Deciphering the mechanics of microtubule networks in mitosis

破译有丝分裂中微管网络的机制

• 批准号: 10637323
• 负责人: Scott Thomas Forth
• 金额: $ 32.11万
• 依托单位: RENSSELAER POLYTECHNIC INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-15 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10637323
• 关键词: Address; Adopted; Alzheimer' s Disease; Anaphase; Assessment tool; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Biological Process; Biophysics; Bundling; C-terminal; CDC2 gene; Cell Cycle; Cell division; Cell physiology; Cells; Chromosome Positioning; Chromosome Segregation; Chromosomes; Code; Complex; Copy Number Polymorphism; Crosslinker; Cyclin B; Cytoskeleton; Data; Defect; Disease; Disease Progression; Exhibits; Failure; Fiber; Filament; Fluorescence Microscopy; Friction; Functional disorder; Goals; Human; Image; In Vitro; Kinesin; Kinetochores; Knowledge; Link; Malignant Neoplasms; Measures; Mechanical Stress; Mechanics; Mediating; Metaphase; Microscopy; Microtubule Bundle; Microtubule-Associated Proteins; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Motion; Motor; Multiprotein Complexes; Mutation; Neurons; PRC1 Protein; Phase; Phenotype; Phosphorylation; Play; Positioning Attribute; Process; Production; Proteins; Publications; Publishing; Regulation; Research; Resistance; Resolution; Role; Sister; Slide; Structure; Techniques; Time; Total Internal Reflection Fluorescent; Viscosity; Work; biophysical analysis; biophysical properties; biophysical techniques; biophysical tools; cell type; crosslink; diagnostic tool; insight; laser tweezer; link protein; live cell imaging; mechanical properties; mutant; nervous system disorder; novel; optic tweezer; protein complex; reconstitution; single molecule; targeted treatment; tool

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.

项目摘要 细胞在各种过程中执行机械任务，包括隔离染色体 细胞分裂。这些任务是通过组织生成细胞骨架网络来完成的。 微米级微管网络需要电动机和非运动蛋白才能移动和组织细丝到 适当的功能机械单元。我们的长期目标是破译基于 这些网络的组装和功能，使用有丝分裂作为模型生物学过程。为了实现这一目标，我们 将采用生化重构，生物物理方法，单分子荧光显微镜和活体 细胞成像。我们将以最新出版物和未发表的初步数据为基础，以关注微管 有丝分裂中的网络机制在以下三个目的中：（1）确定机械和功能 中期中的桥接纤维与动画中的中央纺锤微管网络之间的差异。 具体而言，我们将剖析基本交联的非运动图Prc1的分子机制，即 在有丝分裂主轴内构建不同的主题。这些功能包括连接姐姐的桥接纤维 中期和中央纺锤体中区阵列中的动力学纤维中的纤维。 PRC1是细胞周期调节 通过CDK/细胞周期蛋白B，因此是中期和后期的生化分子。我们将 组装和机械探测细丝网络，以了解主轴如何差异化 在中期和后期移动染色体时产生力并重塑。成像活细胞 在有丝分裂期间，表达突变体Prc1构建体将验证我们的体外发现。 （2）确定分子 地图聚类的机制和地图簇在调节微管组织中的功能作用。 特别是，我们将研究PRC1中内在受干扰的子域对MAP聚类的贡献。 我们发布的初步数据表明，Prc1簇极大地阻碍了细丝滑动，并且 C末端的非结构化域培养了这种效果。我们将使用我们的生物物理和细胞生物学工具 确定减少聚类对微管组织的影响。 （3）确定复合物 电动机和非运动图集体调节微管组织。我们将研究如何 KIF4A/PRC1复合物在微管滑动过程中产生力，以及稳态重叠的排列 产生保持纺锤体中区完整性的电阻。在一起，我们的发现应该推进我们的 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 阐明一个“代码”，该“代码”定义了不同地图的结构和生物化学如何产生蜂窝 可以执行机械工作的机械。由于复制号码，微管网络组件中的错误 基本图中的变异或突变与人类的疾病有关。我们的研究将阐明 将网络失败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的疗法 蛋白质或提供有关评估疾病进展的诊断工具的见解。