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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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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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)
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
- Dynamin II