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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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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.
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RSOB120070F6: 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.

Mentions: To investigate whether Aurora B phosphorylation could affect CHMP4C localization and/or function, we transfected HeLa cells with GFP::CHMP4C mutants containing either phospho-mimic (serine to glutamate; S to E) or non-phosphorylatable (S to A) substitutions at position 210 alone or at positions 210, 214 and 215 (triple mutants). All the mutants accumulated at the midbody with a pattern similar to the wild-type protein (figure 6a). This suggests that the phosphorylation status of CHMP4C does not affect its ability to accumulate at the midbody, with the caveat that phospho-mimic mutants might not exactly reflect the localization and behaviour of the endogenous pool of phosphorylated CHMP4C. We next asked if expression of these GFP::CHMP4C mutants could affect cytokinesis. Over-expression of GFP-tagged wild-type CHMP4C has already been reported to cause cytokinesis failure [6] and we found that transfection of all four GFP::CHMP4C phospho-mutants caused an increase in the number of multi-nucleated cells compared with control cells transfected with GFP alone or cells transfected with wild-type GFP::CHMP4C (figure 6b,c). That the presumptive phospho-mimic mutants behaved like the non-phosphorylatable variant might indicate either that the S to E substitutions cannot successfully mimic Aurora B phosphorylation or that the CHMP4C phosphorylation–dephosphorylation cycle must be tightly regulated for proper CHMP4C function. Taken together, our results indicate that phosphorylation by Aurora B controls the function of CHMP4C during late cytokinesis.Figure 6.


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)

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

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

RSOB120070F6: 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.
Mentions: To investigate whether Aurora B phosphorylation could affect CHMP4C localization and/or function, we transfected HeLa cells with GFP::CHMP4C mutants containing either phospho-mimic (serine to glutamate; S to E) or non-phosphorylatable (S to A) substitutions at position 210 alone or at positions 210, 214 and 215 (triple mutants). All the mutants accumulated at the midbody with a pattern similar to the wild-type protein (figure 6a). This suggests that the phosphorylation status of CHMP4C does not affect its ability to accumulate at the midbody, with the caveat that phospho-mimic mutants might not exactly reflect the localization and behaviour of the endogenous pool of phosphorylated CHMP4C. We next asked if expression of these GFP::CHMP4C mutants could affect cytokinesis. Over-expression of GFP-tagged wild-type CHMP4C has already been reported to cause cytokinesis failure [6] and we found that transfection of all four GFP::CHMP4C phospho-mutants caused an increase in the number of multi-nucleated cells compared with control cells transfected with GFP alone or cells transfected with wild-type GFP::CHMP4C (figure 6b,c). That the presumptive phospho-mimic mutants behaved like the non-phosphorylatable variant might indicate either that the S to E substitutions cannot successfully mimic Aurora B phosphorylation or that the CHMP4C phosphorylation–dephosphorylation cycle must be tightly regulated for proper CHMP4C function. Taken together, our results indicate that phosphorylation by Aurora B controls the function of CHMP4C during late cytokinesis.Figure 6.

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