Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis

Abstract

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes1,2. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction1,2 and has been proposed to attach the cleavage furrow to the intercellular bridge3. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody4. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle3. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

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Figure 1: The C1 domain of MgcRacGAP is required for cytokinesis.
Figure 2: Plasma membrane association of the C1 domain of MgcRacGAP.
Figure 3: Artificial plasma membrane tethering of MgcRacGAP restores cytokinesis in the absence of C1 domain function.
Figure 4: Persistent association of the plasma membrane with the midbody requires the C1 domain of MgcRacGAP.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors for the C1domain of MgcRacGAP have been deposited at the Protein Data Bank (http://www.rcsb.org/pdb) under accession code 4B6D.

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Acknowledgements

We would like to thank W. Bement, W. Earnshaw, J. Gannon, D. Gerlich, M. Glotzer, G. Hammond, M. Hikida, H. Hochegger, R. Irvine, M. Kurosaki, B. Larijani, P. Parker, A. Piekny, R. Prekeris, E. Sahai, K. Samejima and M. Symons for reagents and advice. Work in the Petronczki laboratory is supported by Cancer Research UK and the EMBO Young Investigator Programme. S.L. acknowledges support from an EMBO Long-Term Fellowship.

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Authors

Contributions

M.P. and S.L. designed the experiments. S.L. conducted all experiments with the following exceptions. The imaging data shown in Fig. 4a were recorded by K.-C. S. The structure of the C1 domain was determined by V.E.P. and P.C. Electron microscopy analysis was carried out with contributions from K.B. and L.M.C. The analysis of MgcRacGAP-R385A was carried out by S.S. Lipid interaction assays were carried out by T.T. and N.D. The manuscript was written by M.P.

Corresponding author

Correspondence to Mark Petronczki.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-9, Supplementary References and Supplementary Table 1, which contains data collection, phasing and refinement statistics for the structure determination of MgcRacGAP’s C1 domain by X-ray crystallography. (PDF 9142 kb)

The C1 domain of MgcRacGAP is atypical (related to Figure 2)

This video shows confocal time-lapse series of HeLa Kyoto cells co-expressing mCherry–H2B (red) and a FLAc-tagged version of the C1B domain of human PKCα (white) (left panel) or a FLAc-tagged version of the C1 domain of human MgcRacGAP (white) (right panel). Frames were acquired every 30 seconds. 1 μM TPA was added at t = 0 sec. (MOV 1855 kb)

Live-cell imaging of the plasma membrane and MgcRacGAP dynamics during cytokinesis (related to Figure 4)

This video shows confocal time-lapse series of HeLa Kyoto cell stably co-expressing MyrPalm–mCherry (red) and the indicated MgcRacGAPr–FLAc transgenes (white). Cells were recorded 28 to 48 h after transfection with MgcRacGAP siRNA. Frames were acquired every 2 min. Time point t = 0 min was set to the metaphase-toanaphase transition. (MOV 8773 kb)

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Lekomtsev, S., Su, K., Pye, V. et al. Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis. Nature 492, 276–279 (2012). https://doi.org/10.1038/nature11773

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