Microtubule Dynamics and Chromosome Segregation

描述(申请人提供)：这个项目的主要目标是了解动态微管在细胞分裂过程中是如何控制的，以建立有丝分裂纺锤体，并以高保真的方式分离染色体。本项目旨在研究有丝分裂微管运动蛋白家族的微管运动蛋白调节微管动力学的机制，以促进有丝分裂纺锤体的组装、染色体的分离和染色体数量的长期稳定。我们的方法是跨学科的，从使用全内反射显微镜(TIRF)和纯化组分的单分子生物物理分析，到使用高分辨率定量活细胞成像、突变构建和siRNA耗尽的细胞方法。我们将在生理温度和缓冲条件下使用活的微管的TIRF显微镜来评估有丝分裂动蛋白在微管组装和拆卸中的作用。了解了马达为分离的微管提供的活动后，我们将把我们的研究转移到活的有丝分裂细胞上。使用高分辨率成像，有选择地去除动蛋白和突变类似物，我们将评估如何在有丝分裂纺锤体中使用动蛋白马达活动。这是基本的基础知识，将指导针对有丝分裂激动素的药物的开发，用于癌症治疗。最后，我们有初步证据表明，微管动力学的微小变化在短期内不会对细胞分裂产生深刻影响，但对长期染色体不稳定性有显著影响。此外，已知的肿瘤抑制基因可能对微管动力学产生意想不到的直接或间接影响，这些基因以前没有与微管相连。我们有一个简单的视觉屏幕，它将为我们提供一组与微管动力学变化有关的基因，进而涉及染色体不稳定。这将有助于癌症的分子表征和靶向药物开发。使用这些方法，我们将了解运动动蛋白如何靶向和调节微管末端的聚合物组装和拆卸，以及微管动力学的变化对短期纺锤体组装和染色体分离有什么影响。该项目旨在评估有丝分裂纺锤体中微管动力学的微小变化对染色体不稳定性的影响，并通过这种方式了解肿瘤进展的机制。

DESCRIPTION (provided by applicant): The broad goal of this project is to understand how dynamic microtubules are controlled during cell division to build the mitotic spindle and to segregate chromosomes with high fidelity. This project is designed to investigate the mechanism by which mitotic microtubule motor proteins of the kinesin family regulate microtubule dynamics in order to facilitate mitotic spindle assembly, chromosome segregation and long-term stability of chromosome number. Our approach is interdisciplinary, ranging from single molecule biophysical analyses using total internal reflection microscopy (TIRF) and purified components to cellular approaches using high resolution quantitative live cell imaging, mutant constructs and siRNA depletions. We will use TIRF microscopy of live microtubules at physiological temperature and buffer conditions to evaluate the effect of mitotic kinesins on microtubule assembly and disassembly. Armed with an understanding of the activity the motor provides to microtubules in isolation we will transfer our studies to live mitotic cells. Using hig resolution imaging, selective depletions of kinesins and mutant analogs we will evaluate how kinesin motor activity is used within the mitotic spindle. This is essential basic knowledge which will guide the development of drugs targeted to mitotic kinesins for use in cancer treatment. Finally, we have preliminary evidence that small alterations in microtubule dynamics that do not have a profound impact on cell division in the short term have significant effects on long-term chromosome instability. Furthermore, known tumor suppressor genes which have not previously been linked to microtubules may exhibit unexpected direct or indirect effects on microtubule dynamics. We have a simple visual screen that will provide us with a panel of genes implicated in microtubule dynamics alterations and, by extension, chromosome instability. This will assist in molecular characterization of cancers and targeted drug development. Using these approaches we will learn how motile kinesins target to and regulate polymer assembly and disassembly at microtubule ends and what effect alterations in microtubule dynamics has on short-term spindle assembly and chromosome segregation. This project is designed to evaluate the effect that small changes in microtubule dynamics in the mitotic spindle has on chromosome instability and, in this way, understand the mechanistic forces underlying tumor progression.