Mechanics of Bipolar Mitotic Spindle Assembly

描述(由申请人提供)：两极有丝分裂纺锤体组装对于正确分离复制的基因组至关重要。大多数纺锤体组件现在已经被确定，但它们的自组织、产生和对物理力的反应的方式在基础生物医学研究中仍然是很大程度上未被探索的主题。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差距，因为很少有研究提供关于主轴或它所能产生的力的定量的、系统级的信息。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纺锤体组装的分子和机械方面，重点是基于微管的运动细胞质动力蛋白。这种马达在决定纺锤体形状方面起着至关重要的作用，但它的多功能特性、巨大的尺寸和结构的复杂性使其成为一个难以研究的课题，为原本探索得很好的有丝分裂领域提供了一个小小的前沿。基于初步数据，我们假设依赖于细胞周期的与其他蛋白质的相互作用调节动力蛋白功能，赋予交叉滑动和滑动反平行微管的特定能力。通过表征负责的含动力蛋白的运动复合体的组成和所产生的力，我们希望阐明动力蛋白的有丝分裂功能，从而阐明动力蛋白可以选择性地作为细胞分裂的靶点。为了检验我们的中心假设，我们提出了三个目标。第一种是基于蛋白质组学的筛选，以确定与动力蛋白相互作用的蛋白质，这些蛋白质要么依赖于细胞周期，要么受有丝分裂信号通路调节。在第二个目标中，将使用基于微针的力测量来量化在主轴组装过程中产生的动力蛋白依赖力。最后，第三个目标描述了一种新颖的、遗传编码力探头的设计和校准，用于高分辨率地标测主轴内的滑动细丝力。这些目标中提出的工作的完成有望从根本上促进我们对动力蛋白功能的基本理解，并为最终开发新的抗癌药物确定分子靶点。它还将提供迫切需要的系统级的综合主轴力的表征，以及解决主轴特性(如两极和长度)的冲突模型所需的定量信息。此外，由于它们具有广泛的应用潜力，本文提出的新方法和新途径的发展将极大地扩展未来旨在表征细胞内机械力和力启动信号的研究。 与公共卫生相关：为了准确地分离其染色体，分裂细胞必须首先组装有丝分裂纺锤体。尽管装配过程是机械的，但我们对主轴部件如何在空间中自组织，然后产生并响应物理力，缺乏了解。填补我们知识中的这一空白对人类健康具有重要意义，因为纺锤体组装的错误可能导致非整倍体，这是肿瘤转化的标志，也是染色体出生缺陷的原因。在这个项目中，我们描述了一系列创新的实验，旨在阐明动力蛋白的分子和机械方面，动力蛋白是一种对正确的主轴组装至关重要的分子马达。该项目的完成有望从根本上提高我们对动力蛋白在纺锤体组装过程中功能的基本理解，并提供一份具有良好抗癌药物靶点潜力的动力蛋白调节蛋白清单。此外，由于他们的广泛 应用方面，本文提出的新方法和途径的发展有望极大地扩展未来旨在表征细胞内机械力和力启动信号的研究。

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.