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分子机制。为了实现这些目标，我们正在使用一种独特的方法，即首先在活细胞中标记中心体(通过表达标记有绿色荧光蛋白的蛋白质)，然后在细胞周期的不同阶段通过激光显微手术摧毁它的全部或仅特定部分。用这种方法，我们可以创造出拥有这种细胞器的细胞。在前一批款期内，我们得出了与当前提案相关的两项重要结论。首先，我们发现，与长期存在的教义相反，中心体不是脊椎动物有丝分裂纺锤体形成所必需的。接下来，我们证明了中心体的存在是细胞在间期通过细胞周期所必需的。这里提出的研究代表了我们之前发现的自然进展，并解决了以下问题：第一个目标是只去除一个中心粒，以确定在细胞周期中的进展是否需要整个中心体。在目标2中，我们将确定G1期细胞周期进展所需的中心体存在的确切时间点(例如，在中体分离之前或之后)。目标3是确定中心体在用激光破坏现有中心体后是否能形成从头开始，如果是的话，细胞是否能恢复细胞周期。目的#4是验证一个假设，即正常二倍体细胞对额外中心体的表观遗传会导致形成非整倍体但有活力的后代，并最终形成一群转化的(癌细胞)细胞。最后，目标5是确定即使在体细胞中存在中心体的情况下，是否也可以通过中心体非依赖的途径形成有丝分裂纺锤体。总而言之，从这些研究中获得的知识将定义中心体如何参与细胞增殖和细胞周期控制。由于中心体异常是细胞转化和癌症的标志，这一知识将有助于设计治疗这种疾病的新策略。

The broad objectives of the research proposed here are to elucidate the functions of the centrosome in vertebrate somatic cells, and the molecular mechanisms by which it accomplishes these functions. To achieve these goals we are using a unique approach in which the centrosome is first labeled in living cells (by expressing proteins tagged with Green Fluorescent Protein), so that all or just specific parts of it can then be destroyed by laser microsurgery during different stages of the cell cycle. With this method we can create cells that possess this organelle. During the previous grant period we made two important findings relevant to the current proposal. First we found that, contrary to longstanding dogma, the centrosome is not required for formation of the mitotic spindle in vertebrates. Next we demonstrated that the presence of the centrosome is required for cells to progress through the cell cycle during interphase. The research proposed here represents a natural progression from our previous findings, and address the following questions: Aim#1 is to ablate just one centriole to determine if progression through the cell cycle requires the whole centrosome. In Aim#2 we will determine the exact point during G1 at which the presence of the centrosome is required for cell-cycle progression (e.g., before or after midbody separation). Aim#3 is to determine if a centrosome can form de novo after destroying the existing centrosome with the laser and, if so, whether the cell resumes the cell cycle. Aim#4 is to test the hypothesis that epigenetic inheritance of extra centrosomes by a normal diploid cell leads to formation of aneuploid, but viable, progeny and ultimately to a population of transformed (cancerous) cells. Finally, Aim#5 is to determine if mitotic spindle can be formed via centrosome-independent pathway even in the presence of centrosomes in somatic cells. Together the knowledge obtained from these studies will define how the centrosome is involved in cell proliferation and cell cycle control. Since centrosome abnormalities are a hallmark of cell transformation and cancer, this knowledge will be useful for designing new strategies for the treatment of this disease.