Coupling kinetochore microtubule dynamics to chromosome motion

将动粒微管动力学与染色体运动耦合

• 批准号: 8545869
• 负责人: Ekaterina L Grishchuk
• 金额: $ 29.34万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8545869
• 关键词: Aneuploidy; Animals; Antineoplastic Agents; Biological Assay; Biological Process; Biomechanics; Cell Proliferation; Cell division; Cells; Chromosome Segregation; Chromosome Structures; Chromosomes; Complex; Coupled; Coupling; Data; Development; Ensure; Environment; Figs - dietary; Fostering; Genes; Genetic; Glass; Goals; Health; Homologous Gene; Human; In Vitro; Kinetochores; Label; Lateral; Malignant Neoplasms; Mammalian Cell; Measures; Mechanics; Methodology; Methods; Microspheres; Microtubule Depolymerization; Microtubules; Mission; Mitotic Chromosome; Modeling; Molecular; Molecular Models; Motion; Myosin Rod; Nanostructures; Outcome; Play; Polymers; Process; Property; Protein Dynamics; Proteins; Public Health; Reproduction; Research; Role; Rotation; Saccharomycetales; Slide; Structure; Testing; Work; Yeasts; abstracting; base; chromosome loss; chromosome movement; crosslink; daughter cell; depolymerization; dimer; human disease; improved; innovation; laser tweezer; mathematical model; molecular modeling; nanoscale; nanostructured; novel; prevent; segregation; single molecule

DESCRIPTION (provided by applicant): Coupling kinetochore microtubule dynamics to chromosome motion Abstract: During cell division chromosomes must segregate equally to ensure the health and viability of the daughter cells. It is now well established that accurate chromosome segregation crucially depends on the force- transducing interactions between thread-like polymers (microtubules), and kinetochores, specialized chromosomal structures: when microtubules shorten, the chromosomes are transported to the opposite poles of a dividing cell. Loss of the proper connections between the kinetochores and shortening microtubules leads to a chromosome loss, and is one of the most significant causes of aneuploidy. However, the molecular mechanisms that ensure the stability of these dynamic connections are not known. Our long-term goal is to understand the fundamental biological functions: how the kinetochores of mitotic chromosomes are coupled to the dynamic microtubules ends and how these attachments remain stable under the load. In vitro, depolymerizing microtubules can move objects that are appropriately coupled to their shortening ends. Similar mechanisms are likely to play central role in the pole-directed chromosome movement. To study these processes in the quantitative and mechanistic way we have developed biophysical and single-molecule methods to dissect the interactions between isolated kinetochore proteins and dynamic microtubules in vitro under conditions that mimic aspects of normal kinetochore-microtubule attachments in cells. By using segmented polymers with photoliable plus-end caps we can trigger depolymerization in a highly controlled manner, which enables detailed analysis of disassembly-dependent forces. With these methods, here we seek to understand the molecular mechanisms of the microtubule-dependent coupling carried out by the essential human Ska1- complex, a presumptive functional homolog of the budding yeast Dam1. Our Specific Aims are focused on determining the role of Ska1 oligomerization in assembling the microtubule tip-tracking structures and characterizing their ability to move processively with the shortening ends (Aim 1). We will critically examine how Ska1 captures the energy of microtubule depolymerization, and compare its efficiency with that of the Dam1 ring (Aim 2). To determine how purified Ska1 maintains stable attachment to the shortening polymer, we will use purified Ska1 to couple microtubules to glass microspheres, and examine their motions under a load applied with laser tweezers (Aim 3). This approach is innovative because it focuses sophisticated biophysical methodologies on specific coupling kinetochore complexes, which are essential for accurate inheritance of genetic information. This research is important because it will promote identification of the biomechanical features and specific protein modules that are responsible for a kinetochore's ability to slide along microtubule wall, to withstand counter-forces and to respond to maladaptive conditions in a noisy and stochastic environment of a dividing mammalian cell. Ultimately, this work will facilitate analysis of human diseases, such as cancer, in which accurate chromosome segregation fails.

描述（由申请人提供）：偶联动粒微管动力学染色体运动摘要：在细胞分裂染色体必须隔离平等，以确保子细胞的健康和活力。现在已经确定，准确的染色体分离关键取决于线状聚合物（微管）和动粒（特定的染色体结构）之间的力传递相互作用：当微管缩短时，染色体被运输到分裂细胞的两极。着丝粒和微管之间的正确连接的丢失导致染色体丢失，并且是非整倍体的最重要原因之一。然而，确保这些动态连接稳定性的分子机制尚不清楚。我们的长期目标是了解基本的生物学功能：有丝分裂染色体的动粒如何与动态微管末端偶联，以及这些附件如何在负载下保持稳定。在体外，解聚的微管可以移动与其缩短末端适当偶联的物体。类似的机制可能在极点定向染色体运动中发挥核心作用。为了研究这些过程中的定量和机械的方式，我们已经开发了生物物理和单分子的方法来解剖分离的动粒蛋白和动态微管在体外的条件下，模仿正常的动粒微管附件的方面在细胞中的相互作用。通过使用具有可光致的正末端帽的嵌段聚合物，我们可以以高度受控的方式触发解聚，这使得能够详细分析依赖于解聚的力。通过这些方法，在这里，我们试图了解微管依赖性耦合进行的基本人类Ska1复合物，一个假定的功能同源物的芽殖酵母Dam1的分子机制。我们的具体目标是集中在确定Ska 1寡聚化在组装微管尖端跟踪结构中的作用，并表征其与缩短末端一起移动proceptide的能力（目标1）。我们将严格检查Ska1如何捕获微管解聚的能量，并将其效率与Dam1环（Aim 2）进行比较。为了确定纯化的Ska1如何保持与缩短聚合物的稳定连接，我们将使用纯化的Ska1将微管偶联到玻璃微球上，并检查它们在激光镊子施加的载荷下的运动（目的3）。这种方法是创新的，因为它专注于特定的耦合动粒复合体，这是遗传信息的准确继承所必需的复杂的生物物理方法。这项研究是重要的，因为它将促进识别的生物力学特征和特定的蛋白质模块，负责动粒的能力，沿沿着微管壁滑动，承受反作用力，并在一个嘈杂的和随机的环境中的哺乳动物细胞的适应不良的条件下分裂。最终，这项工作将有助于分析人类疾病，如癌症，其中精确的染色体分离失败。