STRUCTURE AND FUNCTION OF MAMMALIAN KINETOCHORES

着丝点是与着丝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）依赖的运动活动，从它们附着的MT +端添加和移除微管蛋白的能力，以及它们响应MT结合和/或张力发出信号以帮助细胞分裂何时开始后期（有丝分裂检查点）。我们建议研究哺乳动物着丝点在体内和体外的结构和功能，以帮助阐明这些多种功能的机制和它们之间的关系。我们将通过快速冷冻的、冷冻取代的PtK细胞嵌入塑料的EM断层扫描来表征MT-着丝点界面的结构，测试MT端形态与这些聚合物的聚合解聚有关的假设。我们将使用抗体或其他标记方法来定位已知的着丝粒成分，相对于精细的结构标记，如电晕和内外着丝粒板。这种分子解剖将有助于识别可能相互作用的着丝点组分。我们将在体外扩展我们之前的着丝点行为，使用激光镊子操作相对于玻璃结合染色体上的着丝点的Mts，测试着丝点的张力促进着丝点-MT结合的稳定性的假设，以及着丝点相关激酶来测试MAP激酶作为有丝分裂检查点的上游调节剂的假设，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.