Centrosomes are best known for their ability to organize microtubules to form interphase arrays and mitotic spindles. Recent evidence suggests that centrosomes are involved hi cytokinesis and cell cycle progression. However, little is known about the centrosome proteins that contribute to these processes. In the previous funding period, we examined the molecular basis for these under-explored centrosome functions. We identified a novel centrosome protein called centriolin, which is essential for cytokinesis in mammalian cells. Centriolin interacts with proteins implicated or involved in cytokinesis including snapin (SNARE), sec 15 (exocyst), MKLP-1 (centralspindlin) and a novel GTPase activating protein for Rho GTPases. Many of these proteins localize to the midbody in dividing cells and induce cytokinesis defects when downregulated. Based on these and other data, we propose a model in which centriolin serves as a scaffold protein at the midbody to coordinate vesicle fusion, microtubule depolymerization, furrow ingression and cell separation late in cytokinesis. Defective cytokinesis induced by centriolin downregulation was followed by Gl arrest. We unexpectedly found that many centrosome proteins induced GI arrest when downregulated. However, the arrest did not correlate with defects in cytokinesis or other centrosome functions, or in centrosome structure or composition. We postulate that cell cycle arrest is triggered by a checkpoint that monitors defects in a common function not yet identified, or 'centrosome damage' induced by reduction of individual proteins at centrosomes. GI arrest requires p53 and p38 MAP kinase and induces recruitment of activated p53 and p38 to centrosomes. Based on these observations, we propose two specific aims.
In Aim 1 we will determine the role centriolin and associated proteins hi cytokinesis. More specifically, we will test whether centriolin anchors these proteins at the midbody and if a complex of these proteins controls vesicle fusion, microtubule organization and cell separation at the midbody.
Aim 2 focuses on the role of centrosome proteins in cell cycle progression and checkpoint activation. We will determine the mechanism of GI arrest and identify the signal transduction pathway that connects centrosome proteins to cell cycle arrest.
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