STRUCTURE AND FUNCTION OF MAMMALIAN KINETOCHORES

动点是与着丝粒异染色质结合的蛋白质的复杂集合。它们显示微管(MT)依赖的运动活动的两个方向，从它们附着的MT+末端添加和移除微管蛋白的能力，以及它们对MT结合和/或张力的响应信号，以帮助细胞分裂，何时开始后期(有丝分裂检查点)。我们建议在体内和体外研究哺乳动物动点的结构和功能，以帮助阐明这些多重功能的机制和它们之间的关系。我们将通过嵌入塑料中的快速冷冻、冷冻取代的PTK细胞的EM断层扫描来表征MT-动粒界面的结构，验证MT末端形态与这些聚合物的解聚聚合有关的假设。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动粒成分，如日冕和我们外部动粒内板。这一分子解剖学将有助于识别可能相互作用的动粒成分。我们将扩展我们以前在体外对动粒行为的研究，使用激光镊子来操纵相对于玻璃结合染色体上的动粒的MT，测试在动粒-MT键上的张力促进动粒-MT键的稳定性的假设，以及动粒相关的激酶来测试MAP激酶作为有丝分裂检查点的上游调节因子的假设，将MT结合和/或张力耦合到调节后期开始的途径。

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.