Ultrastructural Basis For Mitotic Chromosome Movement in Vertebrates

麦克尤恩9808879 有丝分裂过程中染色体的优雅运动长期以来一直吸引着生物学家，甚至在这些现象对遗传分离的意义被充分认识之前。 这项研究的长期目标是帮助阐明有丝分裂纺锤体上染色体运动的分子机制。 这一目标需要了解染色体如何运动以及它们的运动是如何控制的。 这是有丝分裂研究中一个非常令人兴奋的时刻，从各种分子，遗传，生物化学和功能方法中以快速的速度发现了机制的分子成分。 尽管有这些丰富的新信息，我们仍然没有一个清晰的图片的分子事件，产生和控制染色体运动。 特别是，我们仍然对不同的组件是如何相对于彼此排列的，以及这种排列在染色体运动过程中如何变化知之甚少。目前项目的战略是将光学和电子显微镜的先进技术与分子和生物化学方法联合收割机相结合，以阐明参与染色体/微管相互作用的选定纺锤体组分的三维排列。 具体来说，该项目的第一部分使用激光显微手术和同细胞光和电子显微镜（即，同一细胞的光镜和电镜检查），以确定星体弹射力是否来自微管推动染色体臂，或来自微管马达蛋白将染色体从纺锤体极移开。该项目的第二部分测试的假设，微管的结合着丝粒促进解离的调节蛋白mad 2。 动粒是一种特殊的附件，将染色体附着在纺锤体上，并作为几种关键蛋白质的结合位点。 Mad 2首先使用酵母遗传学鉴定，并且其从动粒的解离与后期细胞周期检查点的释放相关。在项目的第三部分，改进的标本制备和三维重建的方法将被用来修改我们目前的描述动粒架构。 该信息是解释免疫定位数据的先决条件。 本项目的第四部分将在电子显微镜（EM）水平上使用免疫定位来验证着丝粒蛋白F（CENP-F）参与细胞周期中动粒形成的假设。 同样的方法将用于测试新发现的蛋白质Fint 132与CENP-F直接相关的假设。 ***

9808879 McEwen The elegant movements of chromosomes during mitosis have long fascinated biologists, even before the significance of these phenomena for genetic segregation was fully appreciated. The long-term objective of this research is to help elucidate the molecular mechanisms responsible for chromosome movement on the mitotic spindle. This objective requires understanding both how chromosomes move and how their movement is controlled. It is a very exciting time in mitosis research, with molecular components of the mechanism being discovered at a rapid rate from a variety of molecular, genetic, biochemical, and functional approaches. Despite this wealth of new information, we still do not have a clear picture of the molecular events that generate and control chromosome movement. In particular, we still know very little about how different components are arranged relative to one another, and how that arrangement changes during chromosome motion. The strategy of the current project is to combine advanced techniques in light and electron microscopy with molecular and biochemical methods to elucidate the three-dimensional arrangements of selected spindle components that are involved in chromosome/microtubule interactions. Specifically, the first part of the project uses laser microsurgery and same-cell light and electron microscopy (i.e., both light- and electron-microscopical examination of the same cell) to determine whether astral ejection forces arise from microtubules pushing on chromosome arms, or from microtubule motor proteins moving chromosomes away from the spindle pole. The second part of the project tests the hypothesis that the binding of microtubules to the kinetochore promotes dissociation of the regulatory protein mad2. The kinetochore is a specialized appendage that attaches chromosomes to the spindle and serves as the binding site for several key proteins. Mad2 was first identified using yeast genetics, and its dissociation from the kinetochore is correlated with release of the anaphase cell cycle checkpoint. In the third part of the project, improved methods of specimen preparation and three-dimensional reconstruction will be used to revise our current description of the kinetochore architecture. This information is a prerequisite to interpreting immuno-localization data. The fourth part of the project will use immuno-localization at the electron microscopic (EM) level to test the hypothesis that centromere protein F (CENP-F) is involved in kinetochore formation during the cell cycle. The same approach will be used to test the hypothesis that the newly discovered protein Fint 132 is directly associated with CENP-F. ***