FUNCTIONAL PROPERTIES OF CENTROSOMES IN SOMATIC CELLS

本研究的主要目的是阐明脊椎动物体细胞中心体的功能及其实现这些功能的机制。 拟议的工作是可能的成功开发的γ-微管蛋白-GFP融合蛋白，第一次，使中心体清晰可见的活脊椎动物体细胞在整个细胞周期。 目的一是阐明中心体在脊椎动物体细胞有丝分裂过程中纺锤体形成和维持中的作用。 为了做到这一点，我将使用激光显微手术，在有丝分裂的各个阶段，从中期前期到后期，专门破坏一个或两个中心体。 第二个目标是确定在细胞周期的不同时期，在破坏（通过激光显微手术）全部或部分中心体后，细胞是否启动并完成DNA合成和/或进行分裂。 这些实验将揭示是否存在中心体，或在G1中存在仅含有一个中心粒的中心体或单个二倍体体（即，G2期的母/子中心粒对）抑制脊椎动物体细胞中细胞周期的正常进程。 目的3是确定在未处理的细胞中，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改变时（例如，诺考达唑、紫杉醇）。 这些实验将揭示中心体是否仅在需要时招募γ-微管蛋白（例如，在G2/M转换），或者如果它在整个细胞周期中持续募集γ-微管蛋白，但保持其部分处于非活性形式。 这项研究还将提供额外的信息，中心体周期是否继续运行在正常的微管行为的情况下。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，并可能导致开发用于治疗与微管功能相关的疾病状态的新策略（例如，如癌症、老年痴呆症、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.)