MICROTUBULE BASED VESICLE TRANSPORT IN POLARIZED EPITHELIA

极化上皮中基于微管的囊泡运输

• 批准号: 6270727
• 负责人: TRINA A SCHROER
• 金额: $ 15.81万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 1998-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6270727
• 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， 膜细胞器依赖于微管和微管- 基于交通。 微管骨架的排列 沿着简单极化上皮的顶端-基底轴， 这些细丝提供了一个结构框架， 对单层的稳定性，并提供了一种用于传输的基底 of materials材料across横过the cell细胞. 看来跨细胞运动 在基部至顶端的方向，即朝向微管负端， 高度依赖于微管，这表明 蛋白质，细胞质动力蛋白，及其激活剂，动力蛋白是关键 玩家在这个过程中。 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基于动力蛋白的 细胞内运动 了解微管动力学和微管- 基于运动性已显着扩展的应用， 视频增强显微镜技术，因为它们允许评估 复杂亚细胞行为的真实的时间。 细胞内研究 膜运输和内膜动力学同样受益 from this sort分类of analysis分析. 在这个提议中，一系列的实验 旨在阐明WIF-B细胞中的转胞吞途径， 确定细胞质动力蛋白和动力肌动蛋白如何有助于 提出了WIF-B和MDCK的跨细胞运动。 膜 动态将使用一种新的表面标记技术可视化 允许从基底面到顶面的交通， 通过视频增强荧光直接在真实的时间观察 显微镜 一个单独的调查线将探讨的机制， 在WIF-B和MDCK细胞中微管成核。 许多上皮细胞 有丝分裂后和它们的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 不要参与其中。 微管成核的位点将被确定微管成核的位点将被确定 并将确定它们的中心体是否有能力 在体外和体内成核和释放微管。