Project C

着丝粒是一个蛋白质复合体网络，它聚集在着丝粒染色质上，充当染色体和将染色体分离成子细胞的微管之间的连接点。在后期，动点允许染色体跟踪微管的解聚端，这是力产生的主要位置。因此，动点不仅在分解时不会释放微管末端，而且能够利用储存在微管晶格中的能量，并在解聚过程中释放出来，产生染色体到纺锤体极的过程运动。在这份提案中，我们 目的通过检测微管附着部位的一组动粒蛋白复合体，探讨染色体定向运动的分子机制。我们的方法是结合活性分析，电子显微镜和图像分析，以及生物信息学方法，来表征全功能配合物。我们正在研究酵母特异的DAM1复合体，它以微管依赖的方式组装成能够在微管表面扩散并跟踪解聚末端的环结构。我们还鉴定了酵母中保守的NDC80复合体和完整的KMN网络，其中还包括MTW1复合体和Spc105。我们的最终目标是从机制上理解控制着动点与微管的动态连接的分子相互作用。我们的结构模型将使用强大的酵母系统与我们的合作者进行进一步的测试。

The kinetochore is a network of protein complexes that assembles on centromeric chromatin to act as the connection point between chromosomes and the microtubules that segregate them into daughter cells. During anaphase kinetochores allow chromosomes to track the depolymerizing ends of microtubules, which are the primary site of force generation. Thus, kinetochores not only do not let go of microtubule ends as they breakdown, but are able to harness the energy stored in the microtubule lattice and released during depolymerization to produce processive movement of chromosomes to the spindle poles. In this proposal we aim to probe the molecular mechanisms of directed chromosome motion by examining a set of kinetochore protein complexes that are the site of microtubule attachment. Our approach is to combine activity assays, electron microscopy and image analysis, and bioinformatic methods, in the characterization of fully functional complexes. We are studying the yeast-specific DAM1 complex, which assembles in a microtubule-dependent manner into a ring structure that is able to diffuse on the microtubule surface and to track depolymerizing ends. We are also characterizing the conserved NDC80 complex, and the complete KMN network in yeast, which includes also the MTW1 complex and Spc105. Our ultimate objective is to gain a mechanistic understanding of the molecular interactions governing the dynamic attachment of kinetochores to microtubules. Our structural models would be further tested with our collaborators using the powerful yeast system.