Polarized distribution of actin isoforms in gastric parietal cells.
The actin genes encode several structurally similar, but perhaps functionally 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 pool, we purified actin from the cytosol of gastric epithelial cells by DNase I affinity chromatography followed by two-dimensional gel electrophoresis. Actin isoforms were identified by Western blotting with a monoclonal antibody against all actin isoforms and two isoform-specific antibodies against cytoplasmic beta-actin and gamma-actin. Densitometry 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 gastric glandular cells in relation to ezrin, a putative membrane-cytoskeleton linker, we carried out double immunofluorescence using actin-isoform-specific antibodies and ezrin antibody. Immunostaining confirmed that ezrin resides mainly in canaliculi and apical plasma membrane of parietal cells. Staining for the beta-actin isoform was intense along 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 the basolateral membrane. The gamma-actin isoform was distributed heavily near the basolateral membrane of parietal cells, with much less intense staining of parietal cell canaliculi and no staining of apical membranes. Within parietal cells, the cellular localization of beta-actin, 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 membrane extracts in which substantial amounts of actin were co-eluted with ezrin from an anti-ezrin affinity column. The ratio of beta-actin/gamma-actin in the immunoprecipitate (beta/gamma = 2.14 +/- 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that beta- and gamma-actin isoforms are differentially 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 the beta- and gamma-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion.
Yao
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Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Chaponnier
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Gabbiani
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Forte
J G
JG
D000199:Actins / Q000032:analysis* / Q000302:isolation & purification; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D016764:Cell Polarity; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004847:Epithelial Cells; D010295:Parietal Cells, Gastric / Q000737:chemistry*; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification; D011817:Rabbits; D000099149:Ezrin
D000199:Actins; D003598:Cytoskeletal Proteins; D010750:Phosphoproteins; D000099149:Ezrin