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: 10390213
• 负责人: Jawdat MH Al-Bassam
• 金额: $ 7.62万
• 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/10390213
• 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; 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 动态的微管骨架介导细胞内的组织，产生分裂或迁移的力 真核细胞，并形成用于细胞内运输的轨道。微管的基本特性，包括 极化生长和“动态不稳定性”，直接源于微管结构单元的活动， 和β-微管蛋白异二聚体。三个保守的微管蛋白辅因子和专门的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，导致细胞质内的高浓度。 这些组装的机制仍然是神秘的，部分原因是缺乏结构信息。在 此外，我们不了解保守的微管聚合酶如何与肿瘤过表达基因阵列结合， (TOG)结构域募集AB-微管蛋白并加速它们的结合，同时跟踪动态微管末端。 理解这些细胞通路是至关重要的，因为遗传缺陷损害可溶性ab-微管蛋白生物合成或 微管聚合酶与遗传性神经和发育障碍有关， 癌症，分别。该建议探讨了ab-微管蛋白生物合成的生化和物理机制， 微管聚合酶组装及其对微管功能的影响。我们的战略结合了各种方法， 多分辨率尺度，包括纯化蛋白组装体的体外重建，通过冷冻的结构研究， 电子显微镜（cryo-EM），使用体外荧光重建具有微管动力学的组件 基于显微镜的测定，以及活细胞内微管的体内活体成像。 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细胞增殖的功能影响。 αβ-微管蛋白的生物发生和降解。在上一个时期，我们为这些项目建立了重建系统， 与ab-微管蛋白的组装，并描述了导致复杂的中等分辨率结构的冷冻-EM结构研究。 与AB-微管蛋白。1)我们将在多种生化反应中确定ab-微管蛋白生物发生组装体的结构状态， 使用高分辨率cryo-EM来了解这些组件如何催化ab-微管蛋白及其 降解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， 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调节机制。上一 在此期间，我们描述了一个新的模型，ab-微管蛋白募集和聚合的TOG结构域阵列作为微管 聚合酶，基于我们的结构和生物化学研究开发。我们使用体外实验验证了该模型 基于结构的设计缺陷突变体的重建和体内活体成像，揭示了ab-微管蛋白 加速和进行性加端跟踪活动源自TOG域阵列中的独特特征。1)我们将 研究微管聚合酶与其激活剂复合的超复合物的机制， 酸性卷曲螺旋蛋白，在通过使用充分探索的结构，在体外重建和在体内活的成像策略。 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， 使用冷冻EM结构研究的独特五聚体TOG结构域阵列排列，设计者的体外重建 突变体和基于结构的突变体的体内成像方法。我们希望这些研究能够产生新的结构和 生物物理学数据，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， 在微管聚合过程中的募集和掺入。这种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，可能影响一系列患者， 发育和神经系统疾病。