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 IQGAP1_1463–1547. TSG101-AD fusions (rows 2–6) or control AD constructs (row 1, Empty) were coexpressed together with IQGAP1_1463–1547-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 IQGAP1_1463-1547 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.

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-TSG101_154–164A, VPS28, VPS37B, and MVB12A DNA) and FLAG-ALIX_800-802A (0.5 μg DNA) proteins that cannot bind CEP55 are not recruited to midbodies. Microtubule staining (white, α-Tubulin) is also shown for reference.

ALIX and ESCRT-I are required for efficient cytokinesis and abscission. (A) Effects of siRNA depletion of TSG101 and ALIX on cell division. Upper left panel: Western blot showing efficient siRNA depletion of endogenous TSG101 (lane 2) and ALIX (lane 3). Controls show protein levels in HeLa cells treated with an irrelevant siRNA against luciferase (lane 1) and cells depleted of CEP55 (lane 4). Numbering denotes the first nucleotide of the siRNA target site within the gene. α-Tubulin loading controls are shown below. Lower panels: immunofluorescent images of control-treated (left), TSG101-depleted (middle), and ALIX-depleted (right) HeLa cells. Microtubules (red, α-Tubulin) and nuclei (SYTOX green) were stained for reference, and arrowheads highlight multinuclear cells. Upper right panel: quantification of multinucleated HeLa cells following depletion of TSG101, ALIX or CEP55, or treatment with the control siRNA. Measurements were performed in triplicate (n=200 cells each) and error bars denote standard deviations. (B) FACS analyses of the DNA content of cells depleted of TSG101, ALIX, CEP55, or treated with a control siRNA. Peaks corresponding to 2n, 4n, and 8n DNA contents are labeled, and the integrated peak volumes are provided. (C) ALIX is required for successful completion of cytokinesis and abscission. Time-lapse phase-contrast imaging of cell division following ALIX depletion. White arrowheads highlight cells that are starting to divide, black arrowheads highlight arrested midbody structures, and yellow arrowheads highlight cells that have recoalesced to produce multinucleated cells following aborted cytokinesis. The figure illustrates that cells depleted of ALIX either abort cleavage furrow formation at an early stage of cell division to form a multinucleated cell (row 1), or arrest late in cytokinesis and remain tethered via thin membranes before eventually recoalescing to form multinucleated cells (rows 2 and 3). Elapsed times are provided in each panel. Movies showing time-lapse images of the cytokinesis blocks induced by ALIX depletion are provided in Supplementary Figure S9.

VPS4 protein overexpression inhibits cytokinesis. (A) Double labeled immunofluorescence images showing cells expressing VPS4-GFP, VPS4A_K173Q-GFP, or GFP (top row), and co-stained for microtubules (α-Tubulin, red, bottom row). Examples of multinuclear cells and cells in telophase are indicated by yellow and white arrowheads, respectively. (B) Percentages of cells with multiple nuclei (dark bars) or in telophase (light bars). Three fields of 200 cells each were counted, and error bars denote standard deviations.

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 (TSG101_154–164A). 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-ALIX_794–796A (control mutant), but not the binding site mutant FLAG-ALIX_800–802A. (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.

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.

Requirements for different ALIX interactions in cytokinesis. (A) Western blot showing ALIX depletion/re-expression. Upper panel: ALIX levels (anti-ALIX detection) in cells depleted of endogenous ALIX (lanes 2–7) or treated with a control siRNA (lane 1). Cells in lanes 3–7 were cotransfected with vectors expressing WT FLAG-ALIX (lane 3), or the designated FLAG-ALIX mutants. Middle panel: same samples probed with an anti-FLAG antibody. Lower panel: α-Tubulin loading controls. (B) Stacked FACS profiles showing the effects of ALIX depletion/re-expression on cell polyploidy. Samples are the same as in panel A. (C) Percentages of cells with 8n contents following ALIX depletion/re-expression. The plot shows the relative integrated 8n peak volumes in the FACS profiles from panel B. Note that although siRNA-resistant WT ALIX did not fully rescue the polyploid phenotype, the partial rescue phenotypes shown here were highly reproducible (n=11).

ESCRT pathway functions. The schematic model illustrates how ESCRT-I, ALIX, and other ESCRT pathway proteins may be recruited by different adaptor proteins (red) to perform similar roles in the terminal membrane fission events of MVB biogenesis, virus budding, and cytokinesis.