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. ***
