MICROTUBULE BASED VESICLE TRANSPORT IN POLARIZED EPITHELIA

极化上皮中基于微管的囊泡运输

• 批准号: 6301128
• 负责人: TRINA A SCHROER
• 金额: $ 16.08万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301128
• 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 膜细胞器取决于微管和微管 基于运输。 微管细胞骨架的排列 沿着简单偏振上皮的Apico-Basal轴表明 这些细丝提供了一个结构框架，两者都产生 对theNoLayer的稳定性，并为运输提供基板 整个细胞的材料。 看来跨细胞运动 在基础到纯的方向上，即朝着微管负末端， 高度依赖于微管，表明电动机 蛋白质，细胞质动力蛋白，和质量激活剂，dynactin是关键 在此过程中的玩家。 因此，极化上皮是有用的 基于动力蛋白和dynactin的深入研究模型系统 细胞内运动。 了解微管动力学和微管的机制 通过应用大大扩展了基于基于的运动 视频增强显微镜技术，因为它们允许在 复杂的亚细胞行为的实时。 细胞内研究 膜流量和内膜动力学同样受益 从这种分析中。 在此提案中，一系列实验 旨在阐明Wif-B细胞中的跨性途径和 确定细胞质动力蛋白和dynactin如何贡献 提出了Wif-B和MDCK中的跨细胞运动。 膜 动力学将使用新型的表面标记技术可视化 这允许从基础表面到顶部表面的交通 直接通过视频增强荧光直接观察到 显微镜。 单独的调查将探讨 微管核蛋白WIF-B和MDCK细胞。 许多上皮是 有丝分裂后及其微管细胞骨架缺乏中枢性， 提示常规的中心体核定机制可能 不参与。 鉴定微管成核位点 并且将确定其中心体是否有能力 在体外和体内核定并释放微管。