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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。关于新合成的离子转运蛋白在极化上皮细胞中所追求的细胞途径，我们已经了解了很多。将脉冲追踪放射性标记方案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通往质膜的途中所追求的亚细胞路线的性质和持续时间的信息。然而，一般来说，将脉冲标记提供的时间分辨率与显微技术提供的空间分辨率结合起来是不可能的。因此，许多非常有趣的问题仍然超出了实验分析的范围。例如，目前尚不清楚的是，携带新合成膜蛋白的货物囊泡是否能够与整个质膜对接并融合，或者这种功能是否仅限于特定的子结构域。生化实验数据表明，极化上皮细胞中携带基底侧靶向蛋白的囊泡与紧连接下方的质膜融合，与sec 6/8外囊复合物相关。然而，不可能为这一结论提供直接的视觉证据。同样，也不可能确定新合成的膜蛋白是否在递送时在细胞表面迅速分散，或者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再循环机制基本问题的理解。我们将应用FlAsH/ReAsH技术在体内跟踪Na， k - atp酶培养的肾上皮细胞合成后的命运。Na， k - atp酶结构体将在其C端携带四胱氨酸基序。我们已经证明，用GFP的整个序列修饰Na， k - atp酶α亚基的N和C端似乎不会实质性地改变该蛋白的细胞生物学或功能行为，因此我们相信，短四胱氨酸基序的添加至少也是可以容忍的。我们已经产生了稳定转染的MDCK上皮细胞，表达四胱氨酸标记的Na， k - atp酶。泵似乎是活跃的，并适当地定位于基底外侧质膜。我们已经成功地用FlAsH标记了这个结构，我们发现标记模式与抗体染色显示的基底外侧定位相对应。因此，我们现在准备将FlAsH和ReAsH标签结合在脉冲追踪协议中，旨在揭示泵传递的位置以及暂时定义的泵蛋白队列的分布和运输特性。表达四胱氨酸标记Na， k - atp酶的细胞将暴露在含有FlAsH和EDT的溶液中。在第一个标记步骤后的2、4和8小时，细胞将暴露于ReAsH和EDT中，之后将它们固定并通过荧光显微镜检查。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 - atp酶群是否插入到特定的浆体亚结构域，以及它们在传递后是否保持聚集的时间长度。同样的技术也可以应用于其他极化细胞，如神经元，其中Na， k - atp酶类似地定位于细胞表面的特定子域。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 - atp酶的基本的、迄今为止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，以及在脉冲追踪方法中与FlAsH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.