Tuesday, December 5, 2023
12/5/2023
PROJECT SUMMARY After the chromosomes are segregated by the microtubule-based mitotic spindle, cytokinesis completes cell division, partitioning the contents of the mother cell to the two daughter cells. Cytokinesis is accomplished by constriction of an acto-myosin contractile ring that forms on the cortex in an equatorial band encircling the cell equator. Cytokinesis failure generates tetraploid cells that are a common intermediate in the genesis of cancers. Upregulation and mutation of cytokinesis regulators has also been implicated in different cancers, and inhibitors targeting cytokinesis are of interest as potential chemotherapeutic agents, motivating efforts to understand the mechanisms that pattern cortical contractility during cytokinesis. To ensure that each daughter cell receives an equivalent genomic complement, the position of the contractile ring is specified by the anaphase spindle. The spindle sends two superimposed signals to the cortex: (1) a positive signal promoting contractile ring assembly that is generated by set of bundled microtubules, called the central spindle, that forms between the separating chromosomes, and (2) a negative signal generated by the microtubule asters that suppresses the contractility of the non-equatorial cortex. The proposed work focuses on the molecular basis for these two signals that collectively direct contractile ring assembly. A central component of the positive signal promoting contractile ring assembly is centralspindlin, a tetrameric complex formed by a dimer of kinesin-6 and a dimer of CYK4. Centralspindlin is phosphorylated by the mitotic kinase PLK1, which concentrates on the central spindle; it subsequently diffuses to the adjacent plasma membrane where, through mechanisms that remain largely unclear, its CYK4 subunit engages with and activates the ECT2 guanine nucleotide exchange factor (GEF). Active ECT2 in turn generates an equatorial zone enriched for the master regulator of contractile ring assembly, RhoA-GTP. In Aim 1, we address the major open question with respect to positive cytokinesis signaling: the mechanism of ECT2 activation by centralspindlin. To understand how the microtubule asters suppress contractility on the non-equatorial cortex, which occurs at the same time as positive signaling from the central spindle, we developed an assay monitoring clearing of contractile ring proteins from the cell poles in the C. elegans embryo. Using this assay, we identified Aurora A kinase as an essential mediator of aster-based contractility suppression. In Aim 2, we build on this work to identify the Aurora A targets that mediate contractility suppression and extend analysis of this mechanism to human cells. Aim 3 addresses an important gap in understanding how the cell cycle state that supports contractile ring assembly is generated, and assesses whether the duration of cytokinesis, like that of mitosis, is monitored by a p53-based mitotic stopwatch mechanism that eliminates potentially problematic cells from the population.
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