Microtubule Regulation

微管调节

• 批准号: 7931456
• 负责人: Don W Cleveland
• 金额: $ 16.27万
• 依托单位: LUDWIG INSTITUTE FOR CANCER RES LTD
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7931456
• 关键词: Affect; Affinity; Agreement; Anaphase; Aneuploidy; Binding; CDKN2A gene; CENP-E protein; Cell Cycle; Cell Cycle Regulation; Cell Death; Cell division; Cells; Centromere; Chromatids; Chromosomes; Chronic; Complex; Coupling; Cyclin B; Cyclins; DNA; DNA Damage; Dynein ATPase; Electron Microscopy; Elements; Epitopes; Event; Figs - dietary; Genetic; Human; In Vitro; Individual; Kinetochores; Lateral; Malignant Neoplasms; Mammalian Cell; Mediating; Meiosis; Microtubules; Mitosis; Mitotic; Mitotic Checkpoint; Modeling; Modification; Mono-S; Motor; Movement; Paclitaxel; Pathway interactions; Phase; Phosphotransferases; Positioning Attribute; Process; Property; Protein p53; Proteins; Regulation; Research; Role; Signal Pathway; Signal Transduction; Sister; Sister Chromatid; Site; Solid Neoplasm; Structure; Surface; Testing; Therapeutic; Time; Tumor Suppressor Genes; Work; Xenopus; antitumor drug; chromosome movement; cohesin; daughter cell; egg; farnesylation; human PTTG1 protein; inhibitor/antagonist; p19ARF; prevent; public health relevance; reconstruction; residence; segregation; transmission process; tumor; tumor initiation; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): The essential function of mitosis is the delivery of a complete set of chromosomes to each daughter cell. This requires attachment of spindle microtubules to kinetochores, specialized structures formed at centromeres. Kinetochores are more than just structural elements that mediate faithful chromosome inheritance. The unattached kinetochore is also the signal generator for the mitotic checkpoint (also known as the spindle assembly checkpoint), the major cell cycle control mechanism in mitosis. Each unattached kinetochore generates a "wait anaphase" inhibitor that blocks destruction of cyclin B and securin, thereby blocking disjoining of duplicated sister chromosomes and advance into anaphase. We have previously identified a microtubule-dependent motor, CENPE, to be multifunctional these mitotic events. It directly tethers spindle microtubules to kinetochores and it functions as the cyclin-like activator of the essential mitotic checkpoint kinase BubR1. Microtubule capture by CENP-E in turn inactivates BubR1 kinase activity, thereby silencing mitotic checkpoint signaling and permitting anaphase onset. We now propose to determine the properties of CENP-E in microtubule capture at kinetochores (with electron microscopy), the motor properties of CENP-E (including processivity and residence time when microtubule bound), and how CENP-E acts to affect coupling to dynamic microtubules. The functional role of an unusual CENPE modification, farnesylation, will be determined. A major effort will use all purified components to reconstruct mitotic checkpoint signaling in vitro and to identify the "wait anaphase" inhibitor(s) produced by unattached kinetochores. Moreover, recognizing that most human solid tumors are aneuploid, that is, have other than the correct number of 46 chromosomes, we will exploit our discovery that reduced levels of CENP-E generate accelerated rates of such aneuploidy to enable a test of how whole chromosomal aneuploidy affects tumorigenesis initiated by loss of each of the major tumor suppressor genes. This will provide a test of the hypothesis posed more than 100 years ago by the great cytologist Boveri that aneuploidy drives tumorigenesis. Finally, recognizing that successful cancer therapeutics such as taxol yield chronic activation of the mitotic checkpoint, we will examine the mechanisms underlying cell death or adaptation after chrom0ic mitotic checkpoint arrest. PUBLIC HEALTH RELEVANCE: Unattached kinetochores control the cell cycle clock during cell division by generating a "wait" signal, the mitotic checkpoint, which delays cell cycle advance until all chromosomes have successfully attached to spindle microtubules. How this signaling works will be examined by determining how a kinetochore motor protein affects chromosome attachment to spindle microtubules and acts to activate and silence an essential checkpoint kinase. The overall signaling pathway will be determined by reconstructing it from individual components. Finally, since some successful anti-tumor drugs in humans chronically activate the mitotic checkpoint, the mechanisms tha determine cell fate, cell death or adaptation, when so arrested will be determined.

描述（由申请人提供）：有丝分裂的基本功能是将一套完整的染色体传递到每个子细胞。这需要将纺锤体微管附着在动粒上，动粒是在着丝粒处形成的特殊结构。动粒不仅仅是介导忠实染色体遗传的结构元件。未附着的着丝粒也是有丝分裂检查点（也称为纺锤体组装检查点）的信号发生器，有丝分裂中主要的细胞周期控制机制。每个未附着的着丝粒都会产生一种“等待后期”抑制剂，阻止细胞周期蛋白 B 和 securin 的破坏，从而阻止复制姐妹染色体的分离并进入后期。我们之前已经鉴定出一种微管依赖性马达 CENPE，它对这些有丝分裂事件具有多功能性。它直接将纺锤体微管连接到着丝粒，并充当必需的有丝分裂检查点激酶 BubR1 的细胞周期蛋白样激活剂。 CENP-E 捕获的微管反过来会使 BubR1 激酶活性失活，从而沉默有丝分裂检查点信号并允许后期开始。我们现在建议确定 CENP-E 在动粒微管捕获中的特性（使用电子显微镜）、CENP-E 的运动特性（包括微管结合时的持续过程和停留时间），以及 CENP-E 如何作用来影响与动态微管。一种不寻常的 CENPE 修饰——法呢基化——的功能作用将被确定。主要工作将使用所有纯化的成分在体外重建有丝分裂检查点信号，并鉴定由未附着的着丝粒产生的“等待后期”抑制剂。此外，认识到大多数人类实体瘤是非整倍体，即不具有正确数量的 46 条染色体，我们将利用我们的发现，即 CENP-E 水平降低会加速这种非整倍体的发生率，从而能够测试整个染色体如何非整倍性会影响由每个主要抑癌基因缺失引发的肿瘤发生。这将验证 100 多年前伟大的细胞学家 Boveri 提出的非整倍体驱动肿瘤发生的假设。最后，认识到成功的癌症疗法（例如紫杉醇）会导致有丝分裂检查点的慢性激活，我们将研究染色有丝分裂检查点停滞后细胞死亡或适应的机制。公共健康相关性：未附着的动粒通过产生“等待”信号（有丝分裂检查点）来控制细胞分裂过程中的细胞周期时钟，该信号延迟细胞周期前进，直到所有染色体都成功附着到纺锤体微管上。通过确定动粒运动蛋白如何影响染色体与纺锤体微管的附着以及如何激活和沉默重要的检查点激酶，将检查这种信号传导的工作原理。整体信号传导途径将通过从各个成分重建来确定。最后，由于一些成功的人类抗肿瘤药物会长期激活有丝分裂检查点，因此当被阻止时，决定细胞命运、细胞死亡或适应的机制将被确定。