This project will explore the interactions of centrosomes with the cytoskeletal components of microfilaments and microtubules during fertilization, cell division, cell differentiation and embryo development in sea urchin embryos. High pressure freezing will be an ideal choice for fixation as sea urchin eggs and embryos are roughly 80um in diameter and represent a marine reproductive cell system in which cytoskeletal components are very diffficult to preserve by conventional chemical fixation. Additionally, the cell cycles in sea urchin embryos are very rapid, and cytoskeletal changes are extremely dynamic and take place within seconds and minutes after fertilization. These events are likely to be captured best by high pressure freezing. Centrosomes are contributed by the sperm during fertilization and organize the rapidly changing sperm aster to achieve syngamy, and the mitotic apparatus to achieve cell divisions during embryo development. Reorganization of centrosomal material plays a major part in the initiation of cell differentiation. Although the interactions of centrosomes with microtubules are partly understood, very little is known on the mechanisms of centrosome distribution. By using indirect methods (Schatten et al., 1988) microfilaments had been implicated in this process but because of poor preservation by conventional fixation methods, the participation of microfilaments or other cytoskeletal components could previously not be determined directly. Microfilaments are likely to play a role during centrosome separation and distribution throughout embryo development, and intermediate filaments are likely to participate in this process as antibodies against intermediate filaments stained positively around centrosome areas in preliminary immunofluorescence experiments. By using high pressure freezing, this project is likely to contribute to our understanding on the mechanisms of folding and unfolding of centrosomal material and will also contribute to our understanding on centrosorne-cytoskeletal interactions during fertilization, cell division, cell differentiation, and embryo development.
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