Calcineurin activity is required for the completion of cytokinesis

Abstract

Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca2+- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca2+ influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

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Acknowledgments

We wish to thank Sandra L. Schmid for providing the Baculovirus purified dynII protein. Charlotte M. Smith, Maggie Ma, Annie Quan, and Anna Powell are thanked for their technical assistance. We thank Patrick Tam and Roger Reddel for critical reading of the manuscript. This work was supported by grants from the National Health and Medical Research Council (NH&MRC) of Australia (PJR) and the NH&MRC Peter Doherty Fellowship (MC).

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Correspondence to Megan Chircop.

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18_2010_401_MOESM1_ESM.mpg

Movie S1. EGTA blocks cells in anaphase. HeLa cells were treated with RO-3306, a selective small-molecule inhibitor of cdk1 that reversibly arrests human cells at the G2/M border of the cell cycle and allows for effective cell synchronization in early mitosis (Vassilev LT, Tovar C, Chen SQ, Knezevic D, Zhao XL, Sun HM, Heimbrook DC, Chen L (2006) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA 103, 10660–10665). EGTA was added to the cells immediately after RO-3306 washout, when cells were entering mitosis. The movie shows normal progression through mitosis from prophase to anaphase. Cells remained blocked in anaphase, with the cleavage furrow ingressed by approximately 50% for the remainder of the experiment (15 h) (MPG 2206 kb)

18_2010_401_MOESM2_ESM.mpg

Movie S2. Normal mitosis shows rapid abscission. HeLa cells were treated with RO-3306 as in the legend to Movie S1. Cells were released from the G2/M block into fresh medium and mitotic progression was recorded by using time-lapse microscopy. Time-lapse imaging shows that HeLa cells undergo mitosis, rapid ingression, and very rapid abscission to form two independent daughter cells. The arrow indicates the intracellular bridge when it is present. Each frame is 5 min. The time frame selected for movies 2–4 are all the same so they can be directly compared (MPG 316 kb)

Movie S3. EGTA added post-anaphase induces multinucleation. HeLa cells were treated with RO-3306 as in the legend to Movie S1. However, EGTA was added to the cells 1.5 h after RO-3306 washout, when cells were entering cytokinesis. The movie shows that ingression and formation of the midbody occur at a normal rate, but abscission is blocked since the intracellular bridge persists for 160 min (in this example). Finally, this cell regresses to become binucleated, an index of cytokinesis failure. The arrow indicates the intracellular bridge when it is present (MPG 746 kb)

Movie S4. EGTA added post-anaphase delays cytokinesis by delayed abscission. Time-lapse movie of a HeLa cell treated with EGTA as described in the legend to Movie S2. EGTA greatly delayed abscission. This is shown in this cell which exhibited an extremely long and highly persistent intracellular bridge. In this example, the bridge appeared to “snap” after a long delay. This confirms that the point of cytokinesis failure is the abscission step. The arrow indicates the intracellular bridge when it is present (MPG 664 kb)

Movie S5. γ-tubulin assembles into an MR at the midbody. A 3D reconstruction of a representative asynchronously growing HeLa cell undergoing cytokinesis and stained for γ-tubulin. As the dividing cell rotates 360º, γ-tubulin can be seen at the mitotic centrosomes and also at the midbody, where it is structurally arranged into a ring It is important to note that an equivalent movie displaying a 3D reconstruction of the γ-tubulin MR could not be produced of the FMRs for several reasons. Firstly, the size of the FMRs is one third smaller than the γ-tubulin midbody ring and thus is at the limit of the resolution by microscopy. Secondly, the background staining of phospho-dynII in the cytosol interferes with visualization of the FMRs. Lastly, the fact that dynII and CaN are present in two closely positioned rings means that the orientation of the dividing cell does not readily allow visualization down the bore of both rings simultaneously. Hence ring–ring interference prevents a similar movie being made for phospho-dynII or CaN (MPG 2744 kb)

Movie S1.

EGTA blocks cells in anaphase. HeLa cells were treated with RO-3306, a selective small-molecule inhibitor of cdk1 that reversibly arrests human cells at the G2/M border of the cell cycle and allows for effective cell synchronization in early mitosis (Vassilev LT, Tovar C, Chen SQ, Knezevic D, Zhao XL, Sun HM, Heimbrook DC, Chen L (2006) Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA 103, 10660–10665). EGTA was added to the cells immediately after RO-3306 washout, when cells were entering mitosis. The movie shows normal progression through mitosis from prophase to anaphase. Cells remained blocked in anaphase, with the cleavage furrow ingressed by approximately 50% for the remainder of the experiment (15 h) (MPG 2206 kb)

Movie S2.

Normal mitosis shows rapid abscission. HeLa cells were treated with RO-3306 as in the legend to Movie S1. Cells were released from the G2/M block into fresh medium and mitotic progression was recorded by using time-lapse microscopy. Time-lapse imaging shows that HeLa cells undergo mitosis, rapid ingression, and very rapid abscission to form two independent daughter cells. The arrow indicates the intracellular bridge when it is present. Each frame is 5 min. The time frame selected for movies 2–4 are all the same so they can be directly compared (MPG 316 kb)

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Chircop, M., Malladi, C.S., Lian, A.T. et al. Calcineurin activity is required for the completion of cytokinesis. Cell. Mol. Life Sci. 67, 3725–3737 (2010). https://doi.org/10.1007/s00018-010-0401-z

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Keywords

  • Calcineurin
  • Cytokinesis
  • Dephosphorylation
  • Dynamin II