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，以及实现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，首先在活细胞中标记中心体（通过表达带有绿色荧光蛋白标记的蛋白质），这样在细胞周期的不同阶段，中心体的全部或特定部分就可以被激光显微手术破坏。通过这种方法，我们可以创造出具有这种细胞器的细胞。在上一个拨款期间，我们取得了两项与当前建议有关的重要发现。首先，我们发现，与长期以来的教条相反，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进行细胞周期的必要条件。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：目的1是只切除一个中心粒，以确定细胞周期的进展是否需要整个中心体。在目标2中，我们将确定G1期间中心体在细胞周期进程中需要存在的确切点（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摧毁现有中心体后是否可以重新形成，如果可以，细胞是否恢复细胞周期。目的4是验证一个假设，即正常二倍体细胞的额外中心体的表观遗传会导致非整倍体的形成，但可以存活，最终形成转化（癌）细胞群体。最后，目的5是确定即使在体细胞中有中心体存在的情况下，有丝分裂纺锤体是否可以通过中心体独立途径形成。从这些研究中获得的知识将共同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.