Mechanisms controlling the inactivation of microtubule organizing center function at the centrosome

项目总结/摘要 中心体作为微管组织中心（MTOC），协调微管进入有丝分裂细胞。 纺锤体穿过其中心粒周围物质（PCM）。这种活动是双相的，通过组装和 在细胞周期中分解。在细胞分化时，中心体处的MTOC活性通常被抑制。 当MTOC功能被重新分配到非中心体位点以适应 不同的细胞功能尽管中心体MTOC活性过度活跃是某些癌症的标志， 尽管中心体与细胞的侵袭行为有关，但人们对中心体作为MTOC是如何失活的却知之甚少 在有丝分裂退出期间或在分化细胞中维持在非活性状态。我们使用C。优雅作为一个 模型来理解活生物体中MTOC生物学的这些基本知识缺口。我们分析 内源PCM蛋白在C. elegans揭示了PCM由不同的蛋白质区域组成， 组织成一个内部和外部的球体，以不同的速度从中心体中移除， 不同的行为我们发现，磷酸酶反对PCM的有丝分裂激酶，最终 在有丝分裂结束时催化内球PCM蛋白的溶解。PCM的性质似乎 改变，使得剩余的老化PCM外球通过微管破裂成子PCM“包”， 基于皮质拉力的因此，中心体似乎是作为MTOC通过两步失活的。 机制开始于PCM溶解，随后是机械控制的破裂。拟议 研究中，我们将揭示MTOC功能失活的两步模型的机制 在中心体。我们将确定PCM溶解的机制，确定相关的 磷酸酶，它们在中心体的靶点，以及去除这些靶点在解体中的作用 (Aim 1）。然后，我们将具体揭示PCM外层的蛋白质-蛋白质相互作用， 数据包（目标2）。最后，我们将探讨MTOC功能失活的潜在机制， 分化的细胞，并测试中心体失活在细胞分化中的作用（Aim 3）。固有微管 组织对于正常发育和细胞功能以及MTOC功能的过度活跃是必不可少的。 中心体是某些癌症的标志。因此，这些研究中发现的分子可以提供 潜在的治疗靶点，并阐明了这一重要的，但在细胞和 发育生物学

Project Summary/Abstract The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Upon cell differentiation, MTOC activity at the centrosome is often maintained in an inactive state as MTOC function is reassigned to non-centrosomal sites to accommodate different cell functions. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers and has been linked to invasive cell behavior, little is known about how the centrosome is inactivated as an MTOC either during mitotic exit or maintained in an inactive state in differentiated cells. We are using C. elegans as a model to understand these fundamental knowledge gaps in MTOC biology in a live organism. Our analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of distinct protein territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is ruptured into sub-PCM ‘packets’ by microtubule based cortical pulling forces. Thus, the centrosome appears to be inactivated as an MTOC by a two-step mechanism beginning with PCM dissolution, followed by mechanically controlled rupture. In the proposed research, we will uncover the mechanisms underlying this two-step model for the inactivation of MTOC function at the centrosome. We will determine the mechanisms underlying PCM dissolution, identifying the pertinent phosphatases, their targets at the centrosome, and the role of the removal of these targets in disassembly (Aim 1). We will then specifically uncover the protein-protein interactions underlying the PCM outer sphere and packets (Aim 2). Finally, we will probe the mechanisms underlying the inactivation of MTOC function in differentiated cells and test the role of centrosome inactivation in cell differentiation (Aim 3). Proper microtubule organization is essential for normal development and cell function and hyperactive MTOC function at the centrosome is a hallmark of some cancers. Thus, the molecules uncovered in these studies could provide potential therapeutic targets as well as shed light on this important, but understudied topic in cell and developmental biology.