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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

基本信息

批准号：
10625195
负责人：
Jawdat MH Al-Bassam
金额：
$ 15.1万
依托单位：
UNIVERSITY OF CALIFORNIA AT DAVIS
依托单位国家：
美国
项目类别：
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/10625195
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 Homology Modeling 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

项目摘要

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.

项目摘要动态微管细胞骨架介导细胞内组织，产生分裂或分裂的力，迁移真核细胞，并形成细胞内运输的轨道。的基本性质微管，包括极化生长和“动态不稳定性”，直接源于细胞的活动。微管构建块，α-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αβ微管蛋白生物合成和降解的多亚基平台二聚体，导致细胞质内的高浓度。这些集会的机制仍然是大多是神秘的，部分原因是缺乏结构信息。此外，我们不明白如何具有肿瘤过表达基因（TOG）结构域阵列的保守微管聚合酶招募ab- 微管蛋白，并加速它们的掺入，同时跟踪动态微管末端。了解这些细胞途径是至关重要的，因为遗传缺陷损害可溶性AB-微管蛋白的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。这项提案探讨了ab-微管蛋白的生化和物理机制生物发生和微管聚合酶组装及其对微管功能的影响。我们的战略结合了多种分辨率尺度的方法，包括纯化蛋白质的体外重建组件，低温电子显微镜（cryo-EM）结构研究，微管动力学使用体外荧光显微镜为基础的测定，并在体内活成像与活细胞内的微管。首先，我们将确定描述ab-微管蛋白生物发生组装体及其功能的结构转变， αβ-微管蛋白生物发生和降解影响。在前一时期，我们建立了重建系统，并描述了导致介质的冷冻-EM结构研究与ab-微管蛋白复合的分辨率结构。1)我们将确定ab-微管蛋白的结构状态使用高分辨率cryo-EM在多个生化状态下的生物发生组件，以了解这些组件是如何组装体催化AB-微管蛋白的二聚化及其降解。2)我们将剖析当前结构内的结构元件和相互作用，以确定它们在ab-tubulin中的作用使用体外和体内方法的生物发生过程。第二，我们将研究具有TOG结构域阵列的微管聚合酶及其调节机制。上期所描述了通过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物化学研究开发。我们使用中验证了此模型基于结构的设计缺陷突变体的体外重建和体内活体成像，揭示了微管蛋白加速和进行性加末端跟踪活动起源于TOG的独特功能域数组1)我们将研究微管聚合酶超复合物与它们的激活剂，转化酸性卷曲螺旋蛋白，在体外通过使用充分探索的结构，重建和体内活体成像策略。2)确定结构和功能的相关性，具有独特五聚体TOG结构域阵列的哺乳动物微管聚合酶的新模型使用冷冻EM结构研究、设计突变体的体外重建和体内成像进行排列基于结构的突变体的方法。我们希望这些研究能产生新的结构和生物物理数据，这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、募集和微管聚合过程中的掺入。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，可能影响患有一系列发育和神经系统疾病。