Deciphering the mechanics of microtubule networks in mitosis

项目摘要 细胞在广泛的过程中执行机械任务，包括在 细胞分裂。这些任务是通过组织产生力量的细胞骨架网络来完成的。 微米级的微管网络需要运动和非运动蛋白质来移动和组织细丝进入 适当的功能机械装置。我们的长期目标是破译 这些网络的组装和功能，使用有丝分裂作为生物过程的模型。为了实现这一目标，我们 将采用生化重建、生物物理方法、单分子荧光显微镜和活- 细胞成像。我们将在我们最近的出版物和未发表的初步数据的基础上，专注于微管 网络力学在有丝分裂中的三个目标：(1)确定细胞的机械和功能 中期桥接纤维与后期中央纺锤体微管网络的差异。 具体地说，我们将剖析一种重要的交联型非马达图谱Prc1的分子机制，该图谱 在有丝分裂纺锤体内形成不同的图案。这些功能包括连接SISTER的桥接光纤 中期为动粒纤维，后期为中央纺锤体中带排列。Prc1受细胞周期调控 由CDK/Cyclin B结合，因此是中期和后期的生物化学不同分子。我们会 组装并机械地探测细丝网络，以了解主轴如何能够区分 在中期和后期移动染色体的同时产生力量并自我重塑。对活细胞进行成像 在有丝分裂期间，表达突变的PRC1结构将验证我们的体外发现。(2)确定分子 MAP聚集的机制以及MAP聚集在调节微管组织中的功能作用。 特别是，我们将研究PRC1中内在无序的子域如何有助于MAP聚类。 我们公布的和初步的数据表明，Prc1团簇显著阻碍了细丝滑动，并且 C-末端的非结构结构域介导了这一效应。我们将利用我们的生物物理和细胞生物学工具 确定减少聚集对微管组织的影响。(3)确定复合体如何 运动和非运动MAP共同调节微管组织。我们将研究如何 Kif4A/PRC1复合体在微管滑动过程中产生力，以及稳态重叠排列是如何 产生维持主轴中部完整性的阻力。总之，我们的发现应该会推动我们的 了解微米级微管网络如何调节有丝分裂中的染色体运动。我们的目标是 阐明一种定义不同MAP的结构和生化如何导致细胞 能做机械工作的机器。拷贝数导致微管网络组装错误 基本图谱的变异或突变与人类的疾病有关。我们的研究将揭示 将网络故障与疾病状态联系起来的生物物理特性，并可能导致针对这些疾病的治疗 或提供对用于评估疾病进展的诊断工具的洞察。

Project Summary Cells perform mechanical tasks across a wide range of processes including segregating chromosomes during cell division. These tasks are accomplished by the organization of force-generating cytoskeletal networks. Micron-scale microtubule networks need both motor and non-motor proteins to move and organize filaments into proper functional mechanical units. Our long-term goal is to decipher the mechanical code that underlies the assembly and function of these networks, using mitosis as a model biological process. To achieve this goal, we will employ biochemical reconstitution, biophysical methods, single-molecule fluorescence microscopy, and live- cell imaging. We will build on our recent publications and unpublished preliminary data to focus on microtubule network mechanics in mitosis in the following three Aims: (1) Determine the mechanical and functional differences between bridging fibers in metaphase and the central spindle microtubule network in anaphase. Specifically, we will dissect the molecular mechanisms of an essential crosslinking non-motor MAP, PRC1, that builds distinct motifs within the mitotic spindle. These features include bridging fibers that connect sister kinetochore fibers in metaphase and the central spindle midzone array in anaphase. PRC1 is cell cycle regulated by CDK/cyclin B, and therefore is a biochemically distinct molecule in metaphase and anaphase. We will assemble and mechanically probe filament networks to understand how the spindle is able to differentially generate forces and remodel itself while moving chromosomes in metaphase and anaphase. Imaging live cells during mitosis that express mutant PRC1 constructs will validate our in vitro findings. (2) Determine the molecular mechanisms for MAP clustering and the functional role of MAP clusters in regulating microtubule organization. Specially, we will examine how intrinsically disordered subdomains within PRC1 contribute to MAP clustering. Our published and preliminary data suggests that PRC1 clusters significantly impede filament sliding, and that the C-terminal unstructured domain mediates this effect. We will employ our biophysical and cell biological tools to determine the effect that reducing clustering has on microtubule organization. (3) Determine how complexes of motor and non-motor MAPs collectively regulate microtubule organization. We will examine how the Kif4A/PRC1 complex generates forces during microtubule sliding, and how a steady-state overlap arrangement produces resistive forces that maintain spindle midzone integrity. Together, our findings should advance our understanding of how micron-scale microtubule networks regulate chromosome motions in mitosis. We aim to elucidate a ‘code’ that defines how the structure and biochemistry of different MAPs gives rise to cellular machinery that can perform mechanical work. Errors in microtubule network assembly due to copy number variations or mutations in essential MAPs are linked to disease in humans. Our research will shed light on the biophysical properties that link network failure to disease states and may lead to therapies that target these proteins or provide insights into diagnostic tools for assessing disease progression.