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

Capalbo L, Montembault E, Takeda T, Bassi ZI, Glover DM, D'Avino PP - Open Biol (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

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.

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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.
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RSOB120070F1: 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.

Mentions: In a proteomic survey of complexes involved in cell division in Drosophila, we tagged the CPC Borealin-related (Borr) component with two IgG-binding domains of Protein A (PtA) at either its N- or C-terminus. The presence of a tag at either end of Borr did not affect the localization of the protein (see electronic supplementary material, figure S1, and data not shown). Independent Drosophila cell lines stably expressing PtA-tagged Borr proteins were generated, and interacting partners isolated by affinity purification and identified by mass spectrometry (MS). In both purifications, we identified the ESCRT-III component Shrb with a score comparable to or even higher than INCENP (table 2). A reciprocal affinity purification using cells expressing PtA::Shrb also identified all the CPC components (table 2), confirming the association in vivo. Because Borr and INCENP interact directly, these MS data suggested that Borr could also bind to Shrb directly. To confirm this, we used an in vitro glutathione S-transferase (GST) pull-down assay (figure 1a). We generated several GST-tagged Borr fragments in bacteria and tested their ability to pull down a radio-labelled Shrb polypeptide synthesized by in vitro translation (figure 1a). Full-length Borr1–315 and the truncated Borr1–249 fragment pulled down Shrb very efficiently, whereas Borr1–117 did not (figure 1a). Borr118–315 could also efficiently pull down Shrb, whereas both Borr118–249 and the most C-terminal Borr250–315 fragment did not bind to Shrb (figure 1a). We conclude that the Borr118–249 central region is necessary, but not sufficient, for the interaction with Shrb. No direct interaction was observed with two other components of the CPC, INCENP (data not shown) and Aurora B (figure 1b), using a similar assay. To assess if and when CPC colocalized with Shrb during mitosis, we generated a stable cell line stably expressing Shrb tagged with green fluorescent protein (GFP). In late telophase, both Shrb::GFP and Aurora B accumulated at the midbody where the two proteins partially colocalized (figure 1c). However, Shrb::GFP appeared to localize to the midbody ring, whereas Aurora B was more broadly distributed. Finally, during abscission, only Shrb was visible at the midbody (figure 1c). Altogether, these results indicate that Shrb and Borealin interact in late cytokinesis just before abscission.Table 2.


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

Capalbo L, Montembault E, Takeda T, Bassi ZI, Glover DM, D'Avino PP - Open Biol (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376741&req=5

RSOB120070F1: 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.
Mentions: In a proteomic survey of complexes involved in cell division in Drosophila, we tagged the CPC Borealin-related (Borr) component with two IgG-binding domains of Protein A (PtA) at either its N- or C-terminus. The presence of a tag at either end of Borr did not affect the localization of the protein (see electronic supplementary material, figure S1, and data not shown). Independent Drosophila cell lines stably expressing PtA-tagged Borr proteins were generated, and interacting partners isolated by affinity purification and identified by mass spectrometry (MS). In both purifications, we identified the ESCRT-III component Shrb with a score comparable to or even higher than INCENP (table 2). A reciprocal affinity purification using cells expressing PtA::Shrb also identified all the CPC components (table 2), confirming the association in vivo. Because Borr and INCENP interact directly, these MS data suggested that Borr could also bind to Shrb directly. To confirm this, we used an in vitro glutathione S-transferase (GST) pull-down assay (figure 1a). We generated several GST-tagged Borr fragments in bacteria and tested their ability to pull down a radio-labelled Shrb polypeptide synthesized by in vitro translation (figure 1a). Full-length Borr1–315 and the truncated Borr1–249 fragment pulled down Shrb very efficiently, whereas Borr1–117 did not (figure 1a). Borr118–315 could also efficiently pull down Shrb, whereas both Borr118–249 and the most C-terminal Borr250–315 fragment did not bind to Shrb (figure 1a). We conclude that the Borr118–249 central region is necessary, but not sufficient, for the interaction with Shrb. No direct interaction was observed with two other components of the CPC, INCENP (data not shown) and Aurora B (figure 1b), using a similar assay. To assess if and when CPC colocalized with Shrb during mitosis, we generated a stable cell line stably expressing Shrb tagged with green fluorescent protein (GFP). In late telophase, both Shrb::GFP and Aurora B accumulated at the midbody where the two proteins partially colocalized (figure 1c). However, Shrb::GFP appeared to localize to the midbody ring, whereas Aurora B was more broadly distributed. Finally, during abscission, only Shrb was visible at the midbody (figure 1c). Altogether, these results indicate that Shrb and Borealin interact in late cytokinesis just before abscission.Table 2.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

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.

Show MeSH
Related in: MedlinePlus