Centrosomes form the poles of the spindle in animal cells and are involved in microtubule nucleation, chromosome alignment, chromosome movement, and determining the plane of the cleavage furrow. The ways in which the centrosomes are formed, function, and reproduce are poorly understood. 1. We have isolated centrioles from sea urchin sperm and have microinjected them into fertilized sea urchin eggs. This always leads to the formation of numerous supernumerary asters. To test for the role of the putitive centriolar RNA in centrosome formation and reproduction, we will treat the centrioles with various RNAases, a RNA crosslinking compound, or irradiate them with 260nm light in the presence of ethidium bromide. 2. We will study the role of centrosomal RNA in microtubule nucleation in vitro. Isolated centrosomes will be chilled or treated with Colcemid to disassemble existing microtubules. They will then be exposed to 6S tubulin before or after treatment by RNAases, fragments of RNAases, RNA crosslinking and RNA breaking compounds. We will determine the ability of the treated isolates to nucleate microtubules in vitro. 3. Isolated centrosomes will be treated in the ways just described and then microinjected into fertilized eggs. We will test for microtubule nucleation, chromosome attachment, chromosome movement, and reproduction of the centrosomes. 4. We will investigate the functional properties of the pericentriolar material when separated from centrioles in vivo. Portions of the pericentriolar material will be pulled off the centrosome with a microneedle and their behavior followed with a polarizing microscope. We will look for the ability of this pericentrolar material to function as a pole and reproduce with each cell cycle. These experiments should help resolve the controversy over the role of the centriole in the formation of the spindle pole. 5. We will investigate how the poles of acentriolar, anastral spindles are established. We will microinject plant or mouse egg spindles into fertilized eggs. Their behavior in the sea urchin cytoplasm will be determined. This work will provide a better understanding of the mechanisms that control cell division in both normal and cancerous cells.
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