Dissecting the role of TPX2 during cell division

剖析TPX2在细胞分裂过程中的作用，维持稳定的基因组对多细胞生物的生长发育至关重要。保持基因组稳定的两个关键步骤是在细胞分裂过程中准确、无错误地复制和在两个新形成的细胞中均匀分离基因组。未能正确分离基因组会导致非整倍体，这种情况要么会导致细胞死亡，要么会导致细胞调控不当。因此，细胞严格地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。在所有真核细胞中完成细胞分裂需要微管网络的顺序重组。每一种组织状态都具有特定的功能：中期排列染色体，后期分离姊妹染色体，早期胞质分裂刺激沟槽内移，中体组装完成胞质分裂。因此，为了理解一个细胞如何分裂成两个细胞，确定协调这些微管组织状态之间转变的分子事件是至关重要的。由于在有丝分裂过程中几乎没有蛋白质合成，这些微管组织状态之间的转换可能是由于翻译后修饰改变了有丝分裂蛋白质组在不同有丝分裂阶段之间的活性。有丝分裂蛋白质组变化的一个主要因素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，参与了微管的有丝分裂组织。我们的目标是确定在有丝分裂过程中调节TPX2的途径。通过建立在实验室开发的最新数据和技术，将识别以前未知的TPX2调节功能和模式。特殊目的确定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依赖的磷酸化位点。未能在Aurora A位点磷酸化TPX2导致TPX2在细胞内的错误定位，这表明定位TPX2需要Aurora A依赖的磷酸化。接下来，我们将确定哪些TPX2功能受Aurora A依赖的磷酸化调节。我们将集中在有丝分裂的早期阶段，当TPX2和Aurora A定位于类似的微管结构时。在稳定表达可诱导野生型和突变型TPX2(拟磷酸化和非磷酸化突变体)的细胞中，我们将采用RNAi拯救方法，检测Aurora A依赖的TPX2磷酸化在纺锤体组装、纺锤体极组织和动粒介导的微管成核中的作用。我们将使用相同的策略来研究CDK1和MAPK依赖的TPX2磷酸化的作用。这些方法将决定TPX2在细胞分裂过程中的调控方式。确定TPX2在细胞分裂后期的作用：我们在细胞质分裂过程中观察到TPX2在中体微管上的作用，这表明TPX2在细胞质分裂中的作用尚未得到认可。使用新技术来特异性地消耗中期后的TPX2，我们将使用实验室中已经开发的分析方法来剖析细胞质分裂的不同阶段的事件。使用这些方法，我们期望确定TPX2在细胞质分裂中的新角色。摘要：综合这些研究，将确定TPX2如何通过识别新的调节机制和胞质分裂中的新功能来调节细胞分裂。由于TPX2存在于动植物中，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.