Project C

着丝粒是一个蛋白质复合物的网络，它在着丝粒染色质上组装，作为染色体和微管之间的连接点，微管将它们分离成子细胞。在分裂后期，动粒允许染色体追踪微管的解聚末端，微管是力产生的主要场所。因此，动粒不仅在微管末端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并在解聚过程中释放的能量来产生染色体向纺锤体极的进行性运动。在本提案中，我们 目的是通过检测一组作为微管附着位点的动粒蛋白复合物来探索定向染色体运动的分子机制。我们的方法是结合联合收割机活性测定，电子显微镜和图像分析，和生物信息学方法，在全功能复合物的表征。我们正在研究酵母特异性DAM1复合物，它以微管依赖性的方式组装成环状结构，能够在微管表面扩散并跟踪解聚末端。我们还表征了保守的NDC 80复合物，以及酵母中完整的KMN网络，其中还包括MTW 1复合物和Spc 105。我们的最终目标是获得一个机制的理解的动态连接的动粒微管的分子间的相互作用。我们的结构模型将与我们的合作者使用强大的酵母系统进行进一步测试。

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.