Mechanisms of Tubulin Dimer Regulatory Pathways and Their Impact on Microtubule Function

项目概要 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迹。的基本属性 微管，包括极化生长和“动态不稳定性”，直接源于微管的活动 微管构件，α-和β-微管蛋白异二聚体。三个保守的微管蛋白辅因子和 专用 Arf 样 2 G 蛋白形成多亚基平台，用于 αβ-微管蛋白的生物发生和降解 二聚体，导致细胞质内高浓度。这些组件的机制仍然存在 大部分都很神秘，部分原因是缺乏结构信息。另外，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- 微管蛋白并加速其掺入，同时跟踪动态微管末端。了解这些 细胞途径至关重要，因为遗传缺陷会损害可溶性微管蛋白生物发生或微管 聚合酶与遗传性神经系统和发育障碍有关，并且在人类中观察到 分别是癌症。该提案探讨了 ab-tubulin 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。我们的策略 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、通过冷冻电子显微镜 (cryo-EM) 进行结构研究、用 使用基于体外荧光显微镜的测定法和体内实时成像进行微管动力学 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件及其功能的结构转变 αβ-微管蛋白生物发生和降解的影响。前期我们建立了重组 用于这些带有 ab-tubulin 的组件的系统，并描述了导致介质的冷冻电镜结构研究 与 ab-微管蛋白复合物的解析结构。 1) 我们将确定 ab-微管蛋白的结构状态 使用高分辨率冷冻电镜观察多种生化状态下的生物发生组件，以了解这些组件如何 组装体催化 ab-微管蛋白的二聚化及其降解。 2）我们将剖析以下功能角色： 结构元件和当前结构内的相互作用，以确定它们在 ab-微管蛋白中的作用 使用体外和体内方法的生物发生过程。其次，我们要考察其机制 具有 TOG 结构域阵列的微管聚合酶及其调节机制。前段时间，我们 描述了一种通过 TOG 域阵列作为微管进行 ab-微管蛋白招募和聚合的新模型 聚合酶，根据我们的结构和生化研究开发。我们使用以下方法验证了该模型 基于结构的设计师缺陷突变体的体外重建和体内实时成像，揭示了 ab-tubulin 加速和进行性加端追踪活动源自 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复合物的机制 他们的激活剂，转化酸性卷曲螺旋蛋白，通过使用充分探索的结构，在体外 重建和体内实时成像策略。 2）确定我们的结构和功能相关性 哺乳动物微管聚合酶的新模型及其独特的五聚体 TOG 结构域阵列 使用冷冻电镜结构研究、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。我们期望这些研究能够产生新的结构和生物物理数据， 这将完善我们的新模型，加深我们对可溶性微管蛋白生物发生、招募的理解 和微管聚合过程中的掺入。这种理解将反过来指向新的方向 解决微管蛋白生物合成和调节缺陷的策略，可能会影响患有微管蛋白的患者 一系列发育和神经系统疾病。

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.