Microtubule Dynamics and Chromosome Segregation

微管动力学和染色体分离

• 批准号: 7912097
• 负责人: Linda Wordeman
• 金额: $ 9.77万
• 依托单位: UNIVERSITY OF WASHINGTON
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7912097
• 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）：摘要动态微管（MT）的精确调节对于有丝分裂纺锤体组装、染色体分离、细胞运动、信号传导、细胞周期调节和亚细胞模式至关重要。我们已经确定，驱动蛋白 13 家族成员 MCAK（有丝分裂着丝粒相关驱动蛋白）是我 14 年前发现的一种蛋白质，是迄今为止测量到的全局微管动力学最有效的调节因子。我们深入研究了它的作用机制及其在细胞过程中的作用。我们将使用这些数据作为基础来了解调节微管动力学的其他蛋白质的功能。具体来说，我们将研究一种功能上未表征、系统发育上普遍存在的驱动蛋白，它与动物特异性 MCAK 亚家族 Kif24 密切相关。我们还将重点关注驱动蛋白 8 家族成员 kif18A，它同时具有运动活性和 MT 解聚活性，对于染色体大会是不可或缺的。这些驱动蛋白中的每一种都贡献细胞分裂过程中纺锤体组装和染色体分离所需的基本活性。它们代表了抗癌治疗的重要目标。拟议研究的具体目标如下： 具体目标1：研究kinesin-13/MCAK和kinesin-8/Kif18A亚家族的MT解聚机制之间的保守程度。我们将使用 TIRF 显微镜、突变分析、批量 MT 解聚测定和冷冻电镜来确定这些马达的 ATP 酶循环中的持续加工性和头间协调的贡献。与驱动蛋白 13 马达相反，我们假设运动/解聚驱动蛋白 8s 抑制活微管中的 MT 动力学。这与先前发表的稳定 MT 的驱动蛋白 8 的解聚活性的推测相矛盾。我们将研究驱动蛋白 8 家族中保守的 MCAK MT 解聚循环有多少来产生这种不寻常的活性。具体目标 2：定义驱动蛋白 8 和驱动蛋白 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 会增加 K 纤维 MT 周转率，并“松开”着丝粒 MT 的着丝粒。相反，我们假设 Kif18A 抑制染色体运动并减少着丝粒纤维周转。我们将使用 RNAi、嵌合结构和实时成像来研究这两个解聚马达之间的相互作用，这两个马达似乎对染色体的收缩和运动具有拮抗作用。具体目标 3：研究 MT 动态的不同调节因子的表达之间的功能相互关系。我们假设 MT +tip 蛋白（MCAK 是其中之一）水平和平衡的改变将影响肌动蛋白/肌球蛋白细胞骨架的动力学，导致胞质分裂缺陷以及细胞运动性的改变。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这种情况发生的机制。我们进一步假设，改变 MT 动态的单个调节因子的整体水平可能会通过转录和翻译反馈影响其他 MT 调节因子的水平和分布。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 解聚剂 MCAK 变化的反应。公共健康相关性拟议的研究旨在研究调节微管（MT）动态特性的驱动蛋白的作用机制、调节和细胞功能。动态 MT 对于细胞分裂非常重要，即使药物稍微改变其动态特性，通常也会导致细胞周期停滞和细胞快速死亡。许多非常成功的抗癌药物，例如针对 MT 的紫杉醇和长春花碱，都利用了这一特性。此外，此类药物还可用于降低癌细胞的运动性和侵袭潜力。有丝分裂着丝粒相关驱动蛋白 (MCAK) 是微管 (MT) 动力学最有效的调节因子。我们有兴趣探索其活性和细胞功能，以及细胞对 MCAK 水平变化的反应。 MCAK 在多种肿瘤类型中均检测到异常高水平，并且与预后不良和抗癌药物反应不佳的肿瘤相关。针对 MT 的抗癌药物可以通过选择改变 MT 动态的细胞来优先选择高度侵袭性癌症。因此，识别和绘制肿瘤进展过程中 MT 动态调节因子的相互作用，并在确定这些调节因子后寻求专门针对这些调节因子的治疗方法非常重要。要实现这一目标，需要彻底了解它们的作用机制、调节和细胞功能。