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: 8293799
• 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/8293799
• 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. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because rigorous study of the dynamic linkages between spindle microtubules and chromosomal kinetochores will facilitate identification of the specific features of these force-transducing lins, which may be exploited for a selective disruption of kinetochore-microtubule interactions, thereby facilitating development of novel anti-cancer drugs. Thus, the proposed research is relevant to NIH's mission to foster fundamental creative discoveries that would ultimately advance our capacity to protect and improve human health.

描述（由申请人提供）：将动力学微管动力学耦合到染色体运动摘要：在细胞分裂期间，染色体必须平等地分离以确保子细胞的健康和生存能力。现在已经很好地确定，准确的染色体隔离至关重要地取决于螺纹样聚合物（微管）和动脉化合物之间的力转化相互作用，专门的染色体结构：当缩短微管时，染色体将染色体转运到分裂单元相反的电池。动力学和缩短微管之间正确连接的丧失会导致染色体损失，并且是非整倍性的最重要的原因之一。但是，确保这些动态连接的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力学如何耦合到动态微管结束，以及这些附件如何在负载下保持稳定。在体外，去聚合微管可以移动适当耦合到其缩短末端的物体。类似的机制可能在极点导向的染色体运动中起着核心作用。为了以定量和机械方式研究这些过程，我们开发了生物物理和单分子方法，以在模拟细胞中正常动物学细胞体 - 微动物附着的正常方面的条件下，在体外在体外剖析了分离的动力学蛋白与动态微管之间的相互作用。通过使用具有光亮末端盖的分段聚合物，我们可以以高度控制的方式触发去聚合，从而可以详细分析拆卸依赖性力。通过这些方法，我们在这里试图了解由基本人类SKA1-Complex进行的微管依赖性耦合的分子机制，这是萌芽的酵母菌DAM1的假定功能同源物。我们的具体目的是确定SKA1低聚在组装微管尖端跟踪结构中的作用，并表征其在缩短末端进行过程移动的能力（AIM 1）。我们将批判性地研究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（AI​​M 2）。为了确定纯化的SKA1如何保持对缩短聚合物的稳定附着，我们将使用纯化的SKA1将微管将其与玻璃微球搭配在一起，并在用激光镊子施加的负载下检查其运动（AIM 3）。这种方法具有创新性，因为它将复杂的生物物理方法的重点放在特定的耦合动力学配合物上，这对于准确遗传信息的准确遗传至关重要。这项研究很重要，因为它将促进鉴定生物力学特征和特定的蛋白质模块，这些模块负责动力学沿着微管壁滑动，承受反能力性并在分裂的哺乳动物细胞的嘈杂和随机环境中对不良适应性条件做出反应。最终，这项工作将促进对精确染色体隔离失败的癌症等人类疾病的分析。 公共卫生相关性：拟议的研究与公共卫生有关，因为对纺锤体微管与染色体动物学之间的动态联系的严格研究将有助于鉴定这些力传递的lins的特定特征，这可能是对动物学静脉相互作用的选择性颠覆性的，可以利用这是对小动物的选择性型号的造型。因此，拟议的研究与NIH培养基本创造发现的使命有关，这些发现最终将提高我们保护和改善人类健康的能力。