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）：有丝分裂过程中的染色体运动与微管（MT）细丝的解聚和生长有关，其尖端将张力传递到每个染色体上的专用部位，称为动脉化合物。我们的总体目标是了解动力学如何在细胞分裂过程中组织和分离染色体，并拆卸MT组件和拆卸。我们在这里专注于DAM1复合物，这是酵母中动力学的重要组成部分。最近的工作表明，DAM1复合物直接有助于动力学MT的附着，力产生和附着MT的调节，也许是通过形成环绕MT的环。为了检验这些假设，我们开发了一个体外（细胞）运动性测定法，其中大坝1涂层的珠子附着在单个动态MT的尖端上。像动力学一样，珠子保持尖端结合，并进行组装和拆卸驱动的运动。这种使用纯化蛋白质的运动重建已经是一个新的结果，它支持了dAM1复合物在动型-MT附着中的直接作用。此外，它使我们能够应用高级光学诱捕技术来定量评估复合物有助于体内附着和强制产生的潜力，并测试对环假说的关键预测。我们建议（1）确定DAM1复合物是否可以形成负载的载荷附件，（2）确定基于DAM 1的运动性是否取决于包围MT的结构，（3）确定在MT上卷曲的原丝tip在物理上推向DAM1的curling protine prock first fally thragical table tailly dam tailters to dam a dam a dam a dam a andters MT Dynamics in tensions insensens依赖性依赖性依赖性依赖性。这项工作将洞悉动力学和其他尖端跟踪结构的机制，并缩短生长并缩短以产生推动力和拉力以移动细胞器。阐明这些动力学功能的分子基础对于理解癌症的进展至关重要，因为染色体损失经常在癌症中发生，可能是由于弱化的动型-MT附着的突变而引起的。正在开发有希望的新化学治疗剂来靶向有丝分裂机械的组成部分，这些努力将从更完整的知识中受益于特定的动型蛋白的作用和机制。