Mechanistic Analysis of Microtubule Based Motors

描述（由申请人提供）：生物医学研究中的一个主要挑战是定义有丝分裂纺锤体组装的机制以及一系列复杂的马达和微管（MT）相互作用蛋白如何正确地协调染色体分离。有丝分裂缺陷导致出生缺陷和癌症，因此，有丝分裂机制的完整分子理解对人类发育和健康至关重要。有丝分裂驱动蛋白在纺锤体功能的各个方面发挥重要作用-影响染色体的运动和分离，细胞分裂，并调节微管聚合和解聚。虽然驱动蛋白具有共同的结构基序，但关键氨基酸的变化赋予每个驱动蛋白独特的机械化学性质，从而指定其细胞功能。因此，如果我们在体外阐明单个有丝分裂驱动蛋白的酶特性，我们将深入了解其在细胞复杂环境中对纺锤体功能的特定作用，其中有其他分子马达，蛋白质和调节因子。该研究建议评估三个有代表性的驱动蛋白，以解决有关微管晶格上染色体进行性运动的机械要求，MT滑动和纺锤体稳定性的MT-MT交联功能，以及纺锤体组装和动力学的MT缩短和MT伸长的问题。我们将使用稳态前动力学方法结合平衡方法和荧光显微镜来解决以下三个具体目标：1）定义Kar 3Cik 1的机械化学，其在后期的反平行MT的交联功能。2)定义Kar 3Vik 1机械化学，其在MT负端的积累使平行MT在主轴极点交联。3)定义CENP-E过程步进的机械基础。 公共卫生相关性：CENP-E、Eg 5/KSP和驱动蛋白-14对细胞分裂至关重要，因此对人类健康和发育至关重要。它们的选择性抑制可能导致更有效的抗有丝分裂治疗剂用于治疗疾病，如癌症、症状性冠状动脉疾病和增殖性糖尿病视网膜病变。拟议的研究应该导致高通量筛选的新策略，选择化合物来增强化疗后的癌细胞死亡，而不是非整倍体。目前，有许多针对Eg 5/KSP和CENP-E的特异性化疗药物正在I/II期临床试验中。

DESCRIPTION (provided by applicant): A major challenge in biomedical research is to define the mechanisms for mitotic spindle assembly and how a complex array of motors and microtubule (MT) interacting proteins correctly orchestrate chromosome segregation. Defects in mitosis result in birth defects and cancer, and therefore, a full molecular understanding of mitotic mechanisms is critical for human development and health. Mitotic kinesins play essential roles in all facets of spindle function- effecting chromosome movement and segregation, cell cleavage, and regulating microtubule polymerization and depolymerization. While kinesins share common structural motifs, key amino acid changes confer unique mechanochemical properties to each kinesin which specifies its cellular function. Therefore, if we elucidate the enzymatic properties of an individual mitotic kinesin in vitro, we will gain insight into its specific role for spindle function in the complex environment of the cell where there are other molecular motors, proteins, and regulatory factors. The research proposed evaluates three representative kinesins to address questions about the mechanistic requirements for processive movement of chromosomes on the microtubule lattice, MT-MT crosslinking function for MT sliding and spindle stability, and MT shortening and MT elongation for spindle assembly and dynamics. We will use presteady-state kinetic methodologies in combination with equilibrium approaches and fluorescence microscopy to address the following three specific aims: 1) Define the Kar3Cik1 mechanochemistry for its crosslinking function of anti-parallel MTs at anaphase. 2) Define the Kar3Vik1 mechanochemistry for its accumulation at MT minus-ends to crosslink parallel MTs at the spindle poles. 3) Define the mechanistic basis of CENP-E processive stepping. PUBLIC HEALTH RELEVANCE: CENP-E, Eg5/KSP, and Kinesin-14s are essential for cell division and therefore human health and development. Their selective inhibition may lead to more effective anti-mitotic therapeutics for treatment of diseases such as cancer, symptomatic coronary artery disease, and proliferative diabetic retinopathy. The proposed studies should lead to new strategies for high throughput screens that select compounds to enhance cancer cell death rather than aneuploidy after chemotherapy. Presently, there are a number of specific chemotherapeutics targeted to Eg5/KSP and CENP-E in Phase I/II Clinical trials.