The chromosomal passenger complex controls the function of endosomal sorting complex required for transport-III Snf7 proteins during cytokinesis

Abstract

Cytokinesis controls the proper segregation of nuclear and cytoplasmic materials at the end of cell division. The chromosomal passenger complex (CPC) has been proposed to monitor the final separation of the two daughter cells at the end of cytokinesis in order to prevent cell abscission in the presence of DNA at the cleavage site, but the precise molecular basis for this is unclear. Recent studies indicate that abscission could be mediated by the assembly of filaments comprising components of the endosomal sorting complex required for transport-III (ESCRT-III). Here, we show that the CPC subunit Borealin interacts directly with the Snf7 components of ESCRT-III in both Drosophila and human cells. Moreover, we find that the CPC's catalytic subunit, Aurora B kinase, phosphorylates one of the three human Snf7 paralogues-CHMP4C-in its C-terminal tail, a region known to regulate its ability to form polymers and associate with membranes. Phosphorylation at these sites appears essential for CHMP4C function because their mutation leads to cytokinesis defects. We propose that CPC controls abscission timing through inhibition of ESCRT-III Snf7 polymerization and membrane association using two concurrent mechanisms: interaction of its Borealin component with Snf7 proteins and phosphorylation of CHMP4C by Aurora B.

Keywords: Aurora B kinase; Borealin; CHMP4; Shrb; abscission.

Figure 1.

Borr interacts and colocalizes with the ESCRT-III component Shrb in Drosophila cells. (a) The GST::Borr fragments indicated on the schematic in the upper part were purified from bacteria and incubated with Shrb translated and radio-labelled in vitro, and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown in the lower part. The numbers on the left indicate the sizes (kDa) of the molecular mass marker. On the right is shown a schematic of the positive (+) and negative (−) results of the GST pull down. (b) GST::Aurora B (AurB) was purified from bacteria and incubated with Shrb translated and radio-labelled in vitro, and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom. The numbers on the left indicate the sizes (kDa) of the molecular mass marker. (c) Drosophila S2 cells stably expressing Shrb::GFP were fixed and stained to detect Aurora B (red in the merged panel), GFP (green in the merged panel) and DNA (blue in the merged panel). Midbody presence and DNA condensation were used as criteria to stage cells during cytokinesis. The inset shows a 2× magnification of the midbody. Scale bar, 10 µm.

Figure 2.

Human and Drosophila ESCRT-III Snf7 proteins are very well conserved except in their C-terminal tails. CHMP4A, CHMP4B, CHMP4C and Shrb sequences were aligned using the blast program (http://www.ncbi.nlm.nih.gov/) and the Blosum62 colouring scheme (matches are highlighted in dark blue, and positive alignment scores in light blue). The conservation histogram (yellow and brown bars) is shown below. Conservation is measured as a numerical index reflecting the conservation of physico-chemical properties in the alignment: identities (indicated by asterisks) score highest and the next most-conserved group contains substitutions to amino acids lying in the same physico-chemical class. The serine residues phosphorylated by Aurora B are boxed in red.

Figure 3.

Borealin interacts with ESCRT-III CHMP4 proteins in vivo and in vitro. (a) HeLa cells were transfected with constructs expressing PtA::Borealin or PtA alone and one of the plasmids expressing GFP-tagged CHMP4 proteins or GFP alone. After 48 h, cells were harvested, and protein extracts used in a PtA pull-down assay. The extracts and pull downs were then analysed by Western blot to detect GFP (α-GFP) and PtA (α-PtA). The numbers on the left indicate the sizes (kDa) of the molecular mass marker. (b) GST::Borealin (GST::Bor) was purified from bacteria and incubated with CHMP4A, CHMP4B or CHMP4C translated and radio-labelled in vitro and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom. The numbers on the left indicate the sizes (kDa) of the molecular mass marker. (c) The schematic in the upper part illustrates Borealin protein domains and the position of different fragments used in the GST pull-down assay. GST::Borealin (GST::Bor) fragments were purified from bacteria and incubated with CHMP4C translated and radio-labelled in vitro, and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom. The numbers on the left indicate the sizes (kDa) of the molecular mass marker. (d) The schematic in the upper part illustrates CHMP4C protein domains and the two fragments used in the GST pull-down assay. GST::CHMP4C fragments were purified from bacteria and incubated with Borealin translated and radio-labelled in vitro and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom. The numbers on the left indicate the sizes (kDa) of the molecular mass marker.

Figure 4.

CHMP4 proteins colocalize with Borealin to the midbody in HeLa cells. (a) HeLa cells were transfected with constructs expressing GFP::CHMP4A or GFP::CHMP4C for 48 h and then fixed and stained to detect tubulin (red in the merged panels), GFP (green in the merged panels) and DNA (blue in the merged panels). The insets show 2× magnification of the midbody. Scale bars, 10 µm. The presence and thickness of microtubule bundles at the intercellular bridge were used as criteria to stage cells during cytokinesis. (b) HeLa cells were transfected with constructs expressing GFP::CHMP4B or GFP::CHMP4C for 48 h and then fixed and stained to detect Borealin (red in the merged panels), GFP (green in the merged panels) and DNA (blue in the merged panels). Midbody presence and DNA condensation were used as criteria to stage cells during cytokinesis. The insets show magnification of the midbody region at 2.5× (GFP::CHMP4B) and 3× (GFP::CHMP4C). Scale bars, 10 µm. (c) Midbodies were purified from HeLa cells and then fixed and stained to detect CHMP4B (green in the merged panel) and either tubulin or Borealin (red in the merged panel). The arrows mark the CHMP4B dots that colocalize with Borealin. Scale bars, 5 µm.

Figure 5.

Aurora B phosphorylates CHMP4C in vitro and in vivo. (a) GST-tagged CHMP4 proteins, GST alone or the positive control MBP (myelin basic protein) were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-³²P] ATP. The reactions were then separated by SDS-PAGE, and gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. Note that Aurora B is auto-phosphorylated and co-migrated with GST::CHMP4B. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (b) GST-tagged full-length CHMP4C (FL), GST-tagged N-terminal CHMP4Cα12, GST-tagged C-terminal CHMP4Cα345, GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-³²P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (c) GST-tagged wild-type CHMP4Cα345 (WT), the two GST:: CHMP4Cα345 variants containing S to A mutations at position 210 (S210A) or at position 210, 214 and 215 (StripleA), GST alone and the positive control MBP were incubated with (+) or without (−) recombinant Aurora B in the presence of [γ-³²P] ATP. Products of the reactions were then separated by SDS-PAGE and the gels stained with Coomassie Blue, dried and exposed at −80°C. The Coomassie Blue staining of the protein loading is shown at the bottom. The numbers on the right indicate the sizes (kDa) of the molecular mass marker. (d) HeLa cells were transfected with GFP::CHMP4C, synchronized in metaphase with thymidine/nocodazole block and then released into medium containing either ZM447439 or its solvent DMSO as control. Proteins were extracted, separated by SDS-PAGE, transferred onto PVDF membranes and analysed by Western blot to detect the variant of CHMP4C phosphorylated at serine 210, 214 and 215 (phospho-CHMP4C), cyclin B, Borealin, GFP::CHMP4C and tubulin as loading control. The numbers indicate the sizes (kDa) of the molecular mass marker.

Figure 6.

Over-expression of phospho-mimic and non-phosphorylatable CHMP4C mutants leads to cytokinesis failure. (a) HeLa cells were transfected with constructs expressing wild-type GFP::CHMP4C (WT) or one of the GFP::CHMP4C variants containing S to A or S to E substitutions at position 210 (S210A and S210E) or at position 210, 214 and 215 (StripleA and StripleE) for 48 h and then fixed and stained to detect tubulin (red in the merged panel), GFP (green in the merged panel) and DNA (blue in the merged panel). The insets show 2× magnification of the midbody. Scale bars, 10 µm. (b) Examples of multinucleate cells obtained after transfection with GFP::CHMP4C mutants. HeLa cells were transfected with constructs expressing one of the GFP::CHMP4C variants containing S to A substitutions at position 210 (S210A) or at position 210, 214 and 215 (StripleA) or GFP alone as a control for 48 h and then fixed and stained to detect tubulin (red in the merged panel), GFP (green in the merged panel) and DNA (blue in the merged panel). Note that the cells expressing the two GFP::CHMP4C mutants are multinucleated. Scale bars, 10 µm. (c) Percentage of multinucleate cells after treatment for 48 h with the GFP::CHMP4C constructs shown in panel (a). At least 600 cells were counted in each experiment, n = 4. Bars indicate standard errors.

Figure 7.

Model for Aurora B-mediated regulation of CHMP4C during cytokinesis. (a) Schematic of CHMP4C indicating the position of the Aurora B target sites. (b) Interaction with Borealin and phosphorylation by Aurora B keep CHMP4C in its ‘closed’ state at the midbody before abscission. (c) When CPC is no longer at the midbody, CHMP4C can convert into its ‘open’ state and form membrane-associated polymers that mediate abscission.