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ANCHR mediates Aurora-B-dependent abscission checkpoint control through retention of VPS4

Nature Cell Biology volume 16, pages 547–557 (2014)Cite this article

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Abstract

During the final stage of cell division, cytokinesis, the Aurora-B-dependent abscission checkpoint (NoCut) delays membrane abscission to avoid DNA damage and aneuploidy in cells with chromosome segregation defects. This arrest depends on Aurora-B-mediated phosphorylation of CHMP4C, a component of the endosomal sorting complex required for transport (ESCRT) machinery that mediates abscission, but the mechanism remains unknown. Here we describe ANCHR (Abscission/NoCut Checkpoint Regulator; ZFYVE19) as a key regulator of the abscission checkpoint, functioning through the most downstream component of the ESCRT machinery, the ATPase VPS4. In concert with CHMP4C, ANCHR associates with VPS4 at the midbody ring following DNA segregation defects to control abscission timing and prevent multinucleation in an Aurora-B-dependent manner. This association prevents VPS4 relocalization to the abscission zone and is relieved following inactivation of Aurora B to allow abscission. We propose that the abscission checkpoint is mediated by ANCHR and CHMP4C through retention of VPS4 at the midbody ring.

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Figure 1: ANCHR (ZFYVE19) is essential to delay abscission in cells with lagging chromatin.
Figure 2: ANCHR localizes to centrosomes and the midbody, and negatively regulates abscission in a PtdIns(3)P-independent manner.
Figure 3: ANCHR regulates the Aurora-B-dependent abscission checkpoint.
Figure 4: ANCHR interacts with VPS4 at the midbody.
Figure 5: ANCHR-mediated cytokinetic arrest is dependent on its interaction with and retention of VPS4 at the midbody ring.
Figure 6: ANCHR and CHMP4C function in a cooperative and Aurora-B-dependent manner to retain VPS4 at the midbody.
Figure 7: A model for the ANCHR-mediated regulation of VPS4 on abscission checkpoint activation.

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Acknowledgements

A. Engen and H.P. Bjønnes are acknowledged for expert handling of cell cultures, and T. Høiby and C. Herrmann for invaluable technical assistance. O. Mjaavatten at the Proteomics Unit at the University of Bergen (PROBE) and G. de Souza at the Proteomics Core Facility Unit at Oslo University Hospital are acknowledged for performing mass spectrometry analyses of GFP–ANCHR and GFP–VPS4 immunoprecipitates. The confocal microscopy core facility of Oslo University Hospital is acknowledged for providing access to microscopes. We also thank D. Gerlich at the Institute of Molecular Biotechnology (IMBA), Vienna, for supplying plasmids for the generation of GFP–α-tubulin+mCherry–H2B stable cell lines, and J. Martin-Serrano, King’s College London, UK, for advice on NoCut activation. S.B.T. and M.V. are PhD students of the South-Eastern Norway Regional Health Authority. C.R. is a senior researcher and C.C. a postdoctoral fellow of the Norwegian Cancer Society. H.S. was supported by an Advanced Grant from the European Research Council. This work was partly supported by the Research Council of Norway through its Centres of Excellence funding scheme, project number 179571.

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Authors and Affiliations

  1. Centre for Cancer Biomedicine, Faculty of Medicine, Oslo University Hospital, Montebello, N-0379 Oslo, Norway

    Sigrid B. Thoresen, Coen Campsteijn, Marina Vietri, Kay O. Schink, Knut Liestøl, Camilla Raiborg & Harald Stenmark

  2. Department of Biochemistry, Institute for Cancer Research, Oslo University Hospital, Montebello, N-0379 Oslo, Norway

    Sigrid B. Thoresen, Coen Campsteijn, Marina Vietri, Kay O. Schink, Camilla Raiborg & Harald Stenmark

  3. Department of Informatics, University of Oslo, N-0373 Oslo, Norway

    Knut Liestøl

  4. Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense, Denmark

    Jens S. Andersen

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Contributions

S.B.T. generated plasmid constructs, and performed confocal, high-content and live imaging, biochemical work, cell transfections, image processing, data analysis, statistical analyses and preparation of figures. C.C. generated plasmid constructs and stable cell lines, and performed live microscopy, photoconversion experiments, image processing, in vitro kinase assays and data analysis. C.R. carried out and analysed the siRNA screen targeting PtdIns(3)P-binding proteins, and performed cell transfections for epistasis studies. M.V. performed cell transfections, biochemical work and image analysis. K.O.S. generated plasmid constructs and stable cell lines, and performed photoconversion experiments and image processing. K.L. did the statistical analysis of the siRNA screen. J.S.A. performed mass spectrometry analysis of Aurora-B-phosphorylated ANCHR. H.S. coordinated the study and oversaw experiments. S.B.T., C.C. and H.S. wrote the paper. All authors discussed the results and assisted in revising the manuscript.

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Integrated supplementary information

Supplementary Figure 1 The FYVE domain protein ANCHR (ZFYVE19) regulates abscission and prevents cleavage furrow regression in the presence of lagging chromatin.

(a) HeLa cells were transfected with smart-pool siRNAs targeting the PtdIns(3)P-binding FYVE- and PX- domain families of proteins, and after 72 or 96 h fixed and stained for α-tubulin, Aurora B and DNA (Hoechst). Images collected on a high-content ScanR microscope were then scored manually to quantify multinuclear cells. The values obtained are ranked in ascending order according to the frequency of multinuclear cells. Each dot represents the average of four individual observations. Red dots represent PtdIns(3)P-binding proteins, while green and blue dots represent positive and negative controls, respectively. Dashed box highlights ANCHR (ZFYVE19). (b,c) Montage showing sequential images of asynchronous HeLa cells expressing GFP-α-tubulin and mCherry-H2B treated with siRNAs through cytokinesis, with a time interval of 5 min. T = time, T = 0 defined as the point of complete cleavage furrow ingression. Scale bar, 10 μm. (b) Arrows indicate microtubule disassembly. (c) shows furrow regression in ANCHR knock-down cells in the presence of lagging anaphase chromatin (indicated by arrows).

Supplementary Figure 2 ANCHR (ZFYVE19) binds PtdIns(3)P.

(a) Immunoblot showing GST alone or GST-ANCHR incubated with PtdIns(3)P-liposomes. (b) Representative confocal images showing the localization of Myc-ANCHR1 − 133, but not Myc-ANCHR1 − 133R101A to PtdIns(3)P-rich early endosomes. The cells were co-stained with antibodies against the early endosome marker EEA1 and β-Actin. Scale bar, 20 μm. (c) Representative confocal images showing the localization of GFP-ANCHR1 − 133 and GFP-ANCHR R101A to the midbody. The cells were co-stained with antibodies against α-tubulin and DNA (Hoechst). Scale bar, 10 μm.

Supplementary Figure 3 ANCHR interacts with VPS4 at overexpressed and moderate, non-arresting levels.

(a) Coomassie-stained gel of GFP-trap lysates used for mass spectromeric analysis of proteins interacting with overexpressed GFP or GFP-ANCHR. Black arrows indicate GFP and GFP-ANCHR. Red arrows indicate bands specifically detected in the GFP-ANCHR pull down which were excised and sent for mass spec analysis. (b) Western blot analysis of lysates from stable transgenic HeLa lines used for mass spec analysis presented in Supplementary Table 2, probed with anti-ANCHR (left panel) or anti-VPS4 (right panel) antibodies to determine the expression level of transgene compared to endogenous allele. Quantification indicated that the eGFP-ANCHR transgene was overexpressed 6 fold compared to endogenous, whereas eGFP-VPS4 levels were equal to endogenous. (c) GFP-trap immunoprecipitates (IP) from HeLa cells stably expressing GFP-ANCHR analysed by western blotting using antibodies against GFP and VPS4.

Supplementary Figure 4 Localization of ANCHR and VPS4 during interphase and early mitosis.

Localization of ANCHR and VPS4 during interphase and early mitosis. (a) Representative confocal images showing HeLa cells stained with antibodies against endogenous ANCHR and VPS4, and DNA (Hoechst). Scale bar, 10 μm. (b–d) Assessment of VPS4 antibody specificity. HeLa cells were treated with either non-targeting siRNA or VPS4A siRNA for 72 h and stained with antibodies against endogenous ANCHR and VPS4, and DNA (Hoechst). (b) Representative confocal images showing late cytokinetic cells containing chromatin bridges. Circles highlight the midbody ring. Scale bar, 10 μm. (c) Quantification of VPS4 intensities on midbody rings. (D) Western blot from whole cell lysates blotted for VPS4 and β-actin. Arrows indicate VPS4B (top), VPS4A (middle) and an unspecific band (bottom).

Supplementary Figure 5 ANCHR interacts with VPS4 at the midbody.

(a) Representative confocal images showing GFP-ANCHR and endogenous VPS4 at the midbody in a normally segregating cell. The cells were co-stained with Hoechst (DNA). Scale bar, 10 μm. (b) Representative confocal images from HeLa cells stably expressing GFP-VPS4A and mCherry-CEP55 showing their colocalization at the Flemming body (midbody ring). The cells were co-stained with antibodies against α-tubulin and DNA (Hoechst). Scale bar, 10 μm (c) Montage showing sequential stills of a HeLa cell expressing GFP-VPS4 and mCherry-CEP55, imaged at a time interval of 5 min. GFP-VPS4 co-localizes with the mCherry-CEP55-positive Flemming body (midbody ring) before abscission. Arrow indicates severed cytokinetic bridge. Scale bar, 2 μm. (d) The structure of ANCHR showing the defined domains, including the MIM-A and MIM-B. Alignment of the ANCHR MIM-A and MIM-B with other type-1 MIMs from different ESCRTs.

Supplementary Figure 6 The midbody is intact in ANCHR-depleted cells.

(a) Representative confocal images showing ANCHR, VPS4 and RacGAP1 at the midbody in untransfected (left panel) or ANCHR-depleted (right panel) cells. The cells were co-stained with Hoechst (DNA). Scale bar, 10 μm. (b) Quantification of RagGAP1 mean intensity on midbody ring (a.u. = arbitrary units) ± s.d. n = 15.

Supplementary Figure 7 VPS4 interaction with CHMP4C is reduced on Aurora B inhibition.

Quantification of VPS4 from GFP-trap blots illustrated in Fig. 5f. Values are averaged from three independent experiments.

Supplementary Figure 8 In vitro and in vivo phosphorylation of ANCHR.

(a) ANCHR can be phosphorylated by Aurora B at S22 in vitro. In vitro kinase assays using purified recombinant GST or GST-ANCHR, incubated with active Aurora B kinase in the presence of 33P. Incorporation of 33P was visualized by autoradiography, protein loading was assessed by coomassie staining. Mass spectrometry analysis (Supplementary Table 3) of in vitro phosphorylated GST-ANCHR using the non-radioactive phosphorus isotope identified S22 as the dominant phosphorylation site. Subsequent in vitro kinase assays using mutated GST-ANCHR S22A abolished phosphorylation by Aurora B, indicating that S22 is the sole Aurora B target in ANCHR in vitro. (b) ANCHR is not a prominent Aurora B target in vivo. HeLa cells transfected with the indicated constructs were labelled with 33P phosphate overnight, and treated with 2 μm AZD1152 for 2.5 h where indicated. Following lysis, GFP-fusions were immunoprecipitated using GFP antibodies. Incorporation of 33P was evaluated by autoradiography.

Supplementary Figure 9

Uncropped western blots used in this study.

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Supplementary Table 1

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Supplementary Table 2

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Supplementary Table 3

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Supplementary Table 4

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Supplementary Table 5

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Localization of eGFP-Vps4 to the midbody ring before abscission.

HeLa cells stably expressing near-endogenous levels of eGFP-VPS4 and the midbody marker mCherry-CEP55 were imaged as the cell proceeded through abscission. Note that eGFP-VPS4 colocalizes with mCherry-CEP55 before abscission (t = 12 min). (MOV 429 kb)

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Thoresen, S., Campsteijn, C., Vietri, M. et al. ANCHR mediates Aurora-B-dependent abscission checkpoint control through retention of VPS4. Nat Cell Biol 16, 547–557 (2014). https://doi.org/10.1038/ncb2959

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