MICROSCOPIC ANALYSIS OF THE SUBCELLULAR TRAFFICKING OF THE NA,K-ATPASE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。关于极化上皮细胞中新合成的离子转运蛋白所追求的细胞通路，人们已经了解了很多。将脉冲追踪法与各种质膜标记或细胞分离技术相结合的研究提供了这些蛋白质在到达质膜的途中所走的亚细胞路线的性质和持续时间的信息。然而，总的来说，不可能将脉冲标记所提供的时间分辨率与显微技术所提供的空间分辨率相结合。因此，一些非常有趣的问题仍然超出了实验分析的范围。例如，目前尚不清楚携带新合成的膜蛋白的货运小泡是否能够与整个质膜对接和融合，或者这一功能是否仅限于特定的亚区。来自生化实验的数据表明，在极化的上皮细胞中，携带基底面靶向蛋白的囊泡与紧密连接下方的质膜融合，与第6/8节的外囊复合体有关。然而，还不可能为这一结论提供直接的视觉证据。同样，还不可能确定新合成的一组膜蛋白是否在输送后迅速分散在细胞表面，或者它是否保持聚集和离散。这些问题的答案可能会极大地改变我们对膜蛋白靶向和回收机制中基本问题的理解。我们将应用Flash/ReAsH技术跟踪体内培养的Na，K-ATPase培养的肾上皮细胞合成后的命运。将采用的Na，K-ATPase结构在其C末端携带四半胱氨酸基序。我们已经证明，用完整的GFP序列修饰Na，K-ATPaseα亚基的N和C末端似乎不会显著改变该蛋白质的细胞生物学或功能行为，因此我们相信，添加短的四半胱氨酸基序至少也是可以容忍的。我们已经建立了稳定表达四半胱氨酸标记的Na，K-ATPase的MDCK上皮细胞。泵似乎是活跃的，并适当地定位于基侧质膜。我们已经成功地用闪光标记了这个构建物，我们发现标记模式与抗体染色揭示的碱侧定位相对应。因此，我们现在已经准备好在脉冲追逐方案中结合Flash和ReAsH标记，旨在揭示泵送的位置以及时间上定义的泵蛋白队列的分布和运输特性。表达四半胱氨酸标记的Na，K-ATPase的细胞将暴露在含有闪光和EDT的溶液中。在第一个标记步骤后的2、4和8小时，细胞将暴露在ReAsH和EDT中，之后它们将被固定并在荧光显微镜下检查。通过确定绿色和红色信号的相对分布，将有可能评估新合成的Na，K-ATPase队列是否插入特定的质膜亚区，以及它们是否在传递后保持聚集任何长度的时间。同样的技术也可以应用于其他极化细胞，如神经元，在这些细胞中，Na，K-ATPase类似地定位于细胞表面的特定亚域。因此，Flash和ReAsH标记技术的应用将使我们能够在生理相关的环境中定义Na，K-ATPase的基本且至今无法测量的细胞生物学特性。我们目前(2004年3月)正在建立适当的条件，用ReAsH单独标记我们的融合蛋白，以及在脉冲追逐方法中与闪光相结合。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Much has been learned about the cellular pathways pursued by newly synthesized ion transport proteins in polarized epithelial cells. Studies combining pulse chase radiolabeling protocols with various plasma membrane tagging or cell fractionation techniques provide information on the nature and duration of the subcellular itineraries that these proteins pursue en route to the plasma membrane. In general, however, it has not been possible to combine the temporal resolution provide by pulse labeling with the spatial resolution offered by microscopic techniques. Consequently, a number of very interesting issues have remained beyond the reach of experimental analysis. It remains unclear, for example, whether cargo vesicles carrying newly synthesized membrane proteins are able to dock and fuse with the entire plasmalemma or whether this function is restricted to specialized subdomains. Data from biochemical experiments suggest that vesicles carrying basolaterally targeted proteins in polarized epithelial cells fuse with the plasmalemma just below the tight junctions, in association with the sec 6/8 ¿¿exocyst¿¿ complex. It has not been possible, however, to provide direct visual evidence for this conclusion. Similarly, it has not been possible to determine whether a single cohort of newly synthesized membrane proteins disperses rapidly over the cell surface upon delivery, or whether it remains clustered and discrete. The answers to these questions could dramatically alter our understanding of fundamental issues in mechanics of membrane protein targeting and recycling. We will apply the FlAsH/ReAsH technique to follow the post-synthetic fate of Na,K-ATPase cultured renal epithelial cells in vivo. The Na,K-ATPase construct that will be employed carries the tetracysteine motif at its C terminus. We have shown that modifying the N and C termini of the Na,K-ATPase alpha subunit with the entire sequence of GFP does not appear to substantially alter this protein's cell biologic or functional behaviors, and thus we are confident that the addition of the short tetracysteine motif will be tolerated at least as well. We have already generated stably transfected MDCK epithelial cells expressing the tetracysteine-tagged Na,K-ATPase. The pump appears to be active and is localized appropriately to the basolateral plasmalemma. We have successfully labeled this construct with FlAsH, and we find that the labeling pattern corresponds to the basolateral localization revealed by antibody staining. We are now ready, therefore, to combine the FlAsH and ReAsH labels in a pulse chase protocol designed to reveal the site of pump delivery and the distributions and trafficking properties of temporally defined cohorts of pump proteins. Cells expressing tetracysteine-tagged Na,K-ATPase will be exposed to a solution containing FlAsH and EDT. At two, four and eight hours after the first labeling step cells will be exposed to ReAsH and EDT, after which they will be fixed and examined by fluorescence microscopy. By determining the relative distributions of the green and red signals, it will be possible to assess whether newly synthesized cohorts of Na,K-ATPase are inserted into specific plasmalemmal subdomains and whether they remain congregated for any length of time following their delivery. This same technique can also be applied to other polarized cells, such as neurons, in which the Na,K-ATPase is similarly localized to specialized sub-domains of the cell surface. Thus, the application of the FlAsH and ReAsH labeling technique will allow us to define fundamental, and here-to-fore unmeasurable, cell biologic properties of the Na,K-ATPase in physiologically relevant settings. We are currently (March 2004) setting up the appropriate conditions to label our fusion proteins with ReAsH, alone as well as in combination with FlAsH in a pulse-chase approach.