Coordinated regulation of the ESCRT-III component CHMP4C by the chromosomal passenger complex and centralspindlin during cytokinesis

Abstract

The chromosomal passenger complex (CPC)-composed of Aurora B kinase, Borealin, Survivin and INCENP-surveys the fidelity of genome segregation throughout cell division. The CPC has been proposed to prevent polyploidy by controlling the final separation (known as abscission) of the two daughter cells via regulation of the ESCRT-III CHMP4C component. The molecular details are, however, still unclear. Using atomic force microscopy, we show that CHMP4C binds to and remodels membranes in vitro Borealin prevents the association of CHMP4C with membranes, whereas Aurora B interferes with CHMP4C's membrane remodelling activity. Moreover, we show that CHMP4C phosphorylation is not required for its assembly into spiral filaments at the abscission site and that two distinctly localized pools of phosphorylated CHMP4C exist during cytokinesis. We also characterized the CHMP4C interactome in telophase cells and show that the centralspindlin complex associates preferentially with unphosphorylated CHMP4C in cytokinesis. Our findings indicate that gradual dephosphorylation of CHMP4C triggers a 'relay' mechanism between the CPC and centralspindlin that regulates the timely distribution and activation of CHMP4C for the execution of abscission.

Keywords: Aurora B; cell division; centralspindlin; cytokinesis; membrane remodelling; midbody.

Figure 1.

The CPC affects the ability of CHMP4C to bind to and remodel membranes in vitro. (a) Schematic of a lipid bilayer (yellow) assembled on a mica surface (brown). The central portion of the bilayer is flat, whereas the edges of the bilayer are highly curved. (b) Zoomed AFM image of a lipid bilayer incubated with CHMP4C. A height bar is shown on the right. Scale bar, 20 nm. (c) Height relative to the mica surface is plotted along the dotted line shown in (b). The position of the arrowhead (at the bilayer edge) along the line is also shown in (b), which is raised above the height of the bilayer surface, indicating the presence of protein. (d) Sequential AFM images of the same lipid bilayer in the presence of CHMP4C. A height bar is shown on the right. Scale bar, 200 nm. (e) Zoomed AFM image of a lipid bilayer incubated with CHMP4C + mini-CPC. A height bar is shown on the right. Scale bar, 50 nm. (f) Height relative to the mica surface is plotted along the dotted line shown in (e). The position of the arrowhead (at the bilayer edge) along the line is also shown in (e), which shows no rise at the bilayer edge, indicating no protein interaction with the bilayer but rather a concentration of protein on the mica surface. (g) Sequential AFM images of the same lipid bilayer in the presence of CHMP4C + mini-CPC. A height bar is shown on the right. Scale bar, 200 nm. (h) Zoomed AFM image of a lipid bilayer incubated with CHMP4C + Aurora B + ATP. A height bar is shown on the right. Scale bar, 50 nm. (i) Height relative to the mica surface is plotted along the dotted line shown in (h). The position of the arrowhead (at the bilayer edge) along the line is also shown in (h), which is raised above the height of the bilayer surface, indicating the presence of protein. (j) Sequential AFM images of the same lipid bilayer in the presence of CHMP4C + Aurora B + ATP. A height bar is shown on the right. Scale bar, 200 nm. (k) Zoomed AFM image of a lipid bilayer incubated with CHMP4C and Aurora B. A height bar is shown on the right. Scale bar, 20 nm. (l) Height relative to the mica surface is plotted along the dotted line shown in (k). The position of the arrowhead (at the bilayer edge) along the line is also shown in (k), which is raised above the height of the bilayer surface, indicating the presence of protein. (m) Sequential AFM images of the same supported lipid bilayer in the presence of CHMP4C + Aurora B. A height bar is shown on the right. The arrows mark membrane gaps that started closing and then collapsed. Scale bar, 200 nm. (n) Variation in the percentage of the mica surface coated with lipid bilayer in the presence of various proteins.

Figure 2.

Non-phosphorylatable forms of CHMP4C can form spiral structures at the abscission site. (a) HeLa Kyoto cells stably expressing Flag::CHMP4C WT were fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). Insets show two time magnification of the midbody. A three times magnification of the midbody of the cell in abscission is shown at the bottom. Scale bars, 10 µm. (b) HeLa Kyoto cells stably expressing Flag::CHMP4C S201A were fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). Insets show two times magnification of the midbody. A three times magnification of the midbody of the cell in abscission is shown at the bottom. Scale bars, 10 µm. (c) HeLa Kyoto cells stably expressing Flag::CHMP4C StripleA were fixed and stained to detect Flag (red), tubulin (green) and DNA (blue). Insets show two times magnification of the midbody. A three times magnification of the midbody of the cell in abscission is shown at the bottom. Scale bars, 10 µm. (d) HeLa Kyoto cells stably expressing Flag::CHMP4C WT were fixed and stained to detect Flag (green), CHMP4B (red) and tubulin (blue). Insets show two times magnification of the midbody. A three times magnification of the midbody of the cell in abscission is shown at the bottom. Scale bars, 10 µm. In all experiments, the shape and thickness of microtubule bundles were used as criteria to stage telophase cells as described [1,3].

Figure 3.

The two phosphorylated forms of CHMP4C display distinct localization patterns during cytokinesis. (a) HeLa Kyoto cells were fixed and stained to detect tri-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. At the bottom, midbodies were purified form HeLa Kyoto cells and fixed and stained to detect tri-phospho CHMP4C (green) and tubulin (red). Scale bar, 5 µm. (b) HeLa Kyoto cells were fixed and stained to detect tri-phospho CHMP4C (red), Aurora B (green) and tubulin (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. (c) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CHMP4C twice at a 48 h interval and then after 96 h fixed and stained to detect tri-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show a two times magnification of the midbody. Scale bars, 10 µm. (d) HeLa Kyoto cells were fixed and stained to detect mono-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. At the bottom, midbodies were purified form HeLa Kyoto cells and fixed and stained to detect mono-phospho-CHMP4C (green) and tubulin (red). Scale bar, 5 µm. (e) HeLa Kyoto cells were fixed and stained to detect mono-phospho CHMP4C (red), Aurora B (green) and tubulin (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. (f) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or CHMP4C twice at a 48 h interval and after 96 h fixed and stained to detect mono-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show a two times magnification of the midbody. Scale bars, 10 µm. In all experiments, the shape and thickness of microtubule bundles were used as criteria to stage telophase cells as described [1,3].

Figure 4.

The CPC is required for the localization of tri-phospho CHMP4C at the cleavage site. (a) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or KIF20A/MKLP2 and after 48 h fixed and stained to detect tri-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show two times magnifications of the central spindle and midbody. Scale bars, 10 µm. (b) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or KIF20A/MKLP2 and after 48 h fixed and stained to detect mono-phospho CHMP4C (red), tubulin (green) and DNA (blue). Insets show two times magnifications of the central spindle and midbody. Scale bars, 10 µm. (c) Box plot shows the quantification of mono-phospho CHMP4C fluorescence levels at the midbody calculated as described previously [6]; see also Material and methods. More than 50 cells from three separate experiments were counted for each sample. AU, arbitrary unit. p = 0.8 (Student's t-test). (d) HeLa Kyoto cells carrying a doxycycline-inducible GFP-tagged PRC1-Baronase transgene were treated with siRNAs directed against either a random sequence (control) or KIF20A and after 24 h incubated in 2 mM thymidine for a further 20 h. Cells were washed and incubated with or without 1 µg ml⁻¹ doxycycline for 10 h and then fixed and stained to detect tri-phospho CHMP4C (red), PRC1-Baronase (green) and DNA (blue). Scale bars, 10 µm. In all experiments, the shape and thickness of microtubule bundles were used as criteria to stage telophase cells as described [1,3].

Figure 5.

MKLP1 associates with CHMP4C in vivo. (a) Proportional Venn diagram shows the overlap between the proteins pulled down by the Flag tag alone (284) and the proteins that co-purified with Flag::CHMP4C (864). Note that 693 proteins specifically associated with Flag::CHMP4C. (b) Partial list of the CHMP4C-specific interactors identified by affinity purification. A full list can be found in electronic supplementary material, table S1. The bait, CHMP4C, is highlighted in red. (c) HeLa Kyoto cells stably expressing Flag::CHMP4C WT were fixed and stained to detect MKLP1 (red), Flag (green) and DNA (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. (d) HeLa Kyoto cells were fixed and stained to detect mono-phospho CHMP4C (red), MKLP1 (green) and tubulin (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. (e) HeLa Kyoto cells were fixed and stained to detect tri-phospho CHMP4C (red), MKLP1 (green) and tubulin (blue). Insets show two times magnification of the midbody. Scale bars, 10 µm. (f) HeLa cells stably expressing MKLP1::GFP were transfected with plasmids carrying Flag alone, Flag::CHMP4C WT, Flag::CHMP4C S210A or Flag::CHMP4C StripleA. After 48 h, proteins were extracted and subjected to a pull-down assay using GFP-Trap magnetic beads (ChromoTek). The extracts and pull-downs were analysed by western blot to detect Flag and GFP. The numbers on the left indicate the sizes (kDa) of the molecular mass markers.

Figure 6.

MKLP1 binds directly to CHMP4C and is required for its localization to the central spindle during cytokinesis. (a) Schematic diagrams illustrate the protein domains of MKLP1 and CHMP4C. The CHMP4C α-helices are marked at the top. The positions of the different MKLP1 and CHMP4C fragments used for the in vitro pull-down assays are also indicated. CC, coiled coil. (b) The GST::MKLP1 protein fragments shown at the top and GST alone were incubated with the in vitro translated and radiolabelled CHMP4C polypeptides indicated at the right, and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom and the numbers on the left indicate the sizes (kDa) of the molecular mass markers. (c) The GST::CHMP4C protein fragments shown at the top and GST alone were incubated with the in vitro translated and radiolabelled MKLP1 polypeptides indicated at the right, and then pulled down using glutathione beads. The Ponceau S staining of the protein loading is shown at the bottom and the numbers on the left indicate the sizes (kDa) of the molecular mass markers. (d) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or KIF23/MKLP1 and after 48 h fixed and stained to detect triphospho CHMP4C (red), tubulin (green) and DNA (blue). DNA condensation and nuclear shape were used as criteria to stage telophase cells. Insets show two times magnifications of the central spindle and midbody. Scale bars, 10 µm. (e) HeLa Kyoto cells were treated with siRNAs directed against either a random sequence (control) or KIF23/MKLP1 and after 48 h fixed and stained to detect mono-phospho CHMP4C (red), tubulin (green) and DNA (blue). DNA condensation and nuclear shape were used as criteria to stage telophase cells. Insets show two times magnifications of the central spindle and midbody. Scale bars, 10 µm.

Figure 7.

A relay mechanism for the control of CHMP4C by the CPC and centralspindlin. The diagram illustrates the distribution of different forms of CHMP4C and their association with the CPC and centralspindlin at sequential stages of cytokinesis, from mid-telophase to abscission. Full details are given in the text.