Su, K-C; (2013) Control of Cleavage Furrow Formation during Cytokinesis in Human Cells. Doctoral thesis , UCL (University College London).

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Abstract

Cytokinesis is the final stage of the cell cycle. It partitions sister genomes and separates the cytoplasm of nascent daughter cells. Cytokinesis is initiated by the formation of a cleavage furrow whose ingression is powered by an actomyosin network known as the contractile ring. Following furrow ingression, the process of cell separation is completed by a membrane scission reaction. For the accurate inheritance of genetic information, it is crucial that furrow formation is initiated at the cell equator between segregating chromosomes and that this occurs after chromatin has cleared the cleavage plane. In animal cells, the mitotic spindle plays a pivotal role in the formation and placement of the cleavage furrow. The coupling of cytokinesis and chromosome segregation to the mitotic spindle ensures that nuclear and cytoplasmic division are tightly coordinated. The spindle midzone, a structure that is formed at anaphase onset between segregating sister genomes, is thought to play an important instructive role during cleavage furrow formation. How the mitotic spindle controls cytokinetic events at the cell envelope is a key challenge in cell division research. Formation of the cytokinetic furrow in animal cells requires activation of the GTPase RhoA by the conserved guanine nucleotide exchange factor Ect2. How Ect2, which is associated with the spindle midzone, controls RhoA activity at the equatorial cell periphery during anaphase is not understood. Using a genetic complementation system, I have been able to replace the endogenous Ect2 protein with a fluorescently-tagged transgene to study its dynamic localization during cytokinesis. Using live-cell time-lapse microscopy, I found that Ect2 concentrates not only at the spindle midzone but also accumulates at equatorial plasma membrane during cytokinesis. The association of Ect2 with the plasma membrane in vivo involves a pleckstrin homology domain and a polybasic cluster that bind to phosphoinositide lipids in vitro. I further demonstrated that both guanine nucleotide exchange activity and the membrane targeting domains of Ect2 are essential for RhoA activation, contractile ring formation and cleavage furrow ingression in human cells. Membrane localization of Ect2 is spatially confined to the equator by centralspindlin, Ect2’s spindle midzone anchor complex, and is also temporally coordinated with chromosome segregation through the activation state of Cdk1. My results suggest that targeting of Ect2 to the equatorial membrane may represent a key step in the delivery of the cytokinetic signal to the cortex.

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