MICROTUBULE & CENTROSOME ORGANIZATION IN MAMMALIAN CELLS

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Several mammalian cell culture lines (PtK2, PC 12, HeLa) have been utilized to study the structure-function relationship of centrosomal material with microtubule organization during interphase and mitosis employing standard chemical fixation as well as high pressure freezing (BFF). To preserve ultrastructural and immunological details of centrosomes, chromosomes, microtubules, membranes and intermediate filaments several antibodies were used and analyzed with transmission and scanning electron as well as with immunofluorescence microscopy utilizing a human autoinunune antibody against centrosomes (SPJ), a mouse monoconal. antibody against tubulin (M), an intermediate filament antibody against vimentin (Ah-6), and DAR to stain DNA. During interphase in control cells, centrosomal material is closely associated with the nuclear envelope by a fibrous network and becomes gradually dissociated during mitosis where it functions as the microtubule organizing center for the microtubule-based mitotic apparatus. Unlike in sea urchin eggs and embryos where microtubules are needed for cell-cycle specific expansion and compaction of centrosomes (Schatten et al., 1988, Cell Motil. Cytoskel. 11, 248-259), microtubules are not required for cell-cycle specific progression of centrosomes in mammalian cells. These studies extend on previous findings (Joswig and Petzelt, 1990, Cell Motil. Cytoskel. 15, 181-192) and support the notion that different mechanisms for centrosome expansion and compaction behavior are used in different eukaryotic species. 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 centrosome-cytoskeletal interactions during fertilization, cell division, cell differentiation, and embryo development. SCXENTXFXC SUBPROJECT GRANT NUMBER: P41RR00570-27