Microtubule Regulation

描述(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给每个子细胞。这需要纺锤体微管附着在着丝粒上，着丝粒上形成的特殊结构。着丝点不仅仅是调节忠实染色体遗传的结构元素。独立的动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。每个独立的动粒产生一个“等待后期”抑制物，阻止细胞周期蛋白B和Securin的破坏，从而阻止复制的姐妹染色体的分离并进入后期。我们以前已经发现微管依赖的运动，CENPE，是这些有丝分裂事件的多功能分子。它直接将纺锤体微管连接到动点，并作为必要的有丝分裂检查点激酶BubR1的周期素样激活剂发挥作用。由CENP-E捕获的微管反过来使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在运动中心捕获微管中的性质(用电子显微镜)，CENP-E的运动性质(包括微管结合时的加工性和停留时间)，以及CENP-E如何影响与动态微管的耦合。一种不寻常的CENPE修饰，法尼化，其功能作用将被确定。一项主要的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，并鉴定由分离的动点产生的等待后期抑制物(S)。此外，认识到大多数人类实体肿瘤是非整倍体，即具有不同于正确数目的46条染色体，我们将利用我们的发现，即降低CENP-E水平会加速这种非整倍体的发生，从而能够测试整个染色体非整倍体如何影响由每个主要肿瘤抑制基因丢失引发的肿瘤发生。这将对100多年前伟大的细胞学家博弗里提出的非整倍体驱动肿瘤发生的假说进行检验。最后，认识到成功的癌症治疗方法，如紫杉醇，可导致有丝分裂检查点的慢性激活，我们将研究细胞死亡或染色体有丝分裂检查点停止后适应的机制。与公共健康相关：在细胞分裂过程中，独立的动点通过产生“等待”信号来控制细胞周期时钟，即有丝分裂检查点，该信号会延迟细胞周期的推进，直到所有的染色体都成功地附着在纺锤体微管上。通过确定动粒运动蛋白如何影响染色体与纺锤体微管的连接，以及如何激活和沉默必要的检查点激酶，将研究这种信号是如何发挥作用的。整个信号通路将通过从单个组件重建来确定。最后，由于一些成功的人类抗肿瘤药物长期激活有丝分裂检查点，决定细胞命运、细胞死亡或适应的机制将被确定。

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.