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: 10794831
• 负责人: Jessica Lynn Feldman
• 金额: $ 18.63万
• 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/10794831
• 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.

项目总结/摘要 中心体作为微管组织中心（MTOC），协调微管 通过其中心粒周围物质（PCM）进入有丝分裂纺锤体。这种活动是双相的， 在细胞周期中通过组装和分解循环。在细胞分化时， 中心体的MTOC活性通常保持在非活性状态，因为MTOC功能是 重新分配到非中心体位点以适应不同的细胞功能。虽然 过度活跃的中心体MTOC活性是某些癌症的标志， 由于中心体的侵入性细胞行为，人们对中心体作为MTOC如何失活知之甚少 在有丝分裂退出期间或在分化细胞中维持在非活性状态。我们正在使用 C.线虫作为模型来理解MTOC生物学中的这些基本知识缺口， 活的有机体我们对C. elegans透露， PCM由不同的蛋白质区域组成，这些区域被组织成内部和外部球体， 以不同的速率和使用不同的行为从中心体移除。我们发现 磷酸酶对抗有丝分裂激酶添加PCM，最终催化 在有丝分裂结束时内球PCM蛋白的溶解。PCM的性质 似乎发生变化，使得剩余的老化PCM外球破裂成亚PCM 通过基于微管的皮质拉力的“包”。因此，中心体似乎是 作为MTOC通过两步机制灭活，首先是PCM溶解，然后是 通过机械控制破裂。在拟议的研究中，我们将揭示 这种两步模型的机制，MTOC功能的失活，在 中心体我们将确定PCM溶解的机制， 相关磷酸酶、它们在中心体的靶点以及去除这些酶的作用 拆卸目标（目标1）。然后我们将专门揭示蛋白质-蛋白质 PCM外层和包的相互作用（目标2）。最后，我们将探讨 在分化细胞中MTOC功能失活的潜在机制，并测试 中心体失活在细胞分化中的作用（目的3）。正常微管组织 是正常发育和细胞功能以及MTOC功能过度活跃所必需的。 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，并阐明这一重要的，但 在细胞和发育生物学中的研究课题。