Mechanisms of Tubulin dimer Regulatory Pathways and their impact on Microtubule Function

微管蛋白二聚体调控途径的机制及其对微管功能的影响

• 批准号: 10219718
• 负责人: Jawdat MH Al-Bassam
• 金额: $ 1.72万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10219718
• 关键词: Address; Binding; Biochemical; Biogenesis; Biological Assay; Biophysics; Catalysis; Cells; Chromosome Segregation; Complex; Cryoelectron Microscopy; Cytoplasm; Cytoskeleton; Data; Defect; Development; Dimerization; Docking; Elements; Encephalopathies; Enzymes; Eukaryotic Cell; Fission Yeast; Fluorescence Microscopy; Funding; GTP-Binding Proteins; Genes; Growth; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Hereditary Disease; Heterodimerization; Human; Image; Image Analysis; Impairment; In Vitro; Individual; Inherited; Kenny-Caffey syndrome; Lead; Life Cycle Stages; Link; Malignant Neoplasms; Mediating; Methods; Microtubule Polymerization; Microtubules; Modeling; Molecular; Molecular Conformation; Mutation; Orthologous Gene; Pathway interactions; Patients; Physiological; Plus End of the Microtubule; Polymerase; Polymers; Process; Property; Proteins; Public Health; Publishing; Quality Control; Reagent; Regulation; Regulatory Pathway; Resolution; Role; Shapes; Source; Structure; System; Testing; Tubulin; Work; Yeasts; alpha Tubulin; base; beta Tubulin; cofactor; design; developmental disease; dimer; giant axonal neuropathy; imaging approach; in vitro Assay; in vivo; in vivo imaging; lissencephaly; live cell imaging; mutant; neoplastic cell; nervous system disorder; overexpression; particle; polymerization; reconstitution; recruit; self assembly; stem; tool; trafficking; tumor; tumor growth

Project Summary The dynamic microtubule cytoskeleton mediates intracellular organization, generates forces in dividing or migrating eukaryotic cells, and forms tracks for intracellular trafficking. The fundamental properties of microtubules, including polarized growth and “dynamic instability”, stem directly from the activities of the microtubule building blocks, the α- and β-tubulin heterodimers. Three conserved tubulin cofactors and dedicated Arf-like 2 G-protein form multi-subunit platforms for the biogenesis and degradation of αβ-tubulin dimer, leading to a high concentration within the cytoplasm. The mechanisms for these assemblies remain mostly mysterious, due in part to a lack of structural information. In addition, we do not understand how conserved microtubule polymerases with arrays of Tumor Overexpressed Gene (TOG) domains recruit ab-tubulins and accelerate their incorporation while tracking dynamic microtubule ends. Understanding these cellular pathways is critical since genetic defects that impair either soluble ab-tubulin biogenesis or microtubule polymerases are linked to inherited neurological and developmental disorders and are observed in human cancers, respectively. This proposal explores the biochemical and physical mechanisms of ab-tubulin biogenesis and microtubule polymerase assemblies and their impact on microtubule function. Our strategy combines methods across multiple resolution scales, including in vitro reconstitution of purified protein assemblies, structural studies by cryo- electron microscopy (cryo-EM), reconstitution of assemblies with microtubule dynamics using in vitro fluorescence microscopy-based assays, and in vivo live imaging with microtubules within living cells. First, we will determine structural transitions describing ab-tubulin biogenesis assemblies and their functional impact of αβ-tubulin biogenesis and degradation. During the previous period, we established reconstitution system for these assemblies with ab-tubulin and describe cryo-EM structural studies leading to medium resolution structures in complex with ab-tubulins. 1) We will determine structural states for the ab-tubulin biogenesis assemblies in multiple biochemical states using high-resolution cryo-EM to understand how these assemblies catalyze dimerization of ab-tubulin and its degradation. 2) We will dissect functional roles of structural elements and interactions within current structures to determine their role in the ab-tubulin biogenesis process using in vitro and in vivo methods. Second, we will examine the mechanisms of microtubule polymerases with arrays of TOG domains their regulatory mechanisms. In the previous period, we describe a new model for ab-tubulin recruitment and polymerization by TOG domain arrays as microtubule polymerases, developed based on our structural and biochemical studies. We validated this model using in vitro reconstitution and in vivo live imaging of structure-based designer defective mutants, revealing that the ab-tubulin accelerating and processive plus-end tracking activities originate from unique features in TOG domain arrays. 1) We will study mechanisms of super-complexes of microtubule polymerase in complex with their activators, the transforming acidic coiled-coil proteins, in by using well-explored structural, in vitro reconstitution and in vivo live imaging strategies. 2) Determine the structural and functional relevance of our new model to mammalian microtubule polymerases with their unique pentameric TOG domain array arrangement using cryo-EM structural studies, in vitro reconstitution of designer mutants, and in vivo imaging approaches of structure-based mutants. We expect these studies to yield new structural and biophysical data, which will refine our new models will deepen our understanding of soluble ab-tubulin biogenesis, recruitment and incorporation during microtubule polymerization. This understanding will in turn point toward new strategies for addressing defects in tubulin biogenesis and regulation, potentially impacting patients with a range of developmental and neurological disorders.

项目摘要 动态的微管细胞骨架介导细胞内的组织，产生分裂或迁移的力量 真核细胞，并形成细胞内运输的轨迹。微管的基本特性，包括 极化生长和动态不稳定，直接源于微管构建块的活动，α- 和β-微管蛋白杂二聚体。三种保守的微管蛋白辅助因子和特异的Arf样2G蛋白形成多亚基 αβ-微管蛋白二聚体的生物发生和降解平台，导致细胞质内高浓度。 这些集会的机制大多仍是个谜，部分原因是缺乏结构性信息。在……里面 此外，我们不了解微管聚合酶与肿瘤过表达基因阵列如何保守 (TOG)结构域在跟踪动态微管末端的同时招募ab-微管蛋白并加速其并入。 了解这些细胞途径是至关重要的，因为基因缺陷会损害可溶性ab-微管蛋白的生物发生或 微管聚合酶与遗传性神经和发育障碍有关，并在人类中观察到 分别是癌症。本方案探讨了ab-微管蛋白的生物发生和物理机制 微管聚合酶组件及其对微管功能的影响。我们的战略将各种方法结合起来 多分辨率尺度，包括纯化蛋白组件的体外重组，冷冻-凝固法的结构研究。 电子显微镜(冷冻-EM)，利用体外荧光重建具有微管动力学的组件 基于显微镜的分析，以及活细胞内微管的活体成像。 首先，我们将确定描述ab-微管蛋白生物发生组装的结构转变及其对 αβ-微管蛋白的生物发生和降解。在之前的一段时间里，我们为这些人建立了重建制度 具有ab-微管蛋白的组装体，并描述了导致复合体中中等分辨率结构的冷冻-EM结构研究 含ab-微管蛋白。1)我们将在多种生物化学中确定ab-微管蛋白生物发生组件的结构状态 使用高分辨率低温电子显微镜了解这些组件如何催化ab-微管蛋白及其 退化。2)我们将剖析结构元素的功能作用和当前结构中的相互作用，以 使用体外和体内方法确定它们在ab-微管蛋白生物发生过程中的作用。第二，我们将研究 具有TOG结构域阵列的微管聚合酶的机制及其调节机制。在上一次 期间，我们描述了一种利用TOG结构域阵列作为微管的ab-微管蛋白募集和聚合的新模型 聚合酶，基于我们的结构和生化研究而开发。我们使用体外实验验证了这一模型 基于结构的设计缺陷突变体的重组和活体成像，揭示了ab-微管蛋白 加速和过程性正端跟踪活动源自TOG域阵列的独特功能。1)我们会 微管聚合酶与其激活剂形成的超复合体及其转化机制的研究 酸性螺旋卷曲蛋白，通过使用已探索好的结构、体外重建和活体成像策略。 2)确定我们的新模型与哺乳动物微管聚合酶的结构和功能相关性 独特的五聚体TOG结构域阵列的冷冻-EM结构研究，体外重建设计师 突变体，以及基于结构的突变体的体内成像方法。我们预计这些研究将产生新的结构和 生物物理数据，这些数据将完善我们的新模型，将加深我们对可溶性ab-微管蛋白生物发生的理解， 微管聚合过程中的募集和整合。这一理解将反过来指向新的 解决微管蛋白生物发生和调节缺陷的策略，可能影响患者的一系列 发育和神经紊乱。