FUNCTIONAL PROPERTIES OF CENTROSOMES IN SOMATIC CELLS

本研究的主要目的是阐明中心体在脊椎动物体细胞中的功能及其实现这些功能的机制。由于成功开发了一种γ -微管蛋白-绿色荧光蛋白融合蛋白，该蛋白首次在整个细胞周期中清晰地看到活脊椎动物体细胞中的中心体，因此提出的工作成为可能。目的一是阐明在脊椎动物体细胞有丝分裂过程中中心体在纺锤体形成和维持中的作用。为此，我将使用激光显微手术在有丝分裂的各个阶段，从前期中期到后期，特异性地破坏一个或两个中心体。目的二是确定细胞在细胞周期的不同时期（通过激光显微手术）摧毁全部或部分中心体后，是否启动并完成DNA合成，和/或进行分裂。这些实验将揭示，在G1或G2中，中心体的缺失或中心体只含有一个中心粒或单个二倍体（即母亲/女儿中心粒对）的存在，是否会抑制脊椎动物体细胞细胞周期的正常进程。目的3是确定在未经处理的细胞中，在细胞周期的不同时期中心体相关部分和细胞质池之间的γ -微管蛋白交换速率，以及当微管动力学被影响其稳定性的药物（如诺可达唑、紫杉醇）改变时。这些实验将揭示中心体是否仅在需要时（例如，在G2/M转变时）招募γ -微管蛋白，或者是否在整个细胞周期中持续招募γ -微管蛋白，但将其部分保持在非活性形式。这项研究还将提供额外的信息，说明中心体周期是否在没有正常微管行为的情况下继续运行。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治疗的新策略（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.)