Centrosome biogenesis and copy number control

 描述(申请人提供)：我们研究的长期目标是了解控制中心体生物发生的分子机制。中心体是微管核细胞器，在大多数微管相关功能中发挥核心作用，包括细胞运动、细胞内运输和染色体分离。此外，中心体构成了纤毛和鞭毛形成所需的基底体的基础，因此对细胞感知环境和传递信号至关重要。中心体在周期开始时以单拷贝形式存在，在S期复制一次，以确保只有两个拷贝来组织有丝分裂纺锤体的两极。中心体数量的异常在人类癌细胞中很常见，额外的中心体导致染色体分离错误，被认为是导致肿瘤形成的原因。因此，了解细胞实现中心体每周期一次复制的机制是一个与人类健康相当相关的重要基础性问题。Polo-like kinase4(Plk4)是中心体复制的中枢调节因子，具有剂量依赖性。抑制Plk4会抑制中心体复制，而Plk4过表达会导致中心体的产生过多。然而，我们对该激酶的功能知之甚少，尤其是控制中心体生物发生的关键Plk4靶点仍未确定。我们提出的研究试图建立Plk4协调和协调中心体生物发生的机制。之前研究Plk4的努力一直受到阻碍，因为还没有专门和快速操作Plk4功能的工具。在这项应用中，我们通过开发两种互补的方法来克服这一限制，这两种方法允许我们调节活细胞中Plk4的水平和活性。使用这些工具，我们将建立改变Plk4水平/活性的直接影响，并区分Plk4在中心体生物发生中的激酶依赖和支架功能。在目标1中，我们将研究Plk4的快速丢失/抑制对细胞生长和中心体结构的影响。在我们的初步数据中，我们已经确定一个高度保守的中心体蛋白是中心体复制所需的关键Plk4底物。在目标2中，我们建议表征Plk4介导的该底物的磷酸化如何有助于中心体组装。这些研究对于理解中心体组装的正常调节和进一步在癌症治疗中靶向Plk4的持续努力是相关的。

 DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the molecular mechanisms that control centrosome biogenesis. Centrosomes are microtubule-nucleating organelles that play a central role in most microtubule- related functions, including cell motility, intracellular transport and chromosome segregation. In addition, the centrosome forms the basis of the basal body, which is required for the formation of cilia and flagella, and i therefore crucial for cells to both sense their environment and transduce signals. Centrosomes are present as a single copy at the beginning of the cycle and duplicate once during S phase to ensure only two copies are present to organize the poles of the mitotic spindle. Abnormalities in centrosome number are commonly observed in human cancer cells, where extra centrosomes lead to chromosome segregation errors that are thought to drive tumor formation. Understanding the mechanism by which cells achieve the once per cycle duplication of the centrosome is therefore an important fundamental question of considerable relevance to human health. Polo-like kinase 4 (Plk4) has emerged as the central, dose-dependent regulator of centrosome duplication. Suppressing Plk4 inhibits centrosome duplication, while Plk4 overexpression leads to the production of too many centrosomes. However, we understand little about how this kinase functions; and in particular, the critical Plk4 targets that control centrosome biogenesis remain to be identified. Our proposed research seeks to establish the mechanisms through which Plk4 orchestrates and coordinates centrosome biogenesis. Previous efforts to study Plk4 have been hampered because tools to specifically and rapidly manipulate Plk4 function have not been available. In this application we have overcome this limitation by developing two complementary methodologies that allow us to regulate Plk4 levels and activity in living cells. Using these tools we will establish the direct effect of altering Plk4 levels/actiity and distinguish between kinase-dependent and scaffolding functions of Plk4 in centrosome biogenesis. In Aim 1, we will study the effect of rapid loss/inhibition of Plk4 on cell growth and centrosome structure. In our preliminary data we have identified a highly conserved centrosome protein as a key Plk4 substrate required for centrosome duplication. In Aim 2, we propose to characterize how Plk4-mediated phosphorylation of this substrate contributes to centrosome assembly. These studies are relevant for understanding the normal regulation of centrosome assembly and for furthering ongoing efforts to target Plk4 in cancer therapy.