site on TSG101. Left panel: yeast two-hybrid mapping experiments showing that CEP55-DBD constructs bind the proline-rich region (PRR) of TSG101-AD, and that binding requires TSG101 residues 146–163. Middle panels: further yeast two-hybrid mapping experiments showing that the CEP55 binding site maps to TSG101 residues 155–163. Right panels: co-precipitation experiments confirming that OSF-CEP55 co-precipitates wild-type (WT) ESCRT-I complexes that contain either VPS37B or VPS37C subunits, but not analogous ESCRT-I complexes with a mutant TSG101 binding site (TSG101154–164A). Note that Myc-tagged versions of all four ESCRT-I subunits were coexpressed in these experiments. (C) Mapping of the CEP55 binding site on ALIX. Left panel: yeast two-hybrid mapping experiments showing that CEP55-DBD constructs bind the proline-rich region (PRR) of ALIX-AD, and that binding requires ALIX residues 781–810. Middle panels: co-precipitation experiments showing that OSF-CEP55 specifically co-precipitates WT FLAG-ALIX and FLAG-ALIX794–796A (control mutant), but not the binding site mutant FLAG-ALIX800–802A. (D) Summary of CEP55 interactions with TSG101, ALIX, and itself. Mapped binding residues are highlighted in red and the GPP motifs within the two CEP55 binding sites are underlined. Download figure Download PowerPoint CEP55 contains two predicted coiled-coil ‘arms’ separated by a ‘hinge’ (Figure 2A) and the N-terminal arm region reportedly mediates homo-oligomerization (Martinez-Garay et al, 2006; Zhao et al, 2006). Consistent with these observations, we found that two-hybrid constructs corresponding to full-length CEP55 or to just the two arms and the central hinge (CEP5519–385) self-associated in two-hybrid assays (Supplementary Figure S3). Importantly, both ALIX and TSG101 bound the CEP55 hinge alone (Figure 2A), although enhanced ALIX binding was observed for fragments that included arm 1 (Figure 2A and data not shown). In contrast, strong HRS, VPS37C, and VPS37D binding was only detected for longer CEP55 fragments that spanned both arms and the hinge. Two-hybrid and co-immunoprecipitation assays were used to map the CEP55 binding sites on TSG101/ESCRT-I and ALIX (Figures 2B–D). As shown in Figure 2B, CEP55 bound central fragments of TSG101, including the proline-rich region (left panel, PRR, TSG101146–235). Alanine scanning mutations through the TSG101 PRR revealed that the CEP55 binding site spanned TSG101 residues 155–163, because binding was eliminated by the 158PPN160/AAA mutation and reduced by the adjacent 157ATG157/GAA and 161TSY163/AAA mutations (Figure 2B, middle panels). This CEP55 binding site was confirmed in co-immunoprecipitation experiments showing that Ala/Gly mutations across the TSG101 The 154QATGPPNTSYM164 sequence (TSG101154–164A; see Figure 2B) abrogated co-immunoprecipitation of OSF-CEP55 with Myc-tagged ESCRT-I complexes (Figure 2B, right panels, compare lanes 2 and 6 to lanes 4 and 8). These data are in excellent agreement with those of Carlton and Martin-Serrano (2007) mapping the CEP55 binding site to TSG101 residues 158–162. Analogous yeast two-hybrid and co-immunoprecipitation experiments were used to characterize and map the CEP55–ALIX interaction (Figure 2C). Two-hybrid mapping experiments indicated that the hinge region of CEP55 bound ALIX residues 781–810 (Figures 2A and C, lower left panel). ALIX constructs containing mutations that spanned each of the two Pro–Pro tracts in this region were tested for CEP55 binding, which revealed that the ALIX 800GPP802/AAA mutation specifically abrogated CEP55 binding (Figure 2C, right panels). Hence, the CEP55 binding sites on both TSG101 and ALIX were centered about ‘GPP’ sequence elements, suggesting that the CEP55 hinge may recognize a common core sequence motif in both proteins. TSG101 and ALIX mutants that lacked CEP55 binding activities still supported HIV-1 budding, however, arguing against a role for CEP55 in virus release (Supplementary data and Supplementary Figure S4). ALIX and ESCRT-I colocalize with CEP55 at centrosomes and Flemming bodies We next examined the possibility that ALIX and ESCRT-I might function together with CEP55 in the abscission step of cytokinesis. CEP55 concentrates at centrosomes during the G2/M phase of the cell cycle, and then migrates to the midbody to function in abscission during cytokinesis (Fabbro et al, 2005; Martinez-Garay et al, 2006; Zhao et al, 2006). We therefore examined the localization of tagged ALIX and TSG101/ESCRT-I at different stages of the HeLa cell cycle (Figure 3). In non-dividing cells, exogenous ALIX and ESCRT-I were both distributed throughout the cytoplasm, including on endosomal membranes (Welsch et al, 2006). Strikingly, however, both ALIX and TSG101 were also highly enriched together with CEP55 on centrosomes whenever these structures were clearly visible (Figure 3B, rows 2 and 3) (Xie et al, 1998). Centrosomal localization was also confirmed for the endogenous ALIX protein (Supplementary Figure S5, lower row). Thus, both ALIX and ESCRT-I concentrate at the centrosomes of non-dividing cells. Figure 3.CEP55, ALIX, and TSG101/ESCRT-I colocalize at centrosomes and Flemming bodies. (A) Triple labeled immunofluorescence and DIC images showing that FLAG-CEP55 (0.2 μg DNA), GFP-TSG101/ESCRT-I (0.2 μg GFP-TSG101, VPS28, VPS37B, and MVB12A DNA), or GFP-ALIX (0.5 μg DNA) colocalize at the midbodies (arrowheads) of dividing HeLa cells. Microtubule staining (white, α-Tubulin) is also shown for reference in columns 1 and 5. Expanded and merged views of the midbodies from the lower pair of cells in each image are shown in column 5. (B) Double labeled immunofluorescence and DIC images showing that α-Tubulin, FLAG-CEP55, or CEP55-Myc (0.2 or 0.5 μg DNA), Myc-TSG101/ESCRT-I (0.2 μg Myc-TSG101, VPS28, VPS37B, and MVB12A DNA), and FLAG-ALIX (0.5 μg DNA) colocalize at the centrosomes of non-dividing cells (arrowheads). (C) Triple-labeled immunofluorescence and DIC images showing that GFP-CEP55 (0.5 μg DNA) localizes to midbodies (arrowheads) whereas OSF-TSG101/ESCRT-I (0.2 μg GFP-TSG101154–164A, VPS28, VPS37B, and MVB12A DNA) and FLAG-ALIX800-802A (0.5 μg DNA) proteins that cannot bind CEP55 are not recruited to midbodies. Microtubule staining (white, α-Tubulin) is also shown for reference. Download figure Download PowerPoint The distributions of TSG101/ESCRT-I and ALIX in dividing cells were even more striking, as both tagged proteins and endogenous ALIX localized to Flemming bodies, together with CEP55 (Figure 3A; Supplementary Figure S5, upper row). Importantly, however, TSG101/ESCRT-I and ALIX mutants that were unable to bind CEP55 failed to localize to Flemming bodies, although these mutants did not affect the midbody localization of CEP55 (Figure 3C). Similarly, siRNA depletion of endogenous CEP55 blocked the accumulation of ALIX and TSG101/ESCRT-I at Flemming bodies (Supplementary Figures S6A and B, and S7). In contrast, TSG101 depletion did not affect CEP55 localization, and ALIX depletion did not affect CEP55 or TSG101 localization (Supplementary Figures S6C–E). Hence, CEP55 appears to recruit ALIX and TSG101/ESCRT-I to Flemming bodies, where all three proteins are positioned to function together in abscission. CHMP/ESCRT-III and VPS4 concentrate at midbodies Both ESCRT-I and ALIX function together with other ESCRT factors, particularly the ESCRT-III and VPS4 proteins, to facilitate the vesiculation events required for virus budding and MVB biogenesis (Hurley and Emr, 2006; Gill et al, 2007). We therefore tested whether representative proteins from three of the seven different ESCRT-III families were also recruited to the Flemming bodies during cytokinesis. As shown in Figure 4A, C-terminally FLAG-tagged CHMP2A, CHMP4A, and CHMP5 proteins all localized to the midbodies of dividing cells. Moreover, all three proteins were localized into two distinct rings, one on either side of the Flemming body. Hence, ESCRT-III proteins were also recruited to midbodies where they formed distinctive double ring assemblies. Figure 4.ESCRT-III Proteins and VPS4A Concentrate at Flemming bodies. (A) Double labeled immunofluorescence images showing that CHMP2A-FLAG, CHMP4A-FLAG, and CHMP5-FLAG (each 0.5 μg DNA) concentrate in double ring structures (arrowheads) at the midbodies of dividing HeLa cells. Expanded views are shown below each image. Note that these ESCRT-III constructs were employed because they exhibited no (CHMP2A-FLAG and CHMP4A-FLAG) or minimal (CHMP5-FLAG) effects on HIV budding (von Schwedler et al, 2003). Microtubules were also stained for reference (red, α-Tubulin). (B) Double-labeled immunofluorescence images showing that endogenous VPS4A concentrates in double ring structures (arrowheads) within very thin midbodies of dividing HeLa cells. Microtubules were also stained (red, α-Tubulin). Examples of wider midbodies that lacked VPS4A staining are shown in Supplementary Figure S8A, and VPS4 localization is quantified in Supplementary Figure S8B. Download figure Download PowerPoint The localization of VPS4A was also examined, and we found that endogenous VPS4A also formed double-ring structures at the midbodies (Figure 4B). In this case, however, VPS4A localization was only observed when the midbodies were particularly thin, suggesting that VPS4A may only be recruited at the final stages of cytokinesis (see Supplementary Figure S8A). Quantitative analyses revealed that endogenous VPS4A was present at 36% of identifiable midbodies (Supplementary Figure S8B). The efficiency of midbody localization was not affected by TSG101 depletion (35%), but was reduced by ALIX depletion (22%) and eliminated by CEP55 depletion (3%), indicating that ALIX contributes to VPS4 midbody localization and CEP55 is required. Taken together, these experiments demonstrate that many different ESCRT pathway members, including ALIX, ESCRT-I, ESCRT-III subunits, and VPS4A, are recruited to midbodies where they could all function in abscission. ALIX and ESCRT-I are required for efficient cytokinesis To test whether TSG101/ESCRT-I and ALIX are actually required for abscission, we examined cell division in cells depleted of endogenous TSG101 and ALIX (Figure 5). Replicating HeLa cells depleted of TSG101 appeared relatively normal, albeit with a moderate enrichment of multinuclear cells (Figure 5A, lower central panel). In contrast, cells depleted of ALIX were highly aberrant, with most of the cells exhibiting multiple nuclei and/or unusual intercellular connections that appeared to be the remnants of arrested midbodies (Figure 5A, lower right panel). These observations were quantified both by counting multinuclear cells (Figure 5A, upper right) and by using both FACS analyses to measure the relative numbers of cells with 2n, 4n and 8n DNA contents (Figure 5B). As shown in Figure 5B, 9% of HeLa cells treated with an irrelevant control siRNA had 4n DNA contents, and cells with 8n DNA contents were essentially undetectable (<1%). TSG101 depletion doubled the number of cells with a 4n DNA content (18%), but did not detectably increase the numbers of cells with 8n DNA contents. Mo
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