POLARIZED DISTRIBUTION OF ACTIN ISOFORMS IN GASTRIC PARIETAL-CELLS

The actin genes encode several structurally similar, but perhaps funct ionally different, protein isoforms that mediate contractile function in muscle cells and determine the morphology and motility in nonmuscle cells. To reveal the isoform profile in the gastric monomeric actin p ool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel elect rophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specifi c antibodies against cytoplasmic beta-actin and gamma-actin. Densitome try revealed a ratio for beta-actin/gamma-actin that equaled 0.73 +/- 0.09 in the cytosol. To assess the distribution of actin isoforms in g astric glandular cells in relation to ezrin, a putative membrane-cytos keleton linker, we carried out double immunofluorescence using actin-i soform-specific antibodies and ezrin antibody. Immunostaining confirme d that ezrin resides mainly in canaliculi and apical plasma membrane o f parietal cells. Staining for the beta-actin isoform was intense alon g the entire gland lumen and within the canaliculi of parietal cells, thus predominantly near the apical membrane of all gastric epithelial cells, although lower levels of beta-actin were also identified near t he basolateral membrane. The gamma-actin isoform was distributed heavi ly near the basolateral membrane of parietal cells, with much less int ense staining of parietal cell canaliculi and no staining of apical me mbranes. Within parietal cells, the cellular localization of beta-acti n, but not gamma-actin, isoform superimposed onto that of ezrin. In a search for a possible selective interaction between actin isoforms and ezrin, we carried out immunoprecipitation experiments on gastric memb rane extracts in which substantial amounts of actin were co-eluted wit h ezrin from an anti-ezrin affinity column. The ratio of beta-actin/ga mma-actin in the immunoprecipitate (beta/gamma 2.14 +/- 0.32) was sign ificantly greater than that found in the cytosolic fraction. In summar y, we have shown that beta- and gamma-actin isoforms are differentiall y distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between beta-actin and ezrin in gastric parietal cells. Finally, our results suggest that th e beta- and gamma-actin-based cytoskeleton networks might function sep arately in response to the stimulation of acid secretion.