Kinetochores are proteinacious plaques at the outer periphery of centromeric DNA regions of mitotic chromatid pairs.  During the formation of the mitotic spindle, one sister kinetochore becomes attached to the ends of microtubules from one pole, while the other becomes attached to the ends of microtubules from the opposite pole.  The kinetochore is a dynamic structure composed of several constituent proteins, and its attachment to microtubules is known to affect the accumulation of these proteins onto the centromeric region.  The constituent proteins fulfill specific roles within the kinetochore, and changes in the amounts of kinetochore proteins may differ with the timeliness of those roles. The precise pattern of accumulation and depletion is not known for every kinetochore protein, and the aim of this project was to determine that pattern for three different kinetochore proteins/protein groups.  CENP-E is a plus-directed microtubule-motor protein that is involved in attaching microtubule ends to kinetochores and producing motility. Bub1 is a protein kinase that is directly involved in the regulation of the progression through metaphase into anaphase, delaying anaphase until sister kinetochores have become properly attached to microtubules and aligned within the spindle.  Both CENP-E and Bub1 are thought to reside in the outer face of the kinetochores and to be removed from kinetochores as they become attached to microtubules, although there has not been any precise quantification of this event.  Proteins recognized by CREST antibodies are part of the core kinetochore-centromere structure which makes up the inner face of the kinetochore.  It is thought that the CREST proteins are not dislodged from the kinetochore as it becomes attached to microtubules.  Using protein-specific antibodies, with the techniques of immunofluorescent labeling and digital-enhanced microscopy, measurements of protein levels were performed on both fully microtubule-attached PtK? kinetochores and completely unattached kinetochores.  Microtubules were removed from kinetochores by depolymerizing them, using treatment with the drug nocodazole.  In addition, protein levels on kinetochores that had become reattached to microtubules after a period of nocodazole treatment were also measured.  It was found that CENP-E levels were about three times higher on unattached kinetochores than on attached kinetochores, and that reattached kinetochores possessed the same amount of CENP-E as attached kinetochores.  Bub1 levels were also about three times as high on unattached kinetochores as on attached kinetochores, and reattached kinetochores displayed Bub1 levels that were the same as those on attached kinetochores.  The amount of CREST proteins present on kinetochores did not differ with experimental treatments, except for those cells that had been exposed to a long incubation in nocodazole, and was therefore not dependent on microtubule attachment.  These results reveal that CENP-E and Bub1 play roles that are more significant to the pre-metaphase stages of mitosis than to metaphase or anaphase.  On the other hand, CREST proteins likely affect duties that are more essential to the maintenance of kinetochore structure and integrity.