Dissecting the role of TPX2 during cell division

剖析 TPX2 在细胞分裂过程中的作用

• 批准号: RGPIN-2014-04970
• 负责人: Wilde, Andrew
• 金额: $ 3.42万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2015
• 资助国家: 加拿大
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=588128
• 关键词: Dissecting; role; TPX2; during; cell

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.

TPX 2在细胞分裂中的作用 维持稳定的基因组对于多细胞生物的生长和发育至关重要。维持基因组稳定的两个关键步骤是准确无误的复制和细胞分裂期间两个新形成细胞中基因组的均等分离。不能正确分离基因组会导致非整倍性，这是一种导致细胞死亡或细胞失调的情况。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缺乏了解。 所有真核细胞中细胞分裂的完成需要微管网络的顺序重组。每个组织状态执行特定的功能：在中期对齐染色体，在后期分离姐妹染色体，在早期胞质分裂期间刺激沟内移和组装完成胞质分裂的中间体。因此，确定这些微管组织状态之间的转变的分子事件至关重要，以便了解一个细胞如何分裂为两个。由于有丝分裂期间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，这些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一个主要贡献者是蛋白质磷酸化的变化。 TPX 2是一种必需的微管结合蛋白，其被磷酸化并参与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TPX 2的途径。通过建立在实验室开发的最新数据和技术基础上，将确定TPX 2调节的以前未被认识的功能和模式。 具体目标 确定TPX 2磷酸化在细胞分裂中的作用：我们已经确定了TPX 2上保守的Aurora A，Cdk 1和MAP激酶依赖的磷酸化位点。在Aurora A位点不能磷酸化TPX 2导致TPX 2在细胞内的错误定位，表明Aurora A依赖性磷酸化是定位TPX 2所必需的。接下来，我们将确定哪些TPX 2功能受Aurora A依赖性磷酸化的调节。我们将集中在有丝分裂的早期阶段，当TPX 2和极光A定位到相似的微管结构。在稳定表达诱导型野生型和突变型TPX 2（磷酸化模拟和非磷酸化突变体）的细胞中使用RNAi拯救方法，我们将测定Aurora A依赖性TPX 2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。 我们将采用相同的策略来研究Cdk 1和MAP激酶依赖的TPX 2磷酸化的作用。这些方法将确定TPX 2在细胞分裂过程中如何调节。 确定TPX 2在细胞分裂后期的作用：我们已经观察到TPX 2在胞质分裂过程中的微管中，提示TPX 2在胞质分裂中的未被重视的作用。使用新技术在中期后特异性耗尽TPX 2，我们将使用实验室中已经开发的测定来剖析胞质分裂不同阶段的事件。使用这些方法，我们预期识别TPX 2在胞质分裂中的新角色。 总结：结合这些研究将确定如何TPX 2调节细胞分裂，确定新的调控机制和新的功能TPX 2在胞质分裂。由于TPX 2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。