MECHANISM AND REGULATION OF MICROTUBULE TURNOVER

微管是一种动态结构，在真核细胞的几个重要过程中，包括有丝分裂，细胞形状的建立和维持以及细胞运动性。我们工作的总体目标是确定微管在细胞周期和迁移中重塑的机制，以及微管如何有助于有效的细胞运动。 我们实验方法的一个关键特征是活细胞中微管的分析。 为了实现这一点，我们将使用定量数字荧光显微镜，笼状微管蛋白的光活化，荧光模拟细胞化学和GFP标记的蛋白质的光漂白。 主要目的是确定中心体微管负端的动力学特性;测量运动和非运动细胞中中心体微管的成核和释放;确定负责微管运输的机制;确定成纤维细胞样细胞运动期间和细胞迁移到受伤单层期间是否发生中心体重定向;并确定运动细胞中微管周转的调节途径。微管动力学和重塑将在高度能动的角膜细胞和哺乳动物细胞运动的两个模型系统中进行研究：迁移到伤口和成纤维细胞样运动响应生长因子。 这些结果将有助于阐明活细胞中微管更新的机制，并有助于我们理解微管在细胞极性和运动中的作用。 了解正常细胞中的这些过程是分析癌细胞的重要第一步，其中这些过程是异常的。

Microtubules are dynamic structures that are required for several vital processes in eukaryotic cells including mitosis, the establishment and maintenance of cell shape and cell motility. The overall objective of our work is to determine the mechanism(s) by which microtubules are remodelled as cells traverse the cell cycle and locomote and how microtubules contribute to efficient cellular motility. A key feature of our experimental approach is the analysis of microtubules in the living cell. To accomplish this we will use quantitative digital fluorescence microscopy, photoactivation of caged tubulin, fluorescence analog cytochemistry and photobleaching of GFP- tagged proteins. The major aims are to determine the dynamic properties of the minus-ends of centrosomal microtubules; measure nucleation and release of centrosomal microtubules in motile and non-motile cells; determine the mechanism responsible for microtubule transport; determine if centrosome reorientation occurs during motion of fibroblast-like cells and during cell migration into a wounded monolayer; and determine the regulatory pathways that govern microtubule turnover in motile cells. Microtubule dynamics and remodelling will be studied in highly motile keratocytes and in two model systems for mammalian cell motility: migration into a wound and fibroblast-like motion in response to growth factors. The results will elucidate the mechanisms of microtubule turnover in live cells and contribute to our understanding of the role of microtubules in cell polarity and motility. Understanding these processes in normal cells is an important first step towards analysis of cancer cells, where such processes are aberrant.