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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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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.
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RSOB120070F4: 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.

Mentions: We then asked whether Borealin colocalized with CHMP4 proteins during cytokinesis in HeLa cells. We found that all three GFP-tagged CHMP4 proteins accumulated at the midbody in late cytokinesis (figure 4), in agreement with published observations [9,10]. However, both GFP::CHMP4A and GFP::CHMP4B were also found in the nucleus, whereas GFP::CHMP4C was predominantly cytoplasmic (figure 4a,b). Both GFP::CHMP4B and GFP::CHMP4C partially colocalized with Borealin during late cytokinesis (figure 4b) but during abscission only CHMP4 proteins were found at the midbody (figure 4b), a localization pattern similar to that observed in Drosophila cells (figure 1c). To analyse the localization of CHMP4 proteins in more detail, we stained midbodies purified from HeLa cells with antibodies directed against CHMP4B and either tubulin or Borealin (figure 4c). These experiments confirmed that CHMP4B accumulated at the midbody and also showed that the ESCRT-III component localized as discrete dots around the Flemming body to form a structure resembling a pearl necklace (figure 4c). Some of the CHMP4B dots appeared to colocalize with Borealin (figure 4c, arrows). In conclusion, CPC and ESCRT-III Snf7 components associate at the midbody before abscission in both Drosophila and human cells.Figure 4.


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)

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

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Show All Figures
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RSOB120070F4: 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.
Mentions: We then asked whether Borealin colocalized with CHMP4 proteins during cytokinesis in HeLa cells. We found that all three GFP-tagged CHMP4 proteins accumulated at the midbody in late cytokinesis (figure 4), in agreement with published observations [9,10]. However, both GFP::CHMP4A and GFP::CHMP4B were also found in the nucleus, whereas GFP::CHMP4C was predominantly cytoplasmic (figure 4a,b). Both GFP::CHMP4B and GFP::CHMP4C partially colocalized with Borealin during late cytokinesis (figure 4b) but during abscission only CHMP4 proteins were found at the midbody (figure 4b), a localization pattern similar to that observed in Drosophila cells (figure 1c). To analyse the localization of CHMP4 proteins in more detail, we stained midbodies purified from HeLa cells with antibodies directed against CHMP4B and either tubulin or Borealin (figure 4c). These experiments confirmed that CHMP4B accumulated at the midbody and also showed that the ESCRT-III component localized as discrete dots around the Flemming body to form a structure resembling a pearl necklace (figure 4c). Some of the CHMP4B dots appeared to colocalize with Borealin (figure 4c, arrows). In conclusion, CPC and ESCRT-III Snf7 components associate at the midbody before abscission in both Drosophila and human cells.Figure 4.

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