FUNCTIONAL PROPERTIES OF CENTROSOMES IN SOMATIC CELLS

这项研究的主要目标是阐明脊椎动物体细胞中中心体的功能，以及它完成这些功能的机制。这项拟议的工作是由于成功开发了一种伽马-微管蛋白-绿色荧光蛋白融合蛋白，首次使中心体在整个细胞周期中在活的脊椎动物体细胞中清晰可见。第一个目标是阐明中心体在脊椎动物体细胞有丝分裂过程中纺锤体的形成和维持中的作用。为了做到这一点，我将使用激光显微外科手术，在有丝分裂的不同阶段，从中期到后期，专门摧毁一个或两个中心体。第二个目标是确定在细胞周期的不同时期，细胞在破坏(通过激光显微手术)全部或仅部分中心体后，是否启动并完成DNA合成，和/或进行分裂。这些实验将揭示，在脊椎动物体细胞中，中心体的缺失，或者G1中只包含一个中心粒的中心体的存在，或者G2中只包含一个双体(即母子中心粒对)的中心体的存在，是否会抑制脊椎动物体细胞在细胞周期中的正常进展。第三个目标是确定在未经处理的细胞中，在细胞周期的不同时期，以及当微管动力学被影响其稳定性的药物(如诺康唑、紫杉醇)改变时，中心体相关组分和细胞质池之间的伽马微管蛋白交换的速率。这些实验将揭示中心体是否只在需要时(例如，在G2/M转变时)招募伽马微管蛋白，或者是否在整个细胞周期中不断招募伽马微管蛋白，但部分保持不活跃的形式。这项研究还将提供更多关于中心体周期是否在缺乏正常微管行为的情况下继续运行的信息。总之，从这些研究中获得的新数据将使研究人员能够更好地定义中心体如何参与细胞增殖和细胞周期控制，并可能导致开发与微管功能相关的疾病状态的治疗新策略(例如，癌症、阿尔茨海默氏症、关节炎等)。

The broad objectives of this research are to elucidate the functions of the centrosome in vertebrate somatic cells, and the mechanisms by which it accomplishes these functions. The proposed work is made possible by the successful development of a gamma-tubulin-GFP fusion protein that, for the first time, makes the centrosome clearly visible in living vertebrate somatic cells throughout the cell cycle. Aim number 1 is to clarify the role the centrosome in spindle formation and maintenance during mitosis in vertebrate somatic cells. To do this I will use laser microsurgery to specifically destroy one or both of the centrosomes at various stages of mitosis, from mid-prophase through anaphase. Aim number 2 is to determine if cells initiate and complete DNA synthesis, and/or undergo division, after destroying (by laser microsurgery) all or just part of the centrosome during different periods of cell cycle. These experiments will reveal whether 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. Aim number 3 is to determine the rates with which gamma-tubulin exchanges between the centrosome-associated fraction and the cytoplasmic pool during different periods of cell cycle in untreated cells, and when microtubule dynamics is altered by drugs that affect their stability (e.g., nocodazole, taxol). These experiments will reveal if the centrosome recruits gamma-tubulin only when it is needed (e.g., at the G2/M transition), or if it recruits gamma-tubulin continuously throughout the cell cycle but maintains part of it in an inactive form. This study will also provide additional information on whether the centrosome cycle continues to run in the absence of normal microtubule behavior. Together, the novel data obtained from these studies will allow researchers to better define how the centrosome is involved in cell proliferation and cell cycle control, and may lead to the development of new strategies for the treatment of disease states related to microtubule function (e.g., as cancer, Alzheimer's, arthritis, etc.)