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 细胞在广泛的过程中执行机械任务，包括分离染色体， 细胞分裂这些任务是通过组织产生力的细胞骨架网络来完成的。 大规模的微管网络需要运动和非运动蛋白来移动和组织细丝， 适当的功能机械单元。我们的长期目标是破译机械密码， 这些网络的组装和功能，使用有丝分裂作为模型生物过程。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，和活的， 细胞成像我们将建立在我们最近的出版物和未发表的初步数据，重点放在微管 网络力学在有丝分裂中有以下三个目的：（1）确定机械和功能 中期的桥纤维和后期的纺锤体微管网络的差异。 具体来说，我们将剖析一个重要的交联非马达MAP，PRC 1的分子机制， 在有丝分裂纺锤体中构建独特的图案。这些特征包括连接姐妹的桥接纤维 中期有着丝粒纤维，后期有纺锤体中间区排列。PRC 1受细胞周期调节 由CDK/细胞周期蛋白B介导，因此在中期和后期是生物化学上不同的分子。我们将 组装和机械探测细丝网络，以了解纺锤体如何能够差异化地 在中期和后期移动染色体的同时产生力量并重塑自身。活细胞成像 在有丝分裂过程中表达突变型PRC 1构建体将验证我们的体外发现。(2)测定分子 MAP簇的机制和MAP簇在调节微管组织中的功能作用。 特别是，我们将研究如何内在的无序子域PRC 1有助于MAP聚类。 我们发表的和初步的数据表明，PRC 1簇显着阻碍细丝滑动， C-末端非结构化结构域介导这种效应。我们将使用生物物理学和细胞生物学工具 以确定减少聚集对微管组织的影响。(3)确定复合物 运动和非运动MAP共同调节微管组织。我们将研究如何 Kif 4A/PRC 1复合物在微管滑动期间产生力，以及稳态重叠排列如何 产生维持纺锤体中间区完整性的阻力。总之，我们的发现应该会推动我们的 了解微米尺度的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 阐明了一个“代码”，定义了不同MAP的结构和生物化学如何引起细胞凋亡。 能进行机械工作的机器。由于拷贝数导致的微管网络组装错误 必需MAP的变异或突变与人类疾病有关。我们的研究将揭示 这些生物物理特性将网络故障与疾病状态联系起来，并可能导致针对这些疾病的治疗。 蛋白质或为评估疾病进展的诊断工具提供见解。