Dam1 Kinetochore Complex and Dynamic Microtubules

Dam1 动粒复合体和动态微管

• 批准号: 7923677
• 负责人: CHARLES ASBURY
• 金额: $ 26.6万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7923677
• 关键词: Affect; Binding; Binding Proteins; Biological Assay; Cell division; Cells; Chromosomes; Complex; Coupling; Diffusion; Disease; Enzymes; Filament; Goals; Growth; In Vitro; Individual; Kinetochores; Knowledge; Lead; Link; Location; Malignant Neoplasms; Mediating; Methods; Microtubules; Mitosis; Mitotic; Modeling; Molecular; Movement; Mutation; Organelles; Production; Property; Proteins; Recombinants; Regulation; Relative (related person); Research Personnel; Role; Site; Slide; Structure; Techniques; Testing; Time; Weight-Bearing state; Work; Yeasts; base; cell motility; chromosome loss; chromosome movement; depolymerization; in vivo; insight; novel; optical traps; programs; reconstitution; tumor progression

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.

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称为动粒的特殊位点。我们的总体目标是了解动粒如何在细胞分裂过程中利用 MT 组装和拆卸来组织和分离染色体。我们在这里重点关注 Dam1 复合体，它是酵母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围绕 MT 的环，直接有助于动粒-MT 附着、力产生和附着 MT 的调节。为了测试这些假设，我们开发了一种体外（无细胞）运动测定，其中 Dam 1 包被的珠子附着在单个动态 MT 的尖端。与动粒一样，珠子保持尖端结合并经历组装和拆卸驱动的运动。这种使用纯化蛋白重建运动已经是一个新的结果，支持 Dam1 复合物在动粒-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，并测试环假设的关键预测。我们建议（1）确定Dam1复合体是否可以形成动态MT尖端的承载附件，（2）确定基于Dam 1的运动性是否取决于围绕MT的结构，（3）确定MT尖端的卷曲原丝是否物理地推动Dam1以驱动其运动，以及（4）确定Dam1是否以张力依赖的方式改变MT动力学。这项工作将深入了解动粒和其他尖端附着结构利用 MT 生长和缩短来产生推力和拉力来移动细胞器的机制。阐明这些着丝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削弱着丝粒-MT附着的突变引起的。正在开发有前景的新化疗药物来靶向有丝分裂机制的成分，这些努力将从对特定着丝粒蛋白的作用和机制的更完整的了解中受益匪浅。