Fig. 2. Expression of CFP-ezrin constructs in live parietal. (A) Examples of cells infected with rAD-CFP-ezrin WT. (B) Cells infected with rAD-CFP-ezrin T567A. (C) Cells infected with rAD-CFP-ezrin T567D. For all cases images are shown for parietal cells maintained in the resting, nonsecreting, state (rest) and 20-30 minutes after cells had been stimulated to secrete acid (stim) by histamine plus IBMX. For WT and the T567A mutant, CFP-ezrin was mainly targeted to relatively large vacuoles that are derived from the apical plasma membrane when the cells are cultured, thus we refer to them as apical membrane vacuoles. There are lesser amounts of CFP-ezrin WT and T567A displayed on the basolateral membrane (surrounding plasma membrane) and in the cytoplasm. Cells expressing both CFP-ezrin WT and T567A respond well to stimulation with a large expansion of apical membrane vacuoles that fill with secreted acid. Note: the rest/stim images at the lower left are the same cells expressing CFP-ezrin WT at rest and after 20 minutes stimulation. The targeting of CFP-ezrin T567D mutant was totally different. The CFP signal was detected primarily at the basolateral membrane, often in the form of elaborate surface projections, and the cells did not display the normal secretory response to stimulation. Bars, 10 µm.