Dissecting the role of TPX2 during cell division

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Dissecting the role of TPX2 during cell division * Maintaining a stable genome is essential for the growth and development of multicellular organisms. The two key steps in maintaining a stable genome are accurate error free duplication and equal segregation of the genome in the two newly forming cells during cell division. A failure to correctly segregate the genome leads to aneuploidy, a condition that either leads to cell death or mis-regulated cells. The cell therefore tightly regulates cell division to ensure its success; however, at present we still have a poor understanding of the molecular mechanisms that drive and regulate this fundamental process.* Completion of cell division in all eukaryotic cells requires the sequential re-organization of the microtubule network. Each organization state performs specific functions: aligning chromosomes during metaphase, separating sister chromosomes during anaphase, stimulating furrow ingression during early cytokinesis and assembling the midbody for the completion of cytokinesis. Therefore, it is vital to determine the molecular events that orchestrate the transitions between these microtubule organization states in order to understand how one cell divides into two. As there is little protein synthesis during mitosis, the transitions between these microtubule organization states are likely due to post-translational modifications that alter the activity of the mitotic proteome between the different mitotic phases. A major contributor to changes in the mitotic proteome will be changes in protein phosphorylation.* TPX2 is an essential microtubule binding protein that is phosphorylated and involved in the mitotic organization of microtubules. Our goal is to define the pathways that regulate TPX2 during mitosis. By building upon recent data and techniques developed in the lab, previously unappreciated functions and modes of TPX2 regulation will be identified.*SPECIFIC AIMS*Determine the role of TPX2 phosphorylation in cell division: We have identified conserved Aurora A, Cdk1 and MAP kinase dependent phosphorylation sites on TPX2. Failure to phosphorylate TPX2 at the Aurora A sites results in the mislocalization of TPX2 within the cell, suggesting that Aurora A dependent phosphorylation is required for localization TPX2. Next we will determine which TPX2 functions are regulated by Aurora A dependent phosphorylation. We will focus on the early stages of mitosis when TPX2 and Aurora A localize to similar microtubule structures. Using RNAi rescue approaches in cells stably expressing inducible wildtype and mutant TPX2 (phosphomimetic and non-phosphorylatable mutants), we will assay the role of Aurora A dependent TPX2 phosphorylation in spindle assembly, spindle pole organization and kinetochore mediated microtubule nucleation. We will employ the same strategies to study the role of Cdk1 and MAP kinase dependent phosphorylation of TPX2. These approaches will determine how TPX2 is regulated during cell division.**Determine the role of TPX2 in the late stages of cell division: We have observed TPX2 on the microtubules of the midbody during cytokinesis, suggesting unappreciated roles for TPX2 in cytokinesis. Using novel technology to specifically deplete TPX2 after metaphase, we will use assays already developed in the lab to dissect events at different stages of cytokinesis. Using these approaches we anticipate identifying novel roles for TPX2 in cytokinesis. **SUMMARY: Combined these studies will determine how TPX2 regulates cell division by identifying new regulatory mechanisms and new functions for TPX2 in cytokinesis. As TPX2 occurs in plants and animals and has been implicated in apoptosis, these findings will further our understanding of cell division in all eukaryotic cells.