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

项目摘要/摘要 中心体充当微管组织中心(MTOC)，协调微管 通过着丝点周围物质(PCM)进入有丝分裂纺锤体。这个活动是两个阶段的， 在细胞周期中通过组装和拆卸进行循环。在细胞分化时， 中心体上的MTOC活性通常保持在非活动状态，因为MTOC功能 重新分配到非中心体位置以适应不同的细胞功能。虽然 中心体MTOC活性过度活跃是某些癌症的一个标志，并与 侵袭性细胞行为，关于中心体作为MTOC是如何失活的知之甚少 在有丝分裂退出期间或在分化的细胞中保持不活跃状态。我们正在使用 秀丽线虫作为模型来理解MTOC生物学中的这些基本知识差距 一个活生生的有机体。我们对线虫内源PCM蛋白的分析表明， PCM是由不同的蛋白质区域组成的，这些区域被组织成一个内外球体， 以不同的速率和不同的行为从中心体中移除。我们发现 磷酸酶反对有丝分裂酶加成PCM，最终催化 有丝分裂末期内球PCM蛋白溶解。相变材料的性质 似乎发生了变化，以至于剩余的老化的PCM外层球体破裂为子PCM 以微管为基础的皮质拉力所产生的“包”。因此，中心体似乎是 通过两步机制使MTOC失活，首先是PCM溶解，然后是 通过机械控制的破裂。在拟议的研究中，我们将揭示 这一两步模型背后的机制是MTOC功能在 中心体。我们将确定PCM溶解的机制，确定 相关的磷酸酶，它们在中心体的靶标，以及去除这些酶的作用 拆解目标(目标1)。然后我们将专门发现这种蛋白质--蛋白质 PCM外层球体和信息包的相互作用(目标2)。最后，我们将探讨 分化细胞MTOC功能失活的机制及检测 中心体失活在细胞分化中的作用(目标3)。适当的微管组织 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特征。因此，这些研究中发现的分子 可以提供潜在的治疗靶点，并阐明这一重要的，但 细胞和发育生物学中未被研究的课题。

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.