LASER ABLATION OF GFP & ( TUBULIN LABELED CENTROSOMES IN SOMATIC CELLS

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(Supported by GMS R01 40198 to C. L. Rieder) Our recent demonstration that ?-tubulin-GFP (?TGFP) fusion protein specifically labels and delineates the boundaries of the centrosome within in the living cell allows us to reproducibly destroy, by laser microsurgery, the centrosome at any stage of the cell cycle. As we have already demonstrated (first paper below) the disappearance of ?TGFP signal observed during the laser operation corresponds to destruction of the irradiated part of the centrosome. As a result, one can ablate the entire centrosome by targeting all of the centrosome-associated ?TGFP signal, or just a part of it. This novel approach provides an exciting and unique opportunity to selectively destroy, for the first time, all or part of the centrosome in vertebrate somatic cells during interphase and mitosis. Our current line of experiments includes two major sub-projects The first is to clarify the role the centrosome in spindle formation and maint enance during mitosis in vertebrate somatic cells. To do this we will use laser microsurgery to specifically destroy one or both of the centrosomes at various stages of mitosis, from mid-prophase through anaphase. The second is to determine if cells initiate and complete DNA synthesis, and/or undergo division, after destroying all or just part of the centrosome during different periods of cell cycle. These experiments will reveal if the absence of a centrosome, or the presence of a centrosome containing just one centriole in G1 or a single diplosome (i.e., a mother/daughter centriole pair) in G2, inhibits normal progression through the cell cycle in vertebrate somatic cells. Khodjakov, A., R.W. Cole and C. L. Rieder. (late 1997). A synergy of technologies: combining laser microsurgery with green fluorescent protein tagging. Cell Motility and the Cytoskeleton 38:311-317 Khodjakov, A and C. L. Rieder. (1998) A synergy of technologies: Combining laser microsurgery with green fluorescent protein GFP)-tagging. SPIE International Symposium on Biomedical Optics (BIOS'98) Tech. Abs. Digest pp. 156. Khodjakov, A., R.W. Cole and C. L. Rieder. (1998) Destruction of one centrosome by laser microsurgery during prophase down-regulates the activity of the second centrosome and inhibits spindle formation. Molecular Biology of the Cell (in press)