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: 9130191
• 负责人: Ekaterina L Grishchuk
• 金额: $ 30.4万
• 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/9130191
• 关键词: Aneuploidy; Animals; 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; Glass; Goals; Health; Homologous Gene; Human; In Vitro; Kinetochores; Label; Lateral; Malignant Neoplasms; Mammalian Cell; Measures; Mechanics; 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; biophysical techniques; chromosome loss; chromosome movement; crosslink; daughter cell; depolymerization; dimer; genetic information; human disease; improved; innovation; laser tweezer; mathematical model; molecular modeling; nanoscale; nanostructured; novel anticancer drug; 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）：将动粒微管动力学与染色体运动耦合 摘要：在细胞分裂过程中，染色体必须均匀分离，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（特殊的染色体结构）之间的力传导相互作用：当微管缩短时，染色体被运输到分裂细胞的相反两极。动粒之间适当连接的丧失和微管缩短会导致染色体丢失，并且是非整倍性的最重要原因之一。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，以及这些附着物如何在负载下保持稳定。在体外，解聚微管可以移动与其缩短端适当耦合的物体。类似的机制可能在极向染色体运动中发挥核心作用。为了以定量和机制的方式研究这些过程，我们开发了生物物理和单分子方法，在模拟细胞中正常动粒-微管附着的条件下，在体外剖析分离的动粒蛋白和动态微管之间的相互作用。通过使用具有耐光正端盖的分段聚合物，我们可以以高度受控的方式触发解聚，从而能够详细分析与拆卸相关的力。通过这些方法，我们试图了解由人类必需的 Ska1 复合体（芽殖酵母 Dam1 的假定功能同源物）进行的微管依赖性偶联的分子机制。我们的具体目标集中于确定 Ska1 寡聚化在组装微管尖端跟踪结构中的作用，并表征其随着缩短末端不断移动的能力（目标 1）。我们将仔细研究 Ska1 如何捕获微管解聚的能量，并将其效率与 Dam1 环的效率进行比较（目标 2）。为了确定纯化的 Ska1 如何保持与缩短聚合物的稳定附着，我们将使用纯化的 Ska1 将微管与玻璃微球耦合，并检查它们在激光镊子施加的负载下的运动（目标 3）。这种方法具有创新性，因为它将复杂的生物物理方法集中在特定的耦合动粒复合体上，这对于遗传信息的准确遗传至关重要。这项研究很重要，因为它将促进生物力学特征和特定蛋白质模块的识别，这些特征和特定蛋白质模块负责着丝粒沿着微管壁滑动的能力，承受反作用力，并对分裂哺乳动物细胞的嘈杂和随机环境中的适应不良条件做出反应。最终，这项工作将有助于对癌症等人类疾病的分析，在这些疾病中，准确的染色体分离失败。