MECHANISM AND REGULATION OF MICROTUBULE TURNOVER

微管是真核细胞中几个重要过程所必需的动态结构，包括有丝分裂、细胞形状的建立和维持以及细胞运动。我们工作的总体目标是确定微管在细胞穿越细胞周期和运动时被重塑的机制，以及微管如何促进有效的细胞运动。我们实验方法的一个关键特征是对活细胞中的微管进行分析。为了实现这一目标，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标记蛋白的光漂白。主要目的是确定中心体微管负端的动态特性；测量运动细胞和非运动细胞中心体微管的成核和释放；确定微管运输的机制；确定中心体重定向是否发生在成纤维细胞样细胞运动和细胞向受损单层迁移的过程中；并确定运动细胞中控制微管转换的调节途径。微管动力学和重塑将在高度运动的角化细胞和哺乳动物细胞运动的两个模型系统中进行研究：向伤口的迁移和对生长因子的反应的成纤维细胞样运动。这些结果将阐明活细胞中微管转换的机制，并有助于我们理解微管在细胞极性和运动性中的作用。了解正常细胞中的这些过程是分析癌细胞的重要的第一步，在癌细胞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.