STRUCTURE AND FUNCTION OF MAMMALIAN KINETOCHORES

着丝粒是与着丝粒异染色质结合的蛋白质的复杂集合体。它们显示微管（MT）依赖性运动活动的两个方向，从它们附着的MT加末端添加和移除微管蛋白的能力，并且它们响应于MT结合和/或张力而发出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粒的结构和功能，以帮助阐明这些多重功能的机制以及它们之间的关系。我们将通过EM断层扫描的快速冷冻，冷冻取代的PtK细胞，嵌入塑料，测试MT末端形态与这些聚合物的解聚聚合的假设，MT-动粒界面的结构特征。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，如冠和内外动粒板。这种分子解剖学将有助于识别可能相互作用的动粒成分。我们将扩展我们之前的体外动粒行为，使用激光镊子操纵相对于玻璃结合染色体上的动粒的Mts，测试动粒处的张力促进动粒-MT键的稳定性的假设，以及动粒相关激酶来测试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/或张力与调节后期开始的途径偶联。

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.