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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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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.
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RSOB120070F2: 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.

Mentions: We then investigated whether the interaction between Borealin and ESCRT-III Snf7 components had been conserved in human cells. There are three Snf7 paralogues in humans: CHMP4A, CHMP4B and CHMP4C (table 1). The primary sequence of the three CHMP4 proteins and their Drosophila homologue Shrb is very well conserved, although all four proteins diverge considerably in their C-terminal regions (figure 2). To assess whether Borealin could interact with CHMP4 proteins in vivo, we transfected HeLa cells with either PtA-tagged Borealin or PtA alone as control, and each of the three CHMP4 proteins tagged with GFP. PtA::Borealin pulled down GFP::CHMP4A and GFP::CHMP4B very efficiently, but not GFP::CHMP4C or GFP alone (figure 3a). To investigate whether these interactions were direct, we carried out an in vitro GST pull-down assay as described earlier for Borr and Shrb. GST-tagged Borealin purified from bacteria could efficiently pull down all three CHMP4 proteins synthesized by in vitro translation (figure 3b). Our inability to detect an interaction between Borealin and CHMP4C in vivo, in contrast with the positive outcome of the in vitro assay, suggests that the interaction might be regulated by post-translation modifications and/or may occur only in a specific phase of the cell cycle and therefore, not be easily detectable in our unsynchronized cell populations.FigureĀ 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)

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

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

RSOB120070F2: 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.
Mentions: We then investigated whether the interaction between Borealin and ESCRT-III Snf7 components had been conserved in human cells. There are three Snf7 paralogues in humans: CHMP4A, CHMP4B and CHMP4C (table 1). The primary sequence of the three CHMP4 proteins and their Drosophila homologue Shrb is very well conserved, although all four proteins diverge considerably in their C-terminal regions (figure 2). To assess whether Borealin could interact with CHMP4 proteins in vivo, we transfected HeLa cells with either PtA-tagged Borealin or PtA alone as control, and each of the three CHMP4 proteins tagged with GFP. PtA::Borealin pulled down GFP::CHMP4A and GFP::CHMP4B very efficiently, but not GFP::CHMP4C or GFP alone (figure 3a). To investigate whether these interactions were direct, we carried out an in vitro GST pull-down assay as described earlier for Borr and Shrb. GST-tagged Borealin purified from bacteria could efficiently pull down all three CHMP4 proteins synthesized by in vitro translation (figure 3b). Our inability to detect an interaction between Borealin and CHMP4C in vivo, in contrast with the positive outcome of the in vitro assay, suggests that the interaction might be regulated by post-translation modifications and/or may occur only in a specific phase of the cell cycle and therefore, not be easily detectable in our unsynchronized cell populations.FigureĀ 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