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

(由GMS R01 40198至C.L.Rieder支持)我们最近 -微管蛋白-绿色荧光蛋白(？TGFP)融合蛋白的特异性研究 中的中心体的边界 活细胞使我们能够通过激光显微手术重复地摧毁， 细胞周期中任一阶段的中心体。正如我们已经做的 论证(第一篇论文如下)？TGFP信号的消失 在激光操作过程中观察到的对应于 中心体的受辐射部分。因此，人们可以消融 通过靶向所有中心体相关的TGFP来实现整个中心体 信号，或者只是其中的一部分。这种新方法提供了一种 第一次有选择地摧毁的令人兴奋和独特的机会 时间，脊椎动物体细胞中全部或部分中心体 间期和有丝分裂。我们目前的实验系列包括两个 重大子项目一是明确中心体在 主轴的形成和维护 在脊椎动物体细胞有丝分裂期间。要做到这一点，我们将使用 激光显微手术专门摧毁其中一个或两个 有丝分裂不同阶段的中心体，从中期到前期到 后期。第二个步骤是确定细胞是否启动和完成 DNA合成，和/或经历分裂，在摧毁全部或只是 在细胞周期的不同时期，中心体的一部分。这些 实验将揭示，如果没有中心体，或者 在G1或A中只含有一个中心粒的中心体的存在 G2中的单个双体(即母女中心粒对)， 抑制脊椎动物细胞周期的正常进展 体细胞。Khodjakov，A.，R.W.Cole和C.L.Rieder。(迟到 1997年)。技术的协同作用：将激光显微手术与 绿色荧光蛋白标记。细胞运动与细胞骨架 38：311-317 Khodjakov，A和C.L.Rieder。(1998) 技术：将激光显微手术与绿色荧光相结合 蛋白质GFP)-标记。SPIE国际生物医学研讨会 光学(BIOS‘98)技术。腹肌。消化pp。156.Khodjakov，A.，R.W. Cole和C.L.Rieder。(1998)用激光破坏一个中心体 早期显微外科手术下调第二脑区的活动 中心体并抑制纺锤体的形成。分子生物学的研究进展 单元格(在印中)

(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)