Mechanics of Bipolar Mitotic Spindle Assembly

描述（由申请人提供）：双极有丝分裂纺锤体组装对复制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，但它们自组织、产生和响应物理力的方式在基础生物医学研究中仍未得到很大程度的探索。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了关于主轴或它可以产生的力的定量、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、大尺寸和结构复杂性使其成为一个难以研究的课题，在有丝分裂领域提供了一个很小的前沿。基于初步数据，我们假设细胞周期依赖于与其他蛋白质的相互作用调节动力蛋白的功能，赋予交联和滑动反平行微管的特定能力。通过表征含有动力蛋白的运动复合物的组成及其产生的力，我们希望阐明动力蛋白的有丝分裂功能，从而可以选择性地靶向分裂细胞。为了验证我们的中心假设，我们提出了三个目标。第一种方法使用基于蛋白质组学的筛选来鉴定与动力蛋白相互作用依赖于细胞周期或受有丝分裂信号通路调节的蛋白质。在第二个目标中，基于微针的力测量将用于量化主轴装配过程中产生的动力相关力。最后，第三个目标描述了一种新型的、遗传编码的力探针的设计和校准，用于主轴内滑动丝力的高分辨率测绘。在这些目标中提出的工作的完成有望在我们对动力蛋白功能的基本理解方面产生根本性的进展，并为最终开发新的抗癌药物确定分子靶点。它还将提供急需的综合主轴力的系统级表征，以及解决紧急主轴特性（如双极性和长度）的冲突模型所需的定量信息。此外，由于其广泛应用的潜力，本文提出的新方法和方法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。

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.