Dam1 Kinetochore Complex and Dynamic Microtubules

描述(申请人提供)：有丝分裂期间的染色体运动与微管(MT)细丝的解聚和生长有关，微管细丝的尖端将张力传递到每条染色体上的特定位置，称为动点。我们的总体目标是了解动植物在细胞分裂过程中如何利用MT组装和拆解来组织和分离染色体。这里我们关注的是DAM1复合体，它是酵母中动粒的重要组成部分。最近的研究表明，DAM1复合体可能通过形成一个环绕MT的环，直接促进动粒-MT的附着、力的产生和对附着的MT的调节。为了验证这些假设，我们开发了一种体外(无细胞)运动试验，其中Dam 1包被的珠子附着在单个动态MT的尖端。像运动芯片一样，珠子保持尖端绑定，并经历组装和拆卸驱动的运动。使用纯化的蛋白质重建运动已经是一个新的结果，支持Dam1复合体在动粒-MT连接中的直接作用。此外，它还允许我们应用先进的光学捕获技术来定量评估该复合体在体内促进附着和力产生的潜力，并测试环假说的关键预测。我们建议(1)确定Dam1复合体是否可以形成对动态MT尖端的承重附着，(2)确定Dam 1的运动性是否依赖于围绕MT的结构，(3)确定MT尖端的卷曲原丝是否物理地推动Dam1来驱动其运动，以及(4)确定Dam1是否以一种张力依赖的方式改变MT的动力学。这项工作将深入了解动颈和其他顶端附着结构利用MT的生长和缩短来产生推动和拉力移动细胞器的机制。阐明这些动粒功能的分子基础对于了解癌症的进展是至关重要的，因为在癌症中经常发生的染色体丢失可能是由于削弱动粒-MT连接的突变造成的。有希望的新的化疗药物正在开发，以靶向有丝分裂机制的组件，这些努力将大大受益于对特定动粒蛋白的作用和机制的更全面的了解。

DESCRIPTION (provided by applicant): Chromosome movements during mitosis are linked to depolymerization and growth of microtubule (MT) filaments, the tips of which transmit tension to specialized sites on each chromosome, called kinetochores. Our overall goal is to understand how kinetochores harness MT assembly and disassembly to organize and separate chromosomes during cell division. We focus here on the Dam1 complex, an essential component of kinetochores in yeast. Recent work suggests the Dam1 complex contributes directly to kinetochore-MT attachment, force production, and regulation of attached MTs, perhaps by forming a ring encircling the MT. To test these hypotheses, we have developed an in vitro (cell free) motility assay where Dam 1-coated beads attach to the tips of individual dynamic MTs. Like kinetochores, the beads remain tip-bound and undergo assembly- and disassembly-driven movement. This reconstitution of movement using purified proteins is already a novel result supporting a direct role for the Dam1 complex in kinetochore-MT attachment. Moreover, it allows us to apply advanced optical trapping techniques to assess quantitatively the potential for the complex to contribute to attachment and force production in vivo, and to test key predictions of the ring hypothesis. We propose to (1) determine if the Dam1 complex can form load-bearing attachments to dynamic MT tips, (2) determine if Dam 1-based motility depends on a structure encircling the MT, (3) determine if curling protofilaments at the MT tip physically push against Dam1 to drive its movement, and (4) determine if Dam1 alters MT dynamics in a tension-dependent manner. This work will provide insight into the mechanisms by which kinetochores and other tip-attachment structures harness MT growth and shortening to produce pushing and pulling forces to move organelles. Elucidating the molecular basis for these kinetochore functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore-MT attachments. Promising new chemotherapeutics are being developed to target components of the mitotic machinery, and these efforts will benefit substantially from a more complete knowledge of the roles and mechanisms of specific kinetochore proteins.