Centrosomes are the spindle poles in dividing cells and are necessary for microtubule nucleation, chromosome alignment, chromosome movement, and establishment of the plane of the cleavage furrow. The ways in which the centrosomes form, function, and reproduce are poorly understood. We want to learn how these processes are controlled in order to establish a basis for new technologies to control cancer cell proliferation. 1. We isolate centrioles from sea urchin sperm and microinject them into fertilized sea urchin eggs. This leads to the formation of numerous supernumerary asters. To test for the role of the putative centriolar RNA in centrosome formation and reproduction, we will treat the centrioles with various RNAases, and psoralen, a RNA crosslinking compound. 2. We will inject nucleases or psoralen treated centrioles from sea urchin sperm and basal bodies from Chlamydomonas into activated Xenopus eggs to determine if asters from during meiosis or interphase. This should resolve the discrepancy between our existing results and those of another group. 3. To define the similarities and dissimilarities between acentriolar-anastral spindle poles and typical animal centrosomes, we will transfer the anastral- acentriolar spindles from mouse eggs, a mutant Drosophila cell line, and plant cells into fertilized sea urchin eggs. We want to examine the activity of these spindles in the sea urchin cytoplasm. Later we will characterize their ultrastructure to determine if they have undergone any structural changes after one or more cell cycles in the sea urchin egg. 4. We will determine the relative roles of centrioles and pericentriolar material in the reproduction of centrosomes. We will treat eggs with acridine orange and use a focused laser microbeam to ablate centrioles or portions of the pericentiolar material. By serial section ultrastructural analysis, we will correlate the structures damaged with the reproductive capacity of the centrosome. 5. We will determine if centrosome formation and reproduction in sea urchin eggs is under translational control. 6. Several exploratory projects are briefly described.
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