MITD1 is recruited to midbodies by ESCRT-III and participates in cytokinesis

Abstract

Diverse cellular processes, including multivesicular body formation, cytokinesis, and viral budding, require the sequential functions of endosomal sorting complexes required for transport (ESCRTs) 0 to III. Of these multiprotein complexes, ESCRT-III in particular plays a key role in mediating membrane fission events by forming large, ring-like helical arrays. A number of proteins playing key effector roles, most notably the ATPase associated with diverse cellular activities protein VPS4, harbor present in microtubule-interacting and trafficking molecules (MIT) domains comprising asymmetric three-helical bundles, which interact with helical MIT-interacting motifs in ESCRT-III subunits. Here we assess comprehensively the ESCRT-III interactions of the MIT-domain family member MITD1 and identify strong interactions with charged multivesicular body protein 1B (CHMP1B), CHMP2A, and increased sodium tolerance-1 (IST1). We show that these ESCRT-III subunits are important for the recruitment of MITD1 to the midbody and that MITD1 participates in the abscission phase of cytokinesis. MITD1 also dimerizes through its C-terminal domain. Both types of interactions appear important for the role of MITD1 in negatively regulating the interaction of IST1 with VPS4. Because IST1 binding in turn regulates VPS4, MITD1 may function through downstream effects on the activity of VPS4, which plays a critical role in the processing and remodeling of ESCRT filaments in abscission.

FIGURE 1:

MIT domain of MITD1 selectively binds ESCRT-III proteins. (A) Schematic diagram showing the domain architecture of MITD1 and deletion mutants used in this study. Boundary amino acid residues are indicated. (B) Yeast two-hybrid interactions of the MIT domain of MITD1 with C-terminal MIM domains of ESCRT-III proteins were assayed using the HIS3 reporter (sequential 10-fold yeast dilutions are shown). (C) HEK293 cells were cotransfected with the indicated Myc-MITD1 constructs and HA-tagged IST1, CHMP1B, or CHMP2A. Cell lysates were immunoprecipitated (IP) with anti-Myc antibodies and immunoblotted (IB) for HA and Myc epitopes. (D) Interactions of Myc-MITD1 with various HA-IST1 MIM deletions and structure-based mutations were examined using coIP assays with HA-epitope antibodies or control immunoglobulin G (IgG). Immunoblotting was performed with HA- and Myc-epitope antibodies as indicated. An asterisk denotes a probable degradation product. HC, IgG heavy chain; LC, IgG light chain.

FIGURE 2:

During cytokinesis, MITD1 localizes at the periphery of midbodies through its MIT domain. (A) HeLa cells were coimmunostained for endogenous MITD1 (red) and the indicated midbody marker proteins (green). Merged images are at the right, with DAPI nuclear staining in blue. Insets are enlarged images of the midbody region. Bars, 5 μm. (B) Top, schematic diagram showing the domain architecture of MITD1 and deletion mutants used; bottom, HeLa cells were transfected with the indicated Myc-MITD1 constructs and coimmunostained for Myc-epitope (red) and endogenous CHMP2A (green). Merged images with DAPI are at the right. Insets are enlarged images of the midbody region. FL, full length. Bars, 2 μm.

FIGURE 3:

MITD1 can self-assemble through its C-terminal domain. (A) Yeast two-hybrid interactions between the indicated deletion mutants of MITD1 (boundary amino acid residues shown) were tested using the HIS3 reporter (sequential 10-fold yeast dilutions). (B) HEK293 cells were transfected with the indicated MITD1 constructs, and extracts were immunoprecipitated (IP) with anti-FLAG-conjugated beads then immunoblotted (IB) for FLAG or Myc epitopes. (C) HEK293 cells were transfected with the indicated MITD1 constructs and immunoprecipitated with anti-FLAG antibodies or control IgG. Precipitates were then immunoblotted. (D) HEK293 cell lysates were treated with the indicated concentrations of BS³ and subjected to immunoblot analysis as indicated. (E) Cell lysates were prepared under the conditions shown and subjected to immunoblotting using anti-MITD1 antibodies. (F) Gel-exclusion FPLC was performed to determine the native size of endogenous MITD1. Aliquots of each fraction were immunoblotted with anti-MITD1 antibodies. Elution peaks for marker proteins (in kilodaltons) are across the top. (G) HEK293 cells were cotransfected with HA-IST1 and either full-length MITD1 or MITD1 Δ83–126. Cell lysates were immunoprecipitated with anti-Myc antibodies and analyzed by immunoblotting. In B, D, E, and F, sizes of protein standards (in kilodaltons) are indicated.

FIGURE 4:

VPS4 residues required for ESCRT-III interaction are highly conserved in MITD1 and important for MITD1 midbody localization and ESCRT-III interaction. (A) Amino acid sequences of MIT domains of human MITD1 and VPS4A/B were aligned using ClustalW2. An asterisk indicates fully conserved residues. Colon indicates conservation between groups of strongly similar properties. Period indicates conservation between groups of weakly similar properties. (B–D) HEK293 cells were transfected with the indicated constructs and immunoprecipitated (IP) with anti-Myc antibodies. Precipitates were then immunoblotted (IB) with anti–HA- and anti–Myc-epitope antibodies. (E) HeLa cells were transfected with the indicated Myc-MITD1 constructs and immunostained for Myc epitope (red) and CHMP2A (green). Insets are enlarged images of midbody regions. HC, IgG heavy chain; LC, IgG light chain; WT, wild type. Bars, 2 μm.

FIGURE 5:

MIM domains of ESCRT-III proteins regulate midbody localization of MITD1. (A–C) Myc-MITD1 was transfected into HeLa cells along with the indicated HA-tagged CHMP1B (A), CHMP2A (B), or IST1 (C) constructs. Cells were fixed and immunostained for HA (green) and Myc (red) epitopes. Merged images with DAPI staining are to the right. Insets are enlarged images of midbody regions. (D) Among cells expressing HA-tagged ESCRT-III proteins at the midbody, cells with Myc-MITD1 at the midbody were counted (50 cells per group, n = 3 independent experiments). Graphs express means ± SEM. *p < 0.05. WT, wild type. Bars, 10 μm, 2 μm (insets).

FIGURE 6:

MITD1 is involved in cytokinesis. (A) HeLa cells were transfected with control siRNA (siCTL) or three different siRNAs against MITD1 (siMITD1). Cells were lysed and immunoblotted for MITD1. Actin levels were monitored to control for protein loading. (B) After the indicated siRNA transfections, cells were fixed and immunostained for CEP55 (green) and MITD1 (red). Merged images with DAPI staining are to the right. Insets are enlarged images of midbody regions. (C) HeLa cells transfected with the indicated siRNAs were stained with β-tubulin antibodies (green) and DAPI (blue). (D) Cells interconnected by midbodies and multinucleated cells were counted based on DAPI and β-tubulin staining as shown in C. Five hundred cells per group were analyzed in three independent experiments and graphed (means ± SEM). *p < 0.05; **p < 0.01. Bars, 10 μm (B, C), 2 μm (inset in B).

FIGURE 7:

Differential effects of MITD1 on cytokinesis and EGFR degradation. (A) Confocal time-lapse imaging of HeLa cells transfected with control (siCTL) or MITD1 (siMITD1) siRNA. Elapsed times (minutes) are provided in each panel. White arrowheads mark midbodies connecting two cells. Bars, 10 μm. (B) Time required to complete cytokinesis was measured from 65 control and 49 MITD1-depleted cells in three independent experiments (means ± SEM). ****p < 0.0001. (C) HeLa cells transfected with control (siCTL) or MITD1 (siMITD1) siRNA were serum starved and stimulated with EGF for the indicated times. Cell lysates were analyzed by immunoblotting with EGFR, MITD1, and β-tubulin antibodies. (D) Graphical representation of quantified intensities of EGFR protein bands from three independent experiments.

FIGURE 8:

Midbody recruitment of MITD1 is regulated by ESCRT-III. (A) HeLa cells transfected with the indicated siRNAs were stained for endogenous MITD1 (green) and β-tubulin (red). Cells with MITD1 signal enriched at midbodies were counted from 100 cells per group, n = 3 independent experiments. Graphs express means ± SEM. ***p < 0.001. (B, C) HeLa cells were transfected with control (siCTL) or MITD1 (siMITD1) siRNA and costained for CHMP2A (red) along with CHMP1B (B) or IST1 (C) (green). Merged images with DAPI staining are at the right. Insets are enlargements of midbody regions. Bars, 5 μm.

FIGURE 9:

Recruitment of MITD1 to the midbody by ESCRT-III is required for completion of cytokinesis. (A) HeLa cells stably expressing empty vector or the indicated Myc-tagged, siRNA-resistant MITD1 constructs were transfected with either control (siCTL) or MITD1 (siMITD1) siRNA. Cells were lysed 48 h later, and extracts were immunoblotted for MITD1 (endogenous and recombinant) or β-tubulin. An asterisk denotes endogenous MITD1. (B) Localizations of stably expressed recombinant proteins were examined by staining siMITD1-transfected cells with MITD1 (red) and β-tubulin (green) antibodies. For comparisons with endogenous MITD1, cells stably expressing empty vector were transfected with siCTL and stained with indicated antibodies. Merged images with DAPI staining are to the right. (C) HeLa cells stably expressing empty vector or various MITD1 constructs were transfected with siCTL or siMITD1 and stained with β-tubulin (green) antibodies. Arrows indicate multinucleated cells. (D) Cells interconnected by midbodies and multinucleated cells were counted based on DAPI and β-tubulin staining as shown in C. Five hundred cells per group were analyzed in three independent experiments. Graphs express means ± SEM. *p < 0.05; **p < 0.01; n.s., not significant. Bars, 10 μm (B, C), 2 μm (inset in B).

FIGURE 10:

MITD1 competes with VPS4 for binding to IST1. (A, B) HEK293 cells were cotransfected with the indicated constructs. Cell lysates were immunoprecipitated (IP) with anti–HA-epitope antibodies or control IgG. Immunoblotting (IB) was performed with GFP, Myc-epitope, and HA-epitope antibodies as indicated. (C) HA-IST1 and GFP-VPS4 constructs were cotransfected into HEK293 cells with Myc-empty vector or Myc-MITD1 Δ83–126. Molar ratios of Myc-empty vector and Myc-MITD1 Δ83–126 DNA are shown along the top. The amount of GFP-VPS4 DNA was held constant, whereas the amount of Myc-MITD1 Δ83–126 DNA was varied. Cell lysates were immunoprecipitated (IP) with HA-probe antibodies. Immunoblotting (IB) was performed for GFP, Myc epitope, and HA epitope as indicated. An asterisk denotes a probable degradation product or modified form of GFP-VPS4. HC, IgG heavy chain; LC, IgG light chain.