Dissecting the role of TPX2 during cell division

维持一个稳定的基因组对多细胞生物的生长发育至关重要。维持稳定基因组的两个关键步骤是精确的无错误复制和在细胞分裂过程中两个新形成的细胞中基因组的相等分离。正确分离基因组的失败会导致非整倍性，这种情况要么导致细胞死亡，要么导致细胞失调。因此，细胞严格调节细胞分裂以确保其成功；然而，目前我们对驱动和调节这一基本过程的分子机制仍然知之甚少。在所有真核细胞中，细胞分裂的完成需要微管网络的有序重组。每一种组织状态都有特定的功能：在中期排列染色体，在后期分离姐妹染色体，在早期细胞质分裂时刺激沟向内，在完成细胞质分裂时组装中间体。因此，确定在这些微管组织状态之间协调过渡的分子事件是至关重要的，以便了解一个细胞如何分裂成两个。由于在有丝分裂过程中很少有蛋白质合成，这些微管组织状态之间的转变可能是由于翻译后修饰改变了不同有丝分裂阶段之间有丝分裂蛋白质组的活性。有丝分裂蛋白质组变化的主要原因是蛋白质磷酸化的变化。TPX2是一种重要的微管结合蛋白，被磷酸化并参与微管的有丝分裂组织。我们的目标是确定有丝分裂过程中调控TPX2的途径。通过建立在实验室中开发的最新数据和技术，将确定以前未被认识到的TPX2调节的功能和模式。特异性目的：确定TPX2磷酸化在细胞分裂中的作用：我们已经确定了TPX2上保守的Aurora A， Cdk1和MAP激酶依赖性磷酸化位点。如果TPX2在Aurora A位点磷酸化失败，就会导致TPX2在细胞内的错误定位，这表明TPX2的定位需要依赖Aurora A的磷酸化。接下来，我们将确定哪些TPX2功能是由Aurora A依赖性磷酸化调节的。当TPX2和Aurora A定位到类似的微管结构时，我们将重点关注有丝分裂的早期阶段。在稳定表达诱导型野生型和突变型TPX2（拟磷型和非磷酸化突变型）的细胞中使用RNAi拯救方法，我们将分析依赖于Aurora A的TPX2磷酸化在纺锤体组装、纺锤体极组织和着丝点介导的微管成核中的作用。我们将采用相同的策略来研究Cdk1和MAP激酶依赖性TPX2磷酸化的作用。这些方法将确定TPX2在细胞分裂过程中是如何被调节的。确定TPX2在细胞分裂后期的作用：我们在细胞质分裂期间观察到TPX2在中体微管上，提示TPX2在细胞质分裂中未被认识到的作用。使用新技术在中期后特异性地消耗TPX2，我们将使用实验室已经开发的检测方法来解剖细胞分裂不同阶段的事件。利用这些方法，我们期望确定TPX2在细胞分裂中的新作用。综上所述，这些研究将通过鉴定TPX2在细胞分裂中的新调控机制和新功能来确定TPX2如何调控细胞分裂。由于TPX2存在于植物和动物中，并与细胞凋亡有关，这些发现将进一步加深我们对所有真核细胞中细胞分裂的理解。

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 AIMSDetermine 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.