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.

动粒是与着丝粒异染色质结合的蛋白质的复杂组装体。它们表现出两个方向的微管 (MT) 依赖性运动活动，能够从 MT 及其附着的末端添加和去除微管蛋白，并且它们响应 MT 结合和/或张力发出信号，以帮助细胞何时开始分裂后期（有丝分裂检查点）。我们建议在体内和体外研究哺乳动物动粒的结构和功能，以帮助阐明这些多种功能的机制以及它们之间的关系。我们将通过嵌入塑料中的快速冷冻、冷冻替代 PtK 细胞的 EM 断层扫描来表征 MT-着丝粒界面的结构，测试 MT 末端形态与这些聚合物的聚合或解聚相关的假设。我们将使用抗体或其他标记方法来定位与精细结构标记相关的已知着丝粒成分，例如日冕和内部的外着丝粒板。这种分子解剖学将有助于识别可能相互作用的动粒成分。我们将在体外扩展我们之前的着丝粒行为，使用激光镊子操纵 Mts 相对于玻璃结合染色体上的着丝粒，测试着丝​​粒张力促进着丝粒-MT 键稳定性的假设以及要测试的着丝粒相关激酶MAP 激酶作为有丝分裂检查点的上游调节器的假设，将 MT 结合和/或张力与调节有丝分裂检查点的途径耦合起来后期的开始。