STRUCTURE AND FUNCTION OF MAMMALIAN KINETOCHORES

哺乳动物动粒的结构和功能

• 批准号: 6448203
• 负责人: John RICHARD MCINTOSH
• 金额: $ 49.81万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6448203
• 关键词: cell cycle; cell motility; centromere; electron microscopy; freeze etching; immunoelectron microscopy; meiosis; membrane permeability; microtubules; mitogen activated protein kinase; nanotechnology; phosphomonoesterases; phosphorylation; protein binding; protein structure function; structural biology; tissue /cell culture; tomography

Kinetochores are complex assemblies of proteins bound to centromeric heterochromatin. They display two directions of microtubule (MT)- dependent motor activity, the ability to add and remove tubulin from MT plus ends to which they attach, and they signal in response to MT binding and/or tension to help the cell divide when to begin anaphase (the mitotic checkpoint). We propose to study the structure and function of mammalian kinetochores, both in vivo and in vitro, to help elucidate the mechanisms for these multiple functions and the relationships among them. We will characterize the structure of the MT-kinetochore interface by EM tomography of fast-frozen, freeze-substituted PtK cells, embedded in plastic, testing the hypothesis that MT end morphology is related to the polymerization of depolymerization of these polymers. We will use antibodies or other labeling methods to localize known kinetochore components rela6tive to fine structural markers, like the corona and the inner our outer kinetochore plates. This molecular anatomy will help to identify kinetochore components that may interact. We will extend our previous of kinetochore behavior in vitro, using laser tweezers to manipulate Mts relative to kinetochores on glass-bound chromosomes, testing the hypothesis that tension at the kinetochore promotes stability in the kinetochore-MT bond and that kinetochore-associated kinases to test the hypothesis that a MAP kinase serves as an up-stream regulator of the mitotic checkpoint, coupling MT binding and/or tension to the pathway that regulates the onset of anaphase.

动力学是与丝粒异染色质结合的蛋白质的复杂组件。它们显示了两个方向微管（MT） - 依赖运动活动，即从MT加上其附着的末端添加和去除小管蛋白的能力，并且它们响应MT结合和/或张力发出信号，以帮助何时开始后期（有丝分裂检查点）。我们建议研究体内和体外哺乳动物动物学的结构和功能，以帮助阐明这些多种功能以及它们之间的关系的机制。我们将通过嵌入塑料中的快速冻结，冻结取代的PTK细胞的EM层析成像来表征MT-Kinetochore界面的结构，检验了MT终端形态与这些聚合物降聚合的聚合有关的假设。我们将使用抗体或其他标记方法来将已知的动力学组分局部定位到精细的结构标记，例如电晕和我们的外部动物学板。这种分子解剖结构将有助于鉴定可能相互作用的动力学成分。我们将使用激光镊子在玻璃结合的染色体上使用激光镊子在体外进行体外的动力学行为，以操纵MTS，以下假设，即在动力学上的张力促进了动力学MT键的稳定性，并在动力学酶酶上测试了动力学酶，以测试了既定的动力学酶，该酶是构造的。有丝分裂检查点，将MT结合和/或张力耦合到调节后期开始的途径。