Microtubule Dynamics and Chromosome Segregation

微管动力学和染色体分离

• 批准号: 7525817
• 负责人: Linda Wordeman
• 金额: $ 31.32万
• 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/7525817
• 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; Decompression Sickness; Defect; Drug Delivery Systems; Equilibrium; Family; Family member; Feedback; Fiber; Film; Fluorescence Microscopy; Foundations; Frequencies; Goals; Head; Homeostasis; Image; Individual; Invasive; Kinesin; Kinetochores; Lead; Life; Localized; 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; Public Health; Publishing; RNA Interference; Regulation; Research Design; Resolution; Role; Score; Tubulin; Vinblastine; abstracting; cancer cell; cell motility; chromosome movement; depolymerization; dimer; in vitro Assay; interest; outcome forecast; 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.

动态微管（MTs）的精确调控对有丝分裂纺锤体组装、染色体分离、细胞运动、信号传导、细胞周期调控和亚细胞模式形成至关重要。我们已经确定了激酶-13家族成员MCAK（有丝分裂着丝粒相关激酶），一种我在14年前发现的蛋白质，是迄今为止测量到的最有效的全球微管动力学调节剂。我们已经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，以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的、系统发育上普遍存在的与动物特异性MCAK亚家族Kif24密切相关的激酶。我们还将重点关注kinesin-8家族成员kif18A，它具有运动活性和MT解聚活性，是染色体形成不可或缺的基因。在细胞分裂过程中，每一种驱动蛋白都参与纺锤体组装和染色体分离的基本活动。它们是抗癌治疗的重要靶点。拟开展的研究的具体目的如下：具体目的1：研究kinesin-13/MCAK和kinesin-8/Kif18A亚家族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、体MT解聚分析和低温电镜来确定这些马达在atp酶循环中的加工能力和头间协调的贡献。与kinesin-13马达相反，我们假设运动/解聚的kinesin- 8s抑制活微管中的MT动力学。这与先前发表的激酶-8对稳定MT解聚活性的推测相矛盾。我们将研究在激酶-8家族中有多少MCAK - MT解聚循环是保守的，从而产生这种不寻常的活性。具体目标2：确定激酶8和激酶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k -纤维MT的周转，并使着丝粒从着丝粒MT上“松动”。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种解聚合马达之间的相互作用，这两种马达似乎对染色体的聚集和运动具有拮抗作用。具体目标3：研究MT动力学中不同调节因子表达之间的功能相互关系。我们假设MT +尖端蛋白（MCAK是其中之一）的水平和平衡的改变会影响肌动蛋白/肌球蛋白细胞骨架的动力学，导致细胞分裂缺陷，也会改变细胞运动。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动力学单一调控因子的整体水平可能会通过转录和翻译反馈影响其他MT调控因子的水平和分布。我们将使用蛋白质和mRNA定量来评价已知MT调节因子对强效MT解聚剂MCAK变化的反应。拟议的研究旨在研究调节微管（MTs）动态特性的运动蛋白的作用机制、调节和细胞功能。动态mt对细胞分裂非常重要，即使稍微改变其动态特性的药物通常也会导致细胞周期阻滞和细胞快速死亡。许多非常成功的抗癌药物，如针对mt的紫杉醇和长春花碱，都利用了这一特性。此外，这些药物在降低癌细胞的运动性和侵袭性方面也很有用。有丝分裂着丝粒相关运动蛋白（MCAK）是微管（MT）动力学最有效的调节剂。我们感兴趣的是探索其活性和细胞功能，以及细胞对MCAK水平变化的反应。MCAK在多种肿瘤类型中被检测到异常高水平，并且与预后不良和对抗癌药物反应差的肿瘤相关。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袭性癌症。因此，识别和绘制肿瘤进展过程中MT动力学调节因子的相互作用，并在确定这些调节因子后寻求特异性靶向治疗是非常重要的。要实现这一目标，需要对它们的作用机制、调控和细胞功能有透彻的了解。