MICROTUBULE BASED VESICLE TRANSPORT IN POLARIZED EPITHELIA

在许多真核细胞中，亚细胞定位和动力学 膜性细胞器依赖于微管和微管。 以运输为基础。微管细胞骨架的排列 沿简单极化上皮尖-基轴方向提示 这些细丝提供了一个结构框架，既能产生 对单分子层的稳定性，并为运输提供底物 整个细胞中的材料。似乎跨细胞运动 在底部到顶端方向，即朝向微管减去末端， 高度依赖微管，这表明马达 蛋白质、细胞质动力蛋白及其激活剂、动力蛋白是关键 这一过程中的参与者。因此，极化上皮是一种有用的 基于动力蛋白和动力蛋白的深入研究模型系统 细胞内运动性。 对微管动力学和微管机制的认识 基于移动的应用已显著扩展 视频增强的显微技术，因为它们允许在 复杂的亚细胞行为的实时。细胞内环境的研究 膜流量和内膜动力学同样受益 从这类分析来看。在这项提案中，进行了一系列实验 旨在阐明WIF-B细胞中的跨细胞途径，并 确定细胞质动力蛋白和动力蛋白如何参与 提出了WIF-B和MDCK中的跨细胞运动。膜 动力学将使用一种新的表面标注技术进行可视化 其允许从底部表面到顶端表面的流量 通过视频增强荧光直接实时观察 显微镜。 另一项调查将探索 WIF-B和MDCK细胞的微管核化。许多上皮细胞是 有丝分裂后及其微管细胞骨架缺乏中心焦点， 这表明传统的中心体成核机制可能 不参与其中。将确定微管成核的位置 并将确定它们的中心体是否有能力 在体外和体内，微管成核和释放。

In many eukaryotic cells, the subcellular positioning and dynamics of membranous organelles is dependent on microtubules and microtubule- based transport. The arrangement of the microtubule cytoskeleton along the apico-basal axis of simple polarized epithelia suggests that these filaments provide a structural framework that both yields stability to themonolayer and provides a substrate for transport ofmaterials across the cell. It appears that transcellular movement in the basal-to-apical direction, i.e. toward microtubule minus ends, is highly dependent on microtubules, suggesting tht the motor protein, cytoplasmic dynein, andits activator, dynactin are key players in this process. Polarized epithelia are therefore a useful model system for in-depth study of dynein-and dynactin-based intracellular motility. Understanding of mechanisms ofmicrotubule dynamics and microtubule- based motility has been significantly extended by the application of video-enhanced microscopy techniques, as they allow evaulation in real time of complex subcellular behaviors. Studies of intracellular membrane traffic and endomembrane dynamics have benefited equally fromthis sort of analysis. Inthis proposal, a series of experiments designed to elucidate the transcytotic pathway in WIF-B cells and to determine how cytoplasmic dynein and dynactin contribute to transcellular movement in WIF-B and MDCK are proposed. Membrane dynamics will be visualized using a novel surface labeling technique that allow traffic from the basal surface to the apical surface to be observed directly in real time by video-enhanced fluorescence microscopy. A separate line of investigation will explore the mechanism of microtubule nucleationin WIF-B and MDCK cells. Many epithelia are post-mitotic and their microtubule cytoskeletons lack a centralfocus, suggesting that conventional centrosomal nucleating mechanisms may not be involved. Sites for microtubule nucleationwill be identified and it will be determined if their centrosomes have the capacity to nucleate and release microtubules in vitro and in vivo.