A mechanistic study of XMAP215-mediated microtubule polymerization

 描述（由申请人提供）：微管（MT）对于维持细胞结构、形成双极有丝分裂纺锤体和促进细胞内转运/细胞运动至关重要。微管-微管蛋白异二聚体是MT的结构单元。MT末端的微管蛋白的GTP水解依赖性增益和损失是MT动力学的基础。虽然MT在体外是固有动态的，但MT相关蛋白，包括XMAP 215家族，调节细胞中的MT动态，以促进基于MT的复杂动态结构如有丝分裂纺锤体的形成。XMAP 215家族成员使用一系列五个微管蛋白结合肿瘤过表达基因（TOG）结构域来增加MT聚合速率。已经提出了XMAP 215家族的TOG依赖性MT聚合机制的多种模型。环绕模型表明XMAP 215 TOG结构域与微管蛋白以1：1或1：2的XMAP 215：微管蛋白复合物相互作用。相比之下，模板模型提出XMAP 215形成长的线性结构，其促进在MT plu末端添加三个或更多个微管蛋白异二聚体。五聚体TOG结构域阵列的结构分析表明，TOG结构域具有差异的架构。具体地，N-末端TOG结构域在结构上相似，而C-末端TOG结构域具有独特的、不同的结构特征，其在结构上预测接合横向缔合的微管蛋白异二聚体的能力，并且可以 是它们各自在体外促进MT聚合的能力的基础。我假设XMAP 215 TOG结构域模板MT使用结构上不同的功能调整到绑定自由，假聚合，或晶格纳入微管蛋白，这使得他们能够共同促进MT聚合和驱动正确的有丝分裂纺锤体形成。我将测试这一假设（1）生物化学确定TOG结构域如何与不同形式的微管蛋白相互作用，这将有助于阐明XMAP 215：微管蛋白结合化学计量和（2）确定特定的TOG结构域如何有助于XMAP 215依赖的MT聚合活性和细胞培养中的有丝分裂纺锤体结构。总之，这些目标将使我们能够完善XMAP 215如何与微管蛋白相互作用以加速MT聚合并维持双极有丝分裂纺锤体结构的现有模型。

 DESCRIPTION (provided by applicant): Microtubules (MTs) are essential for maintaining cell structure, forming the bipolar mitotic spindle, and facilitating intracellular transport/cellular movement. The -tubulin heterodimer is the building block of MTs. The GTP-hydrolysis dependent gain and loss of -tubulin at MT ends underlies MTs dynamics. While MTs are inherently dynamic in vitro, MT associated proteins, including the XMAP215 family, regulate MT dynamics in cells to facilitate the formation of complex dynamic MT-based structures such as the mitotic spindle. XMAP215 family members use an array of five tubulin-binding tumor overexpressed gene (TOG) domains to increase MT polymerization rates. Multiple models have been proposed for the XMAP215 family's TOG- dependent MT polymerization mechanism. A wrap around model suggests XMAP215 TOG domains interact with tubulin in a 1:1 or 1:2 XMAP215:tubulin complex. In contrast, a templating model proposes that XMAP215 forms a long linear structure that facilitates the addition of three or more tubulin heterodimers at MT plu ends. Structural analysis of pentameric TOG domain arrays suggests that TOG domains have differential architectures. Specifically, the N-terminal TOG domains are structurally similar, whereas the C-terminal TOG domains have unique, divergent architectural features that structurally predict an ability to engage laterally- associated -tubulin heterodimers and may underlie their respective abilities to promote MT polymerization in vitro. I hypothesize that XMAP215 TOG domains template MTs using structurally distinct features tuned to bind free, pseudo-polymerized, or lattice-incorporated tubulin, which allows them to collectively promote MT polymerization and drive proper mitotic spindle formation. I will test this hypothesis by (1) biochemically determining how TOG domains interact with different forms of tubulin, which will help elucidate XMAP215:tubulin binding stoichiometry and (2) determining how specific TOG domains contribute to XMAP215-dependent MT polymerization activity and mitotic spindle structure in cell culture. Together, these aims will allows us to refine the current models of how XMAP215 mechanistically interacts with tubulin to accelerate MT polymerization and maintain bipolar mitotic spindle structure.