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

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、α-和 β-微管蛋白杂二聚体。三个保守的微管蛋白辅助因子和专用的Arf样2G蛋白形成多亚单位平台 对于αβ-微管蛋白二聚体的生物发生和降解，导致细胞质内的高浓度。这个 这些组装的机制大多仍是个谜，部分原因是缺乏结构性信息。此外，我们 不了解微管聚合酶与肿瘤过表达基因(TOG)结构域阵列的保守性 招募αβ-微管蛋白并加速其结合，同时跟踪动态微管末端。了解这些 细胞通路至关重要，因为遗传缺陷会损害可溶性αβ-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，并在人类癌症中观察到， 分别进行了分析。本研究探讨了αβ-微管蛋白生物发生和微管形成的生化和物理机制 聚合酶组件及其对微管功能的影响。我们的策略结合了跨多个分辨率的方法 规模，包括纯化蛋白组件的体外重组，冷冻电子显微镜(冷冻-电子显微镜)的结构研究。 EM)，使用基于体外荧光显微镜的分析方法重建具有微管动力学的组件，以及在 活细胞内的微管活体成像。 首先，我们将确定描述αβ-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αβ-微管蛋白的组装体，并描述了导致复合体中中分辨率结构的冷冻-EM结构研究 含有αβ-微管蛋白。1)我们将确定αβ-微管蛋白生物发生组件在多种生化中的结构状态 使用高分辨率低温电子显微镜了解这些组件如何催化αβ-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，以确定 使用体外和体内方法研究它们在αβ-微管蛋白生物发生过程中的作用。第二，我们将研究这些机制 具有TOG结构域阵列的微管聚合酶及其调节机制。在前一段时间里，我们描述了 一种利用TOG域阵列作为微管聚合酶的αβ-微管蛋白募集和聚合新模型 基于我们的结构和生化研究。我们通过体外重建和活体实验验证了这一模型。 基于结构的设计缺陷突变体的成像，揭示了αβ-微管蛋白加速和进行性正端 跟踪活动源自TOG域阵列中的独特功能。1)我们将研究超络合物的作用机理 微管聚合酶与其激活剂的复合体，转化酸性卷曲蛋白，通过使用井- 探索了结构重建、体外重建和活体成像的策略。2)确定结构和功能 我们的新模型与哺乳动物微管聚合酶及其独特的五聚体TOG结构域阵列的相关性 利用冷冻-EM结构研究、设计突变体的体外重组和活体成像方法进行排列 基于结构的突变体。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αβ-微管蛋白在微管中的生物发生、募集和掺入的理解 聚合反应。这一理解将反过来指向解决微管蛋白生物发生和 监管，可能会影响患有一系列发育和神经障碍的患者。

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 αβ-tubulins and accelerate their incorporation while tracking dynamic microtubule ends. Understanding these cellular pathways is critical since genetic defects that impair either soluble αβ-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 αβ-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 αβ-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 αβ-tubulin and describe cryo-EM structural studies leading to medium resolution structures in complex with αβ-tubulins. 1) We will determine structural states for the αβ-tubulin biogenesis assemblies in multiple biochemical states using high-resolution cryo-EM to understand how these assemblies catalyze dimerization of αβ-tubulin and its degradation. 2) We will dissect functional roles of structural elements and interactions within current structures to determine their role in the αβ-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 αβ-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 αβ-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 αβ-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.