Mechanics of Bipolar Mitotic Spindle Assembly

双极有丝分裂纺锤体组装的力学

• 批准号: 8346684
• 负责人: JESSE C GATLIN
• 金额: $ 26.43万
• 依托单位: UNIVERSITY OF WYOMING
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8346684
• 关键词: Address; Aneuploidy; Antineoplastic Agents; Area; Biological Assay; Biomechanics; Biomedical Research; Calibration; Cell Cycle; Cell physiology; Cells; Characteristics; Chromosome Segregation; Chromosomes; Communities; Complex; Conflict (Psychology); Congenital Abnormality; Data; Development; Dynein ATPase; Equilibrium; Filament; Future; Genome; Goals; Health; Human; Kinesin; Knowledge; Lead; Length; Link; Maps; Measurement; Measures; Mechanics; Mediating; Methods; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Molecular Motors; Molecular Target; Motor; Nature; Neoplastic Cell Transformation; Play; Process; Property; Proteins; Proteomics; Publishing; Regulation; Research; Resolution; Role; Shapes; Signal Pathway; Signal Transduction; Slide; Spatial Distribution; Structure; System; Techniques; Testing; Work; base; cancer therapy; crosslink; design; frontier; genetic regulatory protein; innovation; new therapeutic target; novel; research study; segregation; tool

DESCRIPTION (provided by applicant): Bipolar mitotic spindle assembly is critically important for proper segregation of a duplicated genome. Most spindle components have now been identified, but the ways they self-organize and generate and respond to physical forces remain largely unexplored topics in basic biomedical research. This gap in our understanding of spindle assembly mechanics persists despite years of research in this area because very few studies have provided quantitative, systems-level information about the spindle or the forces it can produce. To address this gap we have proposed an integrated set of experimental approaches to investigate molecular and mechanical aspects of spindle assembly, with a focus on the microtubule-based motor cytoplasmic dynein. This motor plays critically important roles in determining spindle shape, but its multifunctional character, large size and structural complexity have made it a difficult subject to study, providing a small frontier in the otherwise well-explore field of mitosis. Based on preliminary data, we hypothesize that cell cycle-dependent interactions with other proteins regulate dynein function, conferring a specific ability to crosslik and slide antiparallel microtubules. By characterizing the composition of the responsible dynein-containing motor complex and the forces it generates, we hope to elucidate a mitotic function of dynein and, therefore, one that can be selectively targeted in dividing cells. To test our central hypothesis we propose three aims. The first uses a proteomics based screen to identify proteins whose interactions with dynein are either cell-cycle dependent or regulated by mitotic signaling pathways. In the second aim, microneedle-based force measurements will be used to quantify dynein-dependent forces generated during spindle assembly. Lastly, the third aim describes the design and calibration of a novel, genetically encoded force-probe for high-resolution mapping of sliding-filament forces within the spindle. Completion of the work proposed in these aims is expected to produce a fundamental advance in our basic understanding of dynein function and to identify molecular targets for the eventual development of new anti-cancer drugs. It will also provide much needed systems-level characterization of integrated spindle forces as well as quantitative information needed to resolve conflicting models of emergent spindle properties like bipolarity and length. In addition, due to their potential for broad application, development of th new methods and approaches proposed herein will greatly expand future studies aimed at characterizing mechanical forces and force- initiated signaling within cells. PUBLIC HEALTH RELEVANCE: In order to accurately segregate its chromosomes, a dividing cell must first assemble a mitotic spindle. Despite the mechanical nature of the assembly process, our understanding of how spindle components self-organize in space, and then generate and respond to physical forces, is lacking. Filling this gap in our knowledge has important implications in the context of human health, because errors in spindle assembly can lead to aneuploidy, a hallmark of neoplastic transformation and the cause of chromosomal birth defects. In this project, we describe an innovative set of experiments designed to elucidate molecular and mechanical aspects of dynein, a molecular motor critically important for proper spindle assembly. Completion of this project is expected to fundamentally advance our basic understanding of dynein function during spindle assembly and provide a list of dynein regulatory proteins with excellent potential as targets for anti-cancer drugs. In addition, due to their broad applications, the development of new methods and approaches proposed herein is expected to greatly expand future studies aimed at characterizing mechanical forces and force-initiated signaling within cells.

描述（由申请人提供）：双极有丝分裂纺锤体组装对于重复基因组的正确分离至关重要。大多数纺锤体成分现在已经被确定，但它们自我组织、产生和响应物理力的方式在基础生物医学研究中仍然是未探索的课题。尽管在这一领域进行了多年的研究，但我们对主轴装配力学的理解仍然存在这种差距，因为很少有研究提供有关主轴或其可能产生的力的定量系统级信息。为了解决这一差距，我们提出了一套完整的实验方法来研究分子和机械方面的纺锤体组装，重点是基于微管的运动细胞质动力蛋白。该马达在决定纺锤体形状方面起着至关重要的作用，但其多功能特性、大尺寸和结构复杂性使其成为一个难以研究的课题，为有丝分裂领域的其他探索提供了一个小前沿。基于初步的数据，我们假设细胞周期依赖的相互作用与其他蛋白质调节动力蛋白的功能，赋予一个特定的能力，crosslik和幻灯片反平行微管。通过表征负责任的含动力蛋白的运动复合体的组成和它产生的力，我们希望阐明动力蛋白的有丝分裂功能，因此，可以选择性地靶向分裂细胞。为了验证我们的核心假设，我们提出了三个目标。第一个使用蛋白质组学为基础的屏幕，以确定蛋白质的相互作用与动力蛋白是细胞周期依赖或有丝分裂信号通路的调节。在第二个目标中，基于微针的力测量将用于量化主轴组装期间产生的动力蛋白依赖性力。最后，第三个目标描述了一种新的，遗传编码的力探针的设计和校准的高分辨率映射内的主轴滑丝力。完成这些目标中提出的工作，预计将产生一个根本性的进步，我们的动力蛋白功能的基本理解，并确定新的抗癌药物的最终开发的分子靶点。它还将提供急需的系统级表征的综合主轴部队以及定量信息需要解决冲突的模型，如双极性和长度的新兴主轴属性。此外，由于其广泛应用的潜力，本文提出的新方法和途径的开发将极大地扩展旨在表征细胞内的机械力和力引发的信号传导的未来研究。 公共卫生相关性：为了准确分离染色体，分裂细胞必须首先组装有丝分裂纺锤体。尽管组装过程具有机械性质，但我们对主轴组件如何在空间中自组织，然后产生并响应物理力的理解仍然缺乏。填补我们知识中的这一空白在人类健康方面具有重要意义，因为纺锤体组装中的错误可能导致非整倍性，这是肿瘤转化的标志和染色体出生缺陷的原因。在这个项目中，我们描述了一组创新的实验，旨在阐明动力蛋白，一个分子马达的分子和机械方面的正确的主轴组装至关重要。该项目的完成有望从根本上推进我们对动力蛋白在纺锤体组装过程中功能的基本理解，并提供一系列具有良好潜力的动力蛋白调节蛋白作为抗癌药物的靶点。此外，由于其广泛的 由于这些应用，本文提出的新方法和途径的开发有望大大扩展旨在表征细胞内的机械力和力引发的信号传导的未来研究。