Polarized distribution of actin isoforms in gastric parietal cells.

Abstract

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 ,B-actin and -y-actin. Densitometry revealed a ratio for f3-actin/ y-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 ,B-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 ,B-actin were also identified near the basolateral membrane. The y-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 ,B-actin, but not y-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 13-actin/ y-actin in the immunoprecipitate (3/,y = 2.14 + 0.32) was significantly greater than that found in the cytosolic fraction. In summary, we have shown that ,B-and 'y-actin isoforms are differentially distributed in gastric parietal cells. Furthermore, our data suggest a preferential, but not exclusive, interaction between 1B-actin and ezrin in gastric parietal cells.Finally, our results suggest that the ,B-and y-actin-based cytoskeleton networks might function separately in response to the stimulation of acid secretion. INTRODUCTION essential role in a variety of cellular processes such as maintaining cell shape, cell motility, and cytokinesis.

Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo

“…In the gastric parietal cell, there are at least two distinct pools of actin. At the cell cortex, γ-actin predominates whereas β-actin predominates in the canalicular region (Yao et al, 1995) The cortical actin pool is exquisitely sensitive to latrunculin B, which binds monomeric actin, whereas the formation of microvillar filaments appear to be highly resistant to this inhibitor as well as to cytochalasin D, which severs actin filaments (Ammar et al, 2001). Thus, at the cell cortex, a reduced affinity for actin could assist in the rapid remodeling of the actin cytoskeleton (Pollard, 1999).…”

Immunohistochemical Analysis of the Acid Secretion Potency in Gastric Parietal Cells

“…Yao et al [15] revealed that most actin in the gastric gland is present in parietal cells, which are largely globular during the inactive resting secretion phase. However, during the active phase, actin forms a filamentous structure.…”

“…In addition, actin binds to intramembranous CD44 via phosphoryl ezrin in the plasma membrane [12][13][14]. It has previously been suggested that the above-mentioned morphological changes are induced and triggered by this cytoskeletal reorganization of β-actin [15][16][17].…”

Cell Polarity Kinase MST4 Cooperates with cAMP-dependent Kinase to Orchestrate Histamine-stimulated Acid Secretion in Gastric Parietal Cells

“…Gastric glands were isolated from New Zealand White rabbits as modified by Yao et al (5). Briefly, the rabbit stomach was perfused under high pressure with PBS (2.25 mM K 2 HPO 4 , 6 mM Na 2 HPO 4 , 1.75 mM NaH 2 PO 4 , and 136 mM NaCl (pH7.4)) containing 1 mM CaCl 2 and 1 mM MgSO 4 .…”

“…Within the gastric epithelium, ezrin has been localized exclusively to parietal cells and primarily to the apical canalicular membrane of these cells (3,4). Our previous studies have shown that gastric ezrin is co-distributed with the ␤-actin isoform in vivo (5) and preferentially bound to the ␤-actin isoform in vitro (6). It has been postulated that ezrin couples the activation of protein kinase A (PKA) to the apical membrane remodeling associated with parietal cell secretion (3,7).…”