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

控制中心体微管组织中心功能失活的机制

• 批准号: 10670106
• 负责人: Jessica Lynn Feldman
• 金额: $ 31.54万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10670106
• 关键词: Adult; Aging; Alleles; Animals; Behavior; Biology; Caenorhabditis elegans; Carcinoma; Catalytic Domain; Cell Cycle; Cell Differentiation process; Cell physiology; Cells; Cellular biology; Centrioles; Centrosome; Chemicals; Cilia; Data; Development; Developmental Biology; Dissection; Embryo; Excision; Genetic; Gonadal structure; Hyperactivity; Image; Intestines; Knowledge; Link; Malignant Neoplasms; Mammary Neoplasms; Mechanics; Mediating; Metaphase; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Nature; Organism; Phase; Phosphoric Monoester Hydrolases; Phosphotransferases; Population; Process; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Proteomics; RNA Interference; Research; Role; Rupture; Site; Testing; Work; base; cell behavior; in vivo; inhibitor; kinetosome; model organism; neoplastic cell; new therapeutic target; pharmacologic; programs; protein protein interaction; recruit; therapeutic target; tool

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.

项目摘要/摘要 中心体充当微管组织中心(MTOC)，将微管编排成有丝分裂 纺锤穿过其着丝点周围材料(PCM)。这项活动是两阶段的，通过组装和循环 在细胞周期中的分解。在细胞分化时，中心体的MTOC活性通常是 维持在非活动状态，因为MTOC功能被重新分配到非中心体位置以适应 不同的细胞功能。尽管中心体MTOC活性过度活跃是某些癌症和 与侵袭性细胞行为有关，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。我们正在使用线虫作为一种 模型来理解活生物体中MTOC生物学中的这些基本知识差距。我们对此的分析 线虫内源PCM蛋白揭示PCM由不同的蛋白质区域组成 被组织成以不同速率从中心体移除的内球和外球，并使用 不同的行为。我们发现，最终，磷酸酶反对有丝分裂酶增加PCM。 在有丝分裂结束时催化内球PCM蛋白的溶解。出现了PCM的性质 改变以使剩余的老化的PCM外球被微管破裂成亚PCM‘包’ 基于皮质的拉力。因此，中心体似乎通过两个步骤失活为MTOC 机制首先是相变材料的溶解，然后是机械控制的破裂。在建议的 通过研究，我们将揭示MTOC功能失活的两步模型背后的机制 在中心体。我们将确定PCM溶解的机制，确定相关的 磷酸酶，它们在中心体的靶标，以及去除这些靶标在拆卸中的作用 (目标1)。然后，我们将专门发现PCM外层球体下的蛋白质-蛋白质相互作用 信息包(目标2)。最后，我们将探讨MTOC功能失活的机制。 并测试中心体失活在细胞分化中的作用(目标3)。固有微管 组织对于正常的发育和细胞功能以及MTOC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，并阐明了这一重要的，但在细胞和 发育生物学。