Centrosomes for the poles of the mitotic spindle. Since they nucleate all spindle microtubules, centrosomes play a key role in the execution of mitotic events. The duplication, or reproduction, of the interphase centrosome is an important event in the cell's preparations for mitosis. We will characterize the functional properties of the mechanisms that control centrosome reproduction and the mechanisms that coordinate the centrosome cycle with the nuclear cell cycle. This sort of information cannot be gained solely by the biochemical characterization of isolated centrosomes. Our long range goal is to provide the groundwork for new technologies to control the proliferation of cancerous cells. 1. To investigate the how centrosome reproduction is coordinated with the nuclear cell cycle in sea urchin zygotes, we will test the role of maturation promoting factor levels in the reproducing of centrosomes. We will also examine the possibly that the cyclin A-p34cdc2 and cyclin B-p34cdc2 kinase complexes have separate roles in both centrosome reproduction and the nuclear cell cycle. 2. We will examine the mechanisms that control centrosome reproduction using cell-free Xenopus egg extracts. Exogenous controsomes are active in cycling egg extracts. We will determine the extent to which chromosomes can reproduce in extracts that are arrested i mitosis. Next, we will examine the role of the p34cdc2 kinase in the reproduction of centrosomes. 3. We will test the role of the nine triplet microtubules of the centriole in centrosome formation and reproduction by three complementary methods. a. We extract the triplet microtubules from isolated centrioles and show that the remnants organize centrosomes. We will deermine if these centrosomes reproduce in sea urchin zygotes and Xenopus egg extracts and if so, see if daughter centrioles with nine triplet microtubule morphology are assembled. b. To determine if the nine triplet microtubule stucture alone is sufficient to organize a centrosome, we will assay the ability of basal bodies, which may not organize centrosomes in vivo, to form functional centrosomes in Xenopus egg extracts. These experiments will enable us to determine if basal body duplication can be uncoupled from centrosome formation and reproduction. c. We will introduce centrosomes from """"""""acentriolar"""""""" cells into Xenopus egg extracts to determine if they form centrosomes in an animal cytoplasm and if those centrosomes reproduce in a normal fashion. 4. To characterize the mechanisms that control centrosome inheritance in the development, we will detrmine when the matrnal centrosome is suppressed in the development of starfish zygotes. We will transfer meiosis I and meiosis II spindles from maturing eggs to zygotes in first mitosis.
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