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ESCRT-III Governs the Aurora B–Mediated Abscission Checkpoint Through CHMP4C

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Science  13 Apr 2012:
Vol. 336, Issue 6078, pp. 220-225
DOI: 10.1126/science.1217180

To Cut or Not to Cut

During animal cell division, the final separation of daughter cells requires ESCRT-III (endosomal sorting complex required for transport III), the core membrane scission machinery. Carlton et al. (p. 220, published online 15 March; see the Perspective by Petronczki and Uhlmann) report that ESCRT-III modulates abscission timing through one of its subunits, CHMP4C. Depletion of CHMP4C results in faster resolution of the midbody, the cytoplasmic bridge that connects the daughter cells at the end of cytokinesis. This phenotype correlates with a differential spatiotemporal distribution of CHMP4C at the midbody. As CHMP4C is essential for activating the Aurora B–mediated abscission checkpoint, consequently, depletion of CHMP4C results in the accumulation of genetic damage. Thus, the ESCRT machinery protects the cell against genetic damage by coordinating its cytokinetic activity with the abscission checkpoint.

Abstract

The endosomal sorting complex required for transport (ESCRT) machinery plays an evolutionarily conserved role in cytokinetic abscission, the final step of cell division where daughter cells are physically separated. Here, we show that charged multivesicular body (MVB) protein 4C (CHMP4C), a human ESCRT-III subunit, is involved in abscission timing. This function correlated with its differential spatiotemporal distribution during late stages of cytokinesis. Accordingly, CHMP4C functioned in the Aurora B–dependent abscission checkpoint to prevent both premature resolution of intercellular chromosome bridges and accumulation of DNA damage. CHMP4C engaged the chromosomal passenger complex (CPC) via interaction with Borealin, which suggested a model whereby CHMP4C inhibits abscission upon phosphorylation by Aurora B. Thus, the ESCRT machinery may protect against genetic damage by coordinating midbody resolution with the abscission checkpoint.

The final separation of daughter cells during cytokinesis is the ancestral function of the endosomal sorting complex required for transport (ESCRT) machinery (15) which also acts to resolve equivalent membrane scission events in multivesicular body (MVB) formation (6, 7) and human immunodeficiency virus–1 (HIV-1) budding (8, 9). Midbody recruitment of ESCRT-III, the filament-forming scission machinery, is an essential event in cytokinesis that is thought to provide constrictive force during abscission (2, 1012). An Aurora B–dependent abscission checkpoint (NoCut) is thought to retard abscission to prevent damage of lagging chromosomes that are trapped in the midbody (1315) and may function more generally as an abscission timer (13). However, mechanisms that modulate abscission timing remain poorly understood and the involvement of the core abscission machinery in this process is unclear.

Here, we investigated the function of charged MVB proteins CHMP4A, -B, and -C, human homologs of the yeast ESCRT-III subunit Snf7p. For this purpose, specific small interfering RNAs (siRNAs) and antibodies against each of the CHMP4s were developed (16) (fig. S1). We then analyzed ESCRT-dependent endosomal down-regulation of class I major histocompatibility complex (MHC-I) molecules in HeLa cells stably expressing the K3 ubiquitin ligase from Kaposi’s sarcoma–associated herpes virus (KSHV) (17). Similarly to depletion of tumor susceptibility gene 101 (TSG101), depletion of CHMP4B prevented MHC-I degradation, whereas depletion of CHMP4A or CHMP4C had little effect on this process (fig. S2, A and B). As expected (18), depletion of CHMP4A or CHMP4C did not inhibit HIV-1 release, and only depletion of CHMP4B inhibited this ESCRT-dependent process (fig. S3). Furthermore, CHMP4A and CHMP4C were dispensable for completion of cytokinesis, whereas CHMP4B was again the sole paralog required (Fig. 1, A and B). However, in asynchronous cultures of CHMP4C-depleted HeLa cells, fewer cells were connected by midbodies (Fig. 1C), which led us to question whether midbodies were resolved faster in cells lacking CHMP4C.

Fig. 1

CHMP4C negatively regulates cytokinesis. (A) Resolved HeLa cell lysates were examined by blotting with antibodies against CHMP4A, CHMP4B, CHMP4C, or heat shock protein 90 kD (HSP90). (B and C) siRNA-transfected HeLa cells were fixed and stained with α-tubulin. (B) Multinucleate cells (n = 3, ±SD) or (C) cells connected by midbodies (n = 7, ±SD) were scored visually. (D) Resolved lysates from siRNA-transfected HeLa mCh-Tub cells were examined by blotting with antibodies against CHMP4C or HSP90. (E and F) Asynchronous cultures of HeLa mCh-Tub cells were transfected with the indicated siRNA and imaged live, and mitotic durations were quantified. Abscission time was calculated across four independent experiments (luciferase: 93 ± 38 min, n = 96; nontargeting: 94 ± 36 min, n = 94; CHMP4C-1: 59 ± 17 min, n = 88; CHMP4C-2: 61 ± 25 min, n = 100). (G) Resolved cell lysates from HeLa cells stably expressing mCh-Tub and either GFP-CHMP4B or GFP-CHMP4C were examined by blotting with antibodies against HSP90, GFP, CHMP4B, or CHMP4C. (H and I) Cells from (G) were imaged live, and mitotic durations were quantified. The more intense imaging (16) resulted in general abscission delays to 116 ± 45 and 137 ± 61 min for control or GFP-CHMP4B–expressing cells, whereas GFP-CHMP4C–expressing cells took 240 ± 103 min to complete abscission. Data comprise 185 cells per condition from three independent experiments.

We then imaged live HeLa cells stably expressing mCherry-tubulin (HeLa mCh-Tub) (19) to examine mitotic dynamics. CHMP4C-depleted cells (Fig. 1D) showed normal duration of early mitotic phases and centrosome amplification (Fig. 1E and fig. S4A). We next monitored tubulin disassembly at the midbody as a marker that correlates strongly with abscission (15). Cells treated with control siRNAs resolved their midbodies with similar kinetics and depletion of CHMP4C reduced abscission time by ~30 min (Fig. 1F, fig. S4A, and movies S1 to S4). Abscission was also faster in cells codepleted of CHMP4C and Spastin, an adenosine triphosphatase involved in destabilization of midbody microtubules (20, 21), which suggested that CHMP4C and Spastin regulate distinct stages of abscission (fig. S4B). We next used cell lines stably expressing mCh-tubulin and comparable levels of green fluorescent protein (GFP)–tagged CHMP4B or CHMP4C (22) (Fig. 1G) for simultaneous imaging of abscission and midbody recruitment of ESCRT-III. Early phases of mitosis and abscission (Fig. 1, H and I) were similar in control and GFP-CHMP4B–expressing cells. However, expression of GFP-CHMP4C resulted in an abscission delay that could be explained by increased levels of intracellular CHMP4C (Fig. 1I; fig. S5, A to D; and movies S5 and S6). As expected (12), GFP-CHMP4B localized transiently to the midbody arms immediately (21 ± 7 min) before abscission, whereas GFP-CHMP4C localized earlier to the midbody, arriving 176 ± 19 min before abscission (Fig. 2, A to D). During its recruitment, GFP-CHMP4C localized initially to the midbody arms, before being directed to the central region (Flemming body) (Fig. 2, A to D; fig. S5, A to D; and movies S5 and S6). Thus, CHMP4B and CHMP4C exhibit differential spatiotemporal distribution during late cytokinesis.

Fig. 2

Differential spatiotemporal recruitment of CHMP4 paralogs during cytokinesis. (A to D) GFP fluorescence intensities of midbody-localized (A and C) GFP-CHMP4B (n = 14, ±SD) or (B and D) GFP-CHMP4C (n = 9, ±SD) during abscission. Abscission indicated by arrow, time in minutes. (C and D) Selected frames are presented. Initial recruitment of GFP-CHMP4C to midbody arms is marked (arrowhead). (E) ClustalW alignment of the C-terminal regions of CHMP4A, CHMP4B, and CHMP4C, S210 indicated by arrow. MIM2, a motif that binds microtubule interaction and trafficking (MIT) domains. (F) HeLa cells transfected with plasmids encoding the indicated HA-CHMP4 constructs were fixed and stained with antibodies against tubulin and HA. Multinucleate cells were scored (n = 5, ±SD). (G and H) HeLa mCh-Tub cells stably expressing HA-CHMP4CR, HA-CHMP4CRδINS, or HA-CHMP4CR S210A were treated with CHMP4C siRNA, fixed, and stained with HA-specific antibody. The HA-CHMP4C location was scored (n = 3, ±SD). Scale bar is 10 μm.

We sought differences between CHMP4 paralogs that could explain their differential behavior. Alignment of the regulatory region at the C termini of CHMP4s revealed a CHMP4C-specific insertion (INS) at residues 201 to 217 (Fig. 2E). This insertion is expanded in mammals (fig. S6) and contains numerous serine (S) and threonine (T) residues. Transiently overexpressed CHMP4 chimeras containing CHMP4C’s C terminus were more potent inhibitors of cell division (Fig. 2F), and grafting INS into the corresponding region of CHMP4B produced a chimera that inhibited cell division (Fig. 2F), which suggested that INS may be a platform for phosphorylation that inhibits abscission. We next determined the spatial distribution of CHMP4C during abscission by analyzing hemagglutinin (HA)–tagged CHMP4C expressed stably at near-endogenous levels. In early midbodies, HA-CHMP4C localized to the midbody arms, whereas in late midbodies, as observed for GFP-CHMP4C in living cells (Fig. 2B), HA-CHMP4C localized to the Flemming body in an INS-dependent manner (Fig. 2, G and H). In contrast, CHMP4A and CHMP4B were only observed on midbody arms (fig. S5E). We speculated that phosphorylation of residues within INS may have directed CHMP4C’s localization and mapped the determinant of Flemming body localization in late cyokinesis to S210, a residue conforming to the consensus sequence for Aurora B ([R/K](1-3)-X-[S/T] (23) (Fig. 2, G and H, and fig. S5F).

We next wondered whether CHMP4C participated in the Aurora B–dependent NoCut abscission checkpoint. This checkpoint can be activated by partial depletion of nucleoporin 153 kD (NUP153) and is evidenced by an accumulation of cells unable to complete abscission and arrested at the midbody stage (24). NoCut activation was prevented by codepletion of CHMP4C with NUP153 (Fig. 3, A and B), despite phosphorylated Aurora B persisting at midbodies (Fig. 3C). Chromosomes trapped at the midbody may provide an alternative NoCut activation route. We found that HA-CHMP4C preferentially localized to intercellular chromatin bridges illuminated with yellow fluorescent protein (YFP)–tagged lamin-associated protein 2β (LAP2β) (Fig. 3D and fig. S7, A and B). Moreover, HA-CHMP4C colocalized with activated Aurora B at these chromatin bridges (Fig. 3E). We examined intercellular DNA-bridge resolution in CHMP4C-depleted cells and observed that YFP-LAP2β–positive bridge formation and cellular viability were normal (fig. S7, C and D) and also found, similarly to Aurora B–inhibited cells (15), faster resolution of YFP-LAP2β–positive chromatin bridges (Fig. 3F; fig. S7, E to G; and movies S7 to S9). Consequently, stable depletion of CHMP4C resulted in increased levels of histone H2AX phosphorylation (Fig. 3G and fig. S7H), which suggested that deregulation of the abscission checkpoint in these cells results in the accumulation of genetic damage (25).

Fig. 3

CHMP4C regulates the abscission checkpoint. (A to C) (A) Cell lysates from siRNA-transfected HeLa cells were examined by blotting with antibodies against NUP153, CHMP4C, HA, and HSP90. Multinucleate and midbody-connected cells were scored visually (n = 6, ±SD). Alternatively, cells were fixed and stained with antibodies against (B) tubulin or (C) tubulin and pT232 Aurora B. Scale bar is 10 μm. (D and E) HeLa cells stably expressing YFP-LAP2β were transfected with plasmids encoding the indicated HA-CHMP4 constructs. Cells were fixed and stained with antibodies against (D) tubulin and HA or (E) pT232 Aurora B and HA. Scale bar is 10 μm. (F) HeLa cells stably expressing YFP-LAP2β were transfected with the indicated siRNA and imaged live; the duration of LAP2β-bridge resolution was quantified across six independent experiments (luciferase: 576 ± 454 min, n = 116; CHMP4C-1: 321 ± 308 min, n = 112; CHMP4C-2: 291 ± 278 min, n = 103). (G) Cell lysates from clonal short hairpin RNA (shRNA)–transduced HeLa cells were examined by blotting with antibodies against γH2AX, CHMP4C, or HSP90.

Given the essential role of CHMP4C in the regulation of abscission timing and its participation in the Aurora B–dependent abscission checkpoint, we searched for links between components of the chromosomal passenger complex (CPC) and ESCRT-III by yeast two-hybrid screening. We found interactions between Borealin and CHMP2A, CHMP4B, CHMP4C, and CHMP6 (Fig. 4, A and B) and mapped these interactions to the C terminus of Borealin (fig. S8A), a region that recruits adaptor proteins to the CPC. CHMP4C was the strongest interactor with Borealin, and we could coprecipitate CHMP4C with Aurora B (fig. S8B), which confirmed that the ESCRT machinery is able to engage the CPC. Colocalization of HA-CHMP4C and members of the CPC was observed in early, but not late, midbodies (Fig. 4C and fig. S8, C and D). Accordingly, a λ-phosphatase–sensitive, mobility-shifted form of HA-CHMP4C was detected in mitotic lysates and was enriched on a phospho-affinity resin (Fig. 4D and fig. S8E). Similar to centrosomal protein of 55 kD (CEP55) (26, 27), CHMP4C phosphorylation occurred at mitotic onset and reverted in a phosphatase-dependent manner as mitosis progressed (Fig. 4E). An Aurora B inhibitor reduced CHMP4C phosphorylation (Fig. 4F), and the epitope detected by α-CHMP4C, which recognizes INS, was masked in the mobility-shifted fraction and revealed upon λ-phosphatase treatment (Fig. 4D), which suggested that residues within CHMP4C’s insertion were phosphorylated during mitosis. Finally, Aurora B could specifically phosphorylate CHMP4C on S210 within INS, the residue required for Flemming body localization (Fig. 4, G and H, and fig. S8, F and G).

Fig. 4

Aurora B–dependent phosphorylation of CHMP4C S210 activates the NoCut abscission checkpoint. (A) β-Galactosidase assay from yeast cotransformed with the indicated VP16- and GAL4-fused constructs (n = 3, ±SD). (B) Cell lysates and glutathione-bound fractions from human embryonic kidney (HEK) 293T cells transfected with the indicated fusion proteins were examined by Western blotting with antibody against HA. (C) HeLa mCh-Tub cells stably expressing HA-CHMP4CR were fixed and stained with antibodies against HA and Aurora B. (D) Asynchronous and mitotic lysates of HeLa mCh-Tub cells stably expressing HA-CHMP4CR were immunoprecipitated with HA-specific antibody and treated as indicated, then examined by blotting with antibodies against HA, CHMP4C, CHMP4B, CEP55, and HSP90. (E and F). Asynchronous or mitotically arrested HeLa mCh-Tub cells stably expressing HA-CHMP4CR were either (E) released into media containing dimethyl sulfoxide (DMSO) or the phosphatase inhibitor okadaic acid (OA) for the indicated times or (F) treated overnight during the nocodazole arrest with inhibitors of mitogen-activated protein kinase kinase (MEK) (U0126), phosphatidylinositol 3-kinase (LY294002) or Aurora B (ZM447439). Cell lysates were examined by blotting with antibodies against HA and HSP90. (G and H) Proteins were immunoprecipitated from HEK 293T cells with antibody against HA and subjected to an in vitro kinase assay with recombinant Aurora B. Incorporated 32P was visualized by phosphorimaging; blotting with HA-specific antibody allowed detection of immunoprecipitates. (I to K) Asynchronous cultures of HeLa mCh-Tub cells stably expressing HA, HA-CHMP4CR, HA-CHMP4CR δINS, or HA-CHMP4CR S210A were transfected with the indicated siRNA. (I) Resolved cell lysates were examined by blotting with antibodies against CHMP4C, HA, and HSP90. (J and K) Alternatively, cells were imaged live. Abscission times were quantified across seven independent experiments (luciferase, 104 ± 35 min, n = 244; CHMP4C siRNA, 71 ± 37 min, n = 260; CHMP4C siRNA and HA-CHMP4CR, 118 ± 52 min, n = 269; CHMP4C siRNA and HA-CHMP4CR δINS, 81 ± 40 min, n = 264; CHMP4C siRNA and HA-CHMP4CR S210A, 91 ± 38 min, n = 268). (L) HeLa cells stably expressing YFP-LAP2β and HA or HA-CHMP4CR, HA-CHMP4CR δINS, or HA-CHMP4CR S210A were treated with the indicated siRNA and imaged live. The timing of YFP-LAP2β bridge resolution was quantified from two independent experiments (luciferase, 628 ± 382 min, n = 41; CHMP4C siRNA, 413 ± 292 min, n = 41; CHMP4C siRNA and HA-CHMP4CR, 698 ± 332 min, n = 41; CHMP4C siRNA and HA-CHMP4CR δINS, 402 ± 259 min, n = 36; CHMP4C siRNA and HA-CHMP4CR S210A, 421 ± 295 min, n = 40).

To investigate the role of Aurora B phosphorylation of CHMP4C on abscission timing, we used HeLa mCh-Tub cell lines stably expressing HA-tagged, siRNA-resistant versions of CHMP4C (HA-CHMP4CR, HA-CHMP4CR δINS, or HA-CHMP4CR S210A) expressed at similar, near-endogenous levels (Fig. 4I). Interaction of these mutants with known CHMP4C-binding proteins (fig. S8H) was maintained, and early mitotic phases were completed normally in cells depleted of endogenous CHMP4C and reliant on these proteins (fig. S8I). Note that HA-CHMP4CR rescued the faster abscission induced by CHMP4C depletion (Fig. 4J), whereas cells reliant on HA-CHMP4CR δINS and HA-CHMP4CR S210A could not (Fig. 4K). We propose that Aurora B–dependent phosphorylation of S210 allows CHMP4C localization to the Flemming body and acts as a brake on the late stages of cytokinesis. Furthermore, cells stably expressing YFP-LAP2β and HA-CHMP4CR S210A were unable to delay abscission in response to intracellular chromatin bridges, despite the presence of these proteins at the chromatin bridge (fig. S9A), which indicated deregulation of the NoCut checkpoint in these cells (Fig. 4L).

Here, we found that CHMP4C acts as an essential regulator of the Aurora B–mediated abscission checkpoint. In the absence of CHMP4C, cells complete abscission faster. We suggest that Aurora B–dependent phosphorylation of CHMP4C on S210 directs its Flemming body localization and delays abscission through activation of NoCut, possibly by preventing assembly of a productive abscission complex. A phosphomimetic mutation (S210D) had no apparent effect on known CHMP4C interactions (fig. S9B), which suggested that phosphoregulation of abscission timing may involve as-yet-unknown CHMP4C binding partners. That the S210 consensus site is conserved only in mammals suggests that this mechanism might have emerged late in evolution as a safety belt in addition to the interactions of the CPC with CHMP2A and CHMP6. CHMP4C depletion circumvents the NoCut abscission checkpoint, allows faster resolution of chromatin bridges, and induces the accumulation of phosphorylated H2AX. These observations are consistent with a role of CHMP4C in protection against DNA damage accumulation. In this context, as well as being charged MVB proteins (28), CHMPs were originally reported as chromatin-modifying proteins (29) that could associate with condensed chromatin, which suggests that the physical interaction of lagging chromosomes and ESCRT-III at the midbody may trigger activation of NoCut.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1217180/DC1

Materials and Methods

Figs. S1 to S9

References (3032)

Movies S1 to S9

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. Acknowledgments: We thank P. Bieniasz for kind gifts of GFP-CHMP4-expressing cells. J.M.-S was funded by the Medical Research Council (G0802777), the Lister Institute for Preventative Medicine and the EMBO Young Investigators Program. J.M.-S and M.A. were funded by Wellcome Trust grant (WT093056MA). J.G.C. was funded by Wellcome Trust Value in People award (092429/Z/10/Z). We acknowledge the U.K. National Institute for Health Research Comprehensive Biomedical Research Centre at Guy’s and St. Thomas’s National Health Service Foundation Trust and King’s College London for an equipment grant.
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