Cytokinesis is essential for cell proliferation. Failure of cytokinesis leads to aneuploidy or chromosomal instability, which has been associated with human cancers. Successful cytokinesis relies on a dynamic interplay between microtubules, the actin cytoskeleton, and membrane compartments under the control of the cell cycle machinery. In spite of its importance, the molecular mechanism of cytokinesis in animal cells has not yet been fully clarified.

Centralspindlin is an evolutionarily conserved protein complex of a mitotic kinesin and a RhoGAP critical for the assembly of the central spindle. Being in a complex is essential both for the formation of the central spindle and for the in vitro microtubule-bundling activity. When incubated with microtubules, the complex causes the strong bundling of microtubules (arrowheads), while neither the kinesin subunit alone nor the RhoGAP subunit alone does.

We would like to understand cytokinesis more fully, in terms of the dynamic assembly of molecular machinery. The central spindle is a microtubule-based molecular assembly that forms between the segregating chromosomes during anaphase. During telophase, it associates with the ingressing cleavage furrow and matures into the midbody. These microtubule-based structures have crucial roles through all the steps of cytokinesis from initiation to completion. We will address the following questions:

• How is the central spindle/midbody assembled?

• How does the central spindle/midbody contribute to the progression of cytokinesis at the molecular level?

We will focus on centralspindlin, a stable protein complex of a mitotic kinesin-like protein and a Rho-family GTPase-activating protein (RhoGAP), which is crucial for assembly of the central spindle and the midbody. We will characterise the dynamic behaviour of centralspindlin and investigate centralspindlin-interacting proteins by using mammalian cultured cells and Caenorhabditis elegans embryos as model systems. In addition to biochemical and genetical analyses, live imaging both at single molecule level in vitro and at subcellular level in vivo will be performed. We will also develop experimental strategies to (in)activate molecules of interest in vivo in a reasonable time resolution to dissect the molecular mechanism of cytokinesis

Centralspindlin dramatically changes its localisation at metaphase/anaphase transition under the control of a master cell-cycle regulating protein kinase, CDK1. The kinesin subunit is phosphorylated by CDK1 at the sites flanking its catalytic core (P). This phosphorylation reduces the affinity for microtubules and contributes to the prevention of premature formation of the central spindle before anaphase onset, ensuring proper segregation of genetic materials.

Total internal reflection fluorescent microscopy (TIRFM) allows us to directly visualise single molecules of conventional kinesin labeled with green fluorescent protein (GFP) moving along a microtubule immobilised on a coverglass surface. A similar analysis of centralspindlin is underway.

Plain English:
Cytokinesis is the process that divides the cytoplasm into two after chromosomes have been separated to opposite ends of the cell. To ensure that the number of chromosomes is the same from one cell to the next, cytokinesis has to happen at the right time and place; failure to do so can result in cells with abnormal numbers of chromosomes - a defect often associated with cancer. At the molecular level, division of the cytoplasm is performed by reorganising both the lipid membranes that surround the cell, and several fibre- or tube-like protein chains. This is achieved by the co-operative action of a number of proteins, including force generating motors. We are trying to understand how these individual molecular "parts" are assembled into more complicated molecular "machines" for cytokinesis in a highly dynamic and coordinated manner.

Selected publications:

• Guse A, Mishima M and Glotzer MA (2005) Conserved role for Aurora B phosphorylation of ZEN-4/MKLP1 in completion of cytokinesis. Curr Biol 15, 778-86

• Mishima M, Pavicic V, Grüneberg U, Nigg EA, and Glotzer M (2004) Cell cycle regulation of central spindle assembly. Nature 430, 908-13

• Mishima M, and Glotzer M (2004) Cytokinesis. In Encyclopedia of Biological Chemistry (WJ Lennarz & MD Lane eds), Elsevier, Oxford, vol 1, pp. 556-62

• Mishima M and Glotzer M (2003) Cytokinesis: a logical GAP. Curr Biol 13, R589-91

• Mishima M, Kaitna S and Glotzer M (2002) Central spindle assembly and cytokinesis require a kinesin-like protein/RhoGAP complex with microtubule bundling activity. Dev Cell 2, 41-54

Cytokinesis

A C elegans one-cell-stage embryo about to undergo cytokinesis. Following segregation of chromosomes (blue), a bundle structure of microtubules (green) called the central spindle is formed between them. Centralspindlin (red), a critical factor for the formation of this structure, steeply accumulates to the center of the microtubule bundle.