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

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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.