MICROTUBULE BASED VESICLE TRANSPORT IN POLARIZED EPITHELIA

极化上皮中基于微管的囊泡运输

• 批准号: 6564275
• 负责人: TRINA A SCHROER
• 金额: $ 14.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6564275
• 关键词: Golgi apparatus; MDCK cell; apical membrane; basolateral membrane; cellular polarity; centrosome; confocal scanning microscopy; dynein ATPase; electron microscopy; endocytosis; epithelium; fluorescence microscopy; fluorescent dye /probe; intracellular transport; light microscopy; liver cells; lysosomes; membrane proteins; microinjections; microtubules; protein transport; transcytosis; vesicle /vacuole; video microscopy

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.

在许多真核细胞中，亚细胞定位和动力学 膜细胞器依赖于微管和微管 为基础的运输。 微管细胞骨架的排列 沿着单极化上皮细胞的顶端-基底轴表明 这些细丝提供了一个结构框架，既产生 对单层的稳定性并提供运输底物 整个细胞的材料。 看来跨细胞运动 在基部到顶端方向，即朝向微管负端， 高度依赖于微管，表明运动 蛋白质、细胞质动力蛋白及其激活剂动力蛋白是关键 这个过程中的玩家。 因此，极化上皮细胞是一种有用的 用于深入研究动力蛋白和动力蛋白的模型系统 细胞内运动。 了解微管动力学和微管的机制 基于运动性的应用已显着扩展 视频增强显微镜技术，因为它们可以进行评估 复杂的亚细胞行为的实时。 细胞内的研究 膜交通和内膜动力学同样受益 从这样的分析来看。 在这个提案中，进行了一系列的实验 旨在阐明 WIF-B 细胞中的转胞吞途径并 确定细胞质动力蛋白和动力蛋白如何发挥作用 提出了 WIF-B 和 MDCK 中的跨细胞运动。 膜 将使用一种新颖的表面标记技术来可视化动力学 允许从基底表面到顶端表面的交通 通过视频增强荧光直接实时观察 显微镜。 一项单独的调查将探索其机制 WIF-B 和 MDCK 细胞中的微管成核。 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， 表明传统的中心体成核机制可能 不参与。 将确定微管成核位点 并且将确定它们的中心体是否有能力 在体外和体内成核并释放微管。