Cell cycle regulation of central spindle assembly

Abstract

The bipolar mitotic spindle is responsible for segregating sister chromatids at anaphase. Microtubule motor proteins generate spindle bipolarity and enable the spindle to perform mechanical work1. A major change in spindle architecture occurs at anaphase onset when central spindle assembly begins. This structure regulates the initiation of cytokinesis and is essential for its completion2. Central spindle assembly requires the centralspindlin complex composed of the Caenorhabditis elegans ZEN-4 (mammalian orthologue MKLP1) kinesin-like protein and the Rho family GAP CYK-4 (MgcRacGAP). Here we describe a regulatory mechanism that controls the timing of central spindle assembly. The mitotic kinase Cdk1/cyclin B phosphorylates the motor domain of ZEN-4 on a conserved site within a basic amino-terminal extension characteristic of the MKLP1 subfamily. Phosphorylation by Cdk1 diminishes the motor activity of ZEN-4 by reducing its affinity for microtubules. Preventing Cdk1 phosphorylation of ZEN-4/MKLP1 causes enhanced metaphase spindle localization and defects in chromosome segregation. Thus, phosphoregulation of the motor domain of MKLP1 kinesin ensures that central spindle assembly occurs at the appropriate time in the cell cycle and maintains genomic stability.

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Figure 1: ZEN-4/MKLP1 localizes upon anaphase onset and is a Cdk1 substrate in vitro.
Figure 2: Microtubule (MT) binding by ZEN-4 is inhibited by Cdk1/cyclin B phosphorylation.
Figure 3: MKLP1 is phosphorylated on Cdk1/cyclin B sites in metaphase and dephosphorylated during anaphase.
Figure 4: Non-phosphorylatable MKLP1 induces lagging chromosomes, and non-phosphorylatable ZEN-4 localizes in embryos depleted of CDC-14.

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Acknowledgements

This work was supported by a grant from the Austrian Science Foundation and with the support of Boehringer Ingelheim. M.M., V.P. and M.G. would like to thank K. Bartalska for technical assistance, and S. Kaitna and K. Mechtler for help in the initial stages of this project.

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Correspondence to Michael Glotzer.

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Supplementary information

Supplementary Figure 1

Time lapse fluorescence microscopy of rhodamine-labeled, taxol-stabilized microtubules gliding on a surface of ZEN-4MOT (1-434). Elapsed time is shown in min:sec. (MOV 1500 kb)

Supplementary Figure 2

Structural model of human kinesin (based on coordinates 1BG2, Kull et. al.) reveals that the N terminus of conventional kinesin is in the immediate vicinity of loop 12. The highly basic K-loop in KIF1A that interacts with the C-terminal tail of tubulin is located in a loop that corresponds to loop 12 of conventional kinesin. Since the basic N terminus of ZEN-4 extends further that the N- terminus shown in this model, it too could likely interact with the C-terminal tail of tubulin. (MOV 838 kb)

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Mishima, M., Pavicic, V., Grüneberg, U. et al. Cell cycle regulation of central spindle assembly. Nature 430, 908–913 (2004). https://doi.org/10.1038/nature02767

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