Dam1 Kinetochore Complex and Dynamic Microtubules

描述（由申请人提供）：有丝分裂期间的染色体运动与微管（MT）丝的解聚和生长有关，微管丝的尖端将张力传递到每条染色体上的专门位点，称为动粒。我们的总体目标是了解动粒如何利用MT组装和拆卸来组织和分离细胞分裂过程中的染色体。在这里，我们专注于Dam 1复合物，在酵母中的动粒的一个重要组成部分。最近的研究表明，Dam 1复合体可能通过形成一个环绕MT的环，直接促进运动舞蹈MT附着、力的产生和附着MT的调节。为了检验这些假设，我们开发了一种体外（无细胞）运动试验，其中Dam 1包被的珠粒附着在单个动态MT的尖端。像动粒一样，珠子保持尖端束缚并经历组装和拆卸驱动的运动。使用纯化的蛋白质的这种运动重建已经是一个新的结果，支持Dam 1复合物在激动素-MT附着中的直接作用。此外，它使我们能够应用先进的光学捕获技术来定量评估复合物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Dam 1复合体是否可以形成动态MT尖端的承重附件，（2）确定基于Dam 1的运动性是否依赖于环绕MT的结构，（3）确定MT尖端的卷曲原丝是否物理地推动Dam 1以驱动其运动，以及（4）确定Dam 1是否以张力依赖的方式改变MT动力学。这项工作将提供深入了解的机制，其中动粒和其他尖端附件结构利用MT的增长和缩短，以产生推动力和拉力来移动细胞器。阐明这些动粒功能的分子基础对于理解癌症进展至关重要，因为癌症中经常发生的染色体丢失可能是由于削弱动粒-MT附着的突变引起的。正在开发有前途的新化疗药物，以靶向有丝分裂机制的组成部分，这些努力将大大受益于特定动粒蛋白的作用和机制的更完整的知识。

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.