A mechanistic study of XMAP215-mediated microtubule polymerization

 描述(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运输/细胞运动是必不可少的。-微管蛋白异源二聚体是MTS的构建单元。MT末端-微管蛋白依赖于GTP水解酶的得失是MTS动力学的基础。虽然MT在体外天生是动态的，但MT相关蛋白，包括XMAP215家族，调节细胞内MT的动态，以促进复杂的动态MT结构的形成，如有丝分裂纺锤体。XMAP215家族成员使用五个微管蛋白结合肿瘤过表达基因(TOG)结构域来提高MT聚合率。对于XMAP215家族依赖TOG的MT聚合机制，已经提出了多种模型。卷绕模型表明，XMAP215 TOG结构域与微管蛋白以1：1或1：2的XMAP215：微管蛋白复合体相互作用。相反，模板模型认为XMAP215形成了一种长线性结构，有助于在MT Plu末端添加三个或更多微管蛋白异二聚体。对五聚体TOG结构域阵列的结构分析表明，TOG结构域具有不同的结构。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，该结构特征在结构上预测侧向结合的-微管蛋白异二聚体的能力，并且可以 它们各自在体外促进MT聚合的能力。我假设XMAP215 TOG结构域以MTS为模板，利用不同的结构特征进行调节，以结合自由、伪聚合或晶格结合的微管蛋白，这使它们能够共同促进MT聚合并驱动适当的有丝分裂纺锤体的形成。我将通过(1)生化确定TOG结构域如何与不同形式的微管蛋白相互作用来检验这一假设，这将有助于阐明XMAP215：微管蛋白结合化学计量学；(2)确定特定的TOG结构域如何影响细胞培养中依赖XMAP215的MT聚合活性和有丝分裂纺锤体结构。综上所述，这些目标将使我们能够改进目前的模型，了解XMAP215如何与微管蛋白机械相互作用，以加速MT聚合并保持两极有丝分裂纺锤体结构。

 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.