Centrosome biogenesis and copy number control

 描述（由申请人提供）：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。中心体是微管成核细胞器，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，中心体形成了基体的基础，这是纤毛和鞭毛形成所必需的，因此对于细胞感知它们的环境和信号至关重要。中心体在周期开始时作为单个拷贝存在，并且在S期期间复制一次，以确保仅存在两个拷贝来组织有丝分裂纺锤体的两极。在人类癌细胞中通常观察到中心体数量的减少，其中额外的中心体导致染色体分离错误，被认为是驱动肿瘤形成的原因。因此，了解细胞实现中心体每周期复制一次的机制是一个与人类健康相当相关的重要基本问题。Polo样激酶4（Plk 4）已成为中心体复制的中心、剂量依赖性调节因子。抑制Plk 4可抑制中心体复制，而Plk 4过表达则会导致产生过多的中心体。然而，我们对这种激酶的功能知之甚少;特别是，控制中心体生物发生的关键Plk 4靶标仍有待确定。我们提出的研究旨在建立Plk 4编排和协调中心体生物发生的机制。以前研究Plk 4的努力受到阻碍，因为没有特异性和快速操纵Plk 4功能的工具。在本申请中，我们通过开发两种互补的方法克服了这一限制，使我们能够调节活细胞中Plk 4的水平和活性。使用这些工具，我们将建立改变Plk 4水平/活性的直接影响，并区分Plk 4在中心体生物发生中的激酶依赖性和支架功能。在目标1中，我们将研究Plk 4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，我们已经确定了一个高度保守的中心体蛋白作为中心体复制所需的关键Plk 4底物。在目的2中，我们提出了如何Plk 4介导的磷酸化的基板有助于中心体组装的特点。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Plk 4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.