Split timing

The microtubular central spindle is a molecular assembly that participates in the regulation of cytokinesis. And, the centralspindlin complex — which consists of the kinesin-like protein ZEN-4 and the Rho-family GTPase-activating protein CYK-4 in Caenorhabditis elegans (and their orthologues MKLP1 and MgcRacGAP in mammalian cells) — bundles the individual microtubules together and so is essential for the assembly of this machine at anaphase. But how is the timing of this assembly regulated?

Reporting in Nature, Mishima et al. started to dissect this problem by identifying the mechanism that regulates centralspindlin activity and, therefore, central-spindle assembly. They showed, using in vitro assays, that the amino-terminal motor domains of ZEN-4 (ZEN-4^MOT) and MKLP1 are phosphorylated by the cyclin-B–CDK1 complex, which is active during metaphase and becomes inactivated at the metaphase–anaphase transition. Deletion of an N-terminal basic extension that contained one of the cyclin-B–CDK1 phophporylation sites of ZEN-4^MOT also reduced its ATPase activity and its ability to cause microtubule movement — both vital functions of the kinesin.

Phosphorylation of a conserved threonine residue in the isolated ZEN-4^MOT reduced the ATPase and microtubule-mobility functions of the domain, which indicates that this protein might be controlled by phosphoregulation. In agreement with this, when a mutated, unphosphorylatable form of MKLP1 (MKLP1-AA) was expressed in cultured mammalian cells, the protein localized inappropriately to the spindle and the genomic material failed to segregate properly. Furthermore, by using a phospho-threonine-proline antibody and comparing chromosome-segregation events in normal and MKLP1-AA-expressing cells, Mishima et al. showed that MKLP1 is phosphorylated during metaphase — which prevents its association with the spindle — and is dephosphorylated during anaphase when it becomes localized to the central spindle.

Finally, the CDC14 phosphatase was shown to dephosphorylate both MKLP1 and ZEN-4 in vitro. And in C. elegans, depletion of CDC-14 prevented the localization of a tagged form of ZEN-4, but not that of an unphosphorylatable mutant of ZEN-4, to the central spindle in anaphase. So, it seems that cell-cycle kinase and phosphatase activities control the timing of central-spindle formation and, consequently, cell division.