Microtubule Dynamics and Chromosome Segregation

微管动力学和染色体分离

• 批准号: 7664466
• 负责人: Linda Wordeman
• 金额: $ 32.95万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7664466
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Actins; Affect; Animals; Antineoplastic Agents; Biological Assay; Cell Cycle; Cell Cycle Arrest; Cell Cycle Regulation; Cell Death; Cell division; Cell physiology; Cells; Centromere; Chimera organism; Chromosome Segregation; Chromosomes; Cytokinesis; Cytoskeleton; Data; Defect; Drug Delivery Systems; Equilibrium; Family; Family member; Feedback; Fiber; Film; Fluorescence Microscopy; Foundations; Frequencies; Goals; Head; Homeostasis; Image; Individual; Kinesin; Kinetochores; Lead; Life; Malignant Neoplasms; Maps; Measures; Messenger RNA; Methods; Microtubule Depolymerization; Microtubules; Mitotic; Mitotic spindle; Modification; Motor; Motor Activity; Myosin ATPase; Paclitaxel; Pattern; Pharmaceutical Preparations; Polymers; Power stroke; Principal Investigator; Property; Proteins; Proteomics; Publishing; RNA Interference; Regulation; Research Design; Resolution; Role; Tubulin; Vinblastine; abstracting; cancer cell; cell motility; chromosome movement; depolymerization; dimer; in vitro Assay; interest; outcome forecast; public health relevance; response; tumor; tumor progression

DESCRIPTION (provided by applicant): Abstract Precise regulation of dynamic microtubules (MTs) is essential for mitotic spindle assembly, chromosome segregation, cell motility, signaling, cell cycle regulation and subcellular patterning. We have established that the kinesin-13 family member MCAK (Mitotic Centromere-associated kinesin), a protein that I discovered 14 years ago, is the most potent regulator of global microtubule dynamics so far measured. We have intensively studied its mechanism of action and its role in cellular processes. We will use these data as a foundation to understand the function of other proteins that modulate microtubule dynamics. Specifically we will investigate a functionally uncharacterized, phylogenetically ubiquitous kinesin that is closely related to the animal-specific MCAK subfamily, Kif24. We will also focus on kinesin-8 family member kif18A, which possesses both motor activity and MT depolymerizing activity and is indispensable for chromosome congression. Each of these kinesins contributes essential activities required for spindle assembly and chromosome segregation during cell division. They represent vital targets for anti-cancer theurapeutics. Specific aims of the proposed studies are as follows: Specific Aim 1: Investigate the degree of conservation between the MT depolymerization mechanisms of kinesin-13/MCAK and kinesin-8/Kif18A subfamilies. We will use TIRF microscopy, mutational analysis, bulk MT depolymerization assays and cryo-EM to determine the contribution of processivity, and inter-head coordination in the ATPase cycle for these motors. In contrast to kinesin-13 motors, we hypothesize that the motile/depolymerizing kinesin- 8s suppress MT dynamics in live microtubules. This contradicts the conjectures drawn from the previously published depolymerization activity of kinesin-8s for stabilized MTs. We will investigate how much of the MCAK MT depolymerization cycle is conserved in the kinesin-8 family to produce this unusual activity. Specific Aim 2: Define the role of kinesin-8 and kinesin-13 family members in controlling chromosome movement and MT spindle fiber dynamics. We hypothesize that MCAK increases K-fiber MT turnover and "loosens" kinetochores from the kinetochore MTs. In contrast, we hypothesize that Kif18A suppresses chromosome movement and decreases kinetochore fiber turnover. We will use RNAi, chimeric contructs and live imaging to investigate the interplay between these two depolymerizing motors that appear to have antagonistic effects on chromosome congression and motility. Specific Aim 3: Investigate the functional interrelationship between the expression of different regulators of MT dynamics. We hypothesize that alterations in the levels and balance of MT +tip proteins (of which MCAK is one) will affect the dynamics of the actin/myosin cytoskeleton resulting in cytokinesis defects and also in modifications to cell motility. We will use high resolution filming, protein depletions and proteomic methods to investigate the mechanism by which this occurs. We further hypothesize that altering the global levels of single regulators of MT dynamics may affect the levels and distributions of other MT regulators via transcriptional and translational feedback. We will use protein and mRNA quantification to score the response of known MT regulators to changes in the potent MT depolymerizer MCAK. PUBLIC HEALTH RELEVANCE The proposed studies are designed to investigate the mechanism of action, regulation and cellular function of kinesins that regulate the dynamic properties of microtubules (MTs). Dynamic MTs are so important for cell division that drugs that alter their dynamic properties even slightly, will usually lead to cell cycle arrest and rapid cell death. Many highly successful anti-cancer drugs, such as paclitaxel and vinblastine that target MTs, exploit this property. In addition, such drugs have been useful in decreasing the motility and invasive potential of cancer cells. Mitotic Centromere-associated Kinesin (MCAK) is the most potent regulator of microtubule (MT) dynamics thus identified. We are interested in exploring its activity and cellular function and also the cellular response to changes in MCAK levels. MCAK has been detected at abnormally high levels in a wide variety of tumor types and is correlated with tumors with an unfavorable prognosis and poor response to anti-cancer drugs. Anti-cancer drugs that target MTs can lead to preferential selection of highly invasive cancers by selecting for cells that modify MT dynamics. Thus, it is extremely important to identify and map the interplay of regulators of MT dynamics during tumor progression and to seek therapies that specifically target these regulators once they are identified. A thorough understanding of their mechanism of action, regulation and cellular function is required to realize this goal.

描述（由申请人提供）：动态微管（MTS）的抽象精确调节对于有丝分裂主轴组件，染色体隔离，细胞运动，信号传导，细胞周期调节和亚细胞图案至关重要。我们已经确定，我14年前发现的一种蛋白质是动力蛋白13家族成员MCAK（有丝分裂共粒相关的驱动蛋白），是迄今为止测量的最有效的全球微管动力学调节剂。我们深入研究了其作用机理及其在细胞过程中的作用。我们将使用这些数据作为基础，以了解调节微管动力学的其他蛋白质的功能。具体而言，我们将研究一种与动物特异性MCAK亚科KIF24密切相关的功能性无形式，系统发育无处不在的驱动蛋白。我们还将重点关注运动蛋白-8家族成员KIF18A，它具有运动活性和MT解放活性，对于染色体会议而言是必不可少的。这些驱动器中的每一个都贡献了纺锤体组件和细胞分裂期间染色体隔离所需的重要活动。它们代表了抗癌神经药物的重要目标。拟议的研究的具体目的如下：具体目标1：研究驱动蛋白13/MCAK的MT解聚机制之间的保护程度，并研究了驱动蛋白-8/KIF18A亚科。我们将使用TIRF显微镜，突变分析，大量MT去聚合测定和冷冻EM来确定这些电机在ATPase循环中在ATPase周期中的贡献。与驱动蛋白13电动机相反，我们假设运动/去聚合驱动蛋白8S抑制了活微管中的MT动力学。这与稳定MTs的驱动蛋白8s的抑制作用相矛盾。我们将研究在动力蛋白8家族中保守的MCAK MT去聚合周期有多少，以产生这种异常活性。特定目标2：定义驱动蛋白8和动力蛋白13家族成员在控制染色体运动和MT纺锤体纤维动力学中的作用。我们假设MCAK增加了KineTochore MT的K纤维MT流动和“松动”动力学。相比之下，我们假设KIF18A抑制了染色体运动并降低了动脉纤维的转换。我们将使用RNAi，嵌合构造和实时成像来研究这两个解聚机的相互作用，这些电动机似乎对染色体会议和运动性具有拮抗作用。特定目标3：研究MT动力学不同调节剂的表达之间的功能相互关系。我们假设MT +TIP蛋白的水平和平衡发生变化（MCAK是一个）将影响肌动蛋白/肌球蛋白细胞骨架的动力学，从而导致细胞因子缺陷以及细胞运动的修饰。我们将使用高分辨率拍摄，蛋白质消耗和蛋白质组学方法来研究发生这种情况的机制。我们进一步假设，改变MT动态的单个调节剂的全球水平可能会通过转录和翻译反馈影响其他MT调节器的水平和分布。我们将使用蛋白质和mRNA定量来评估已知MT调节剂对有效MT脱聚合剂MCAK变化的响应。公共卫生相关性拟议的研究旨在研究调节微管（MTS）动态特性的动力素的作用，调节和细胞功能的机理。动态MT对细胞分裂非常重要，以至于稍微改变其动态特性的药物通常会导致细胞周期停滞和快速细胞死亡。许多非常成功的抗癌药物，例如针对MTS的紫杉醇和vinblastine，都利用了该特性。此外，此类药物对降低癌细胞的运动性和侵入性潜力很有用。有丝分裂的丝粒相关驱动蛋白（MCAK）是因此鉴定出的最有效的微管（MT）动力学调节剂。我们有兴趣探索其活性和细胞功能以及对MCAK水平变化的细胞反应。 MCAK在多种肿瘤类型的异常高水平中发现了MCAK，并且与肿瘤的预后不良和对抗癌药物的反应不佳相关。靶向MT的抗癌药物可以通过选择修饰MT动力学的细胞来优先选择高度侵入性癌症。因此，识别和绘制肿瘤进展过程中MT动态调节剂的相互作用并寻求专门针对这些调节剂后的疗法非常重要。需要对其作用机理，调节和细胞功能的透彻理解才能实现这一目标。