Laboratory  of  John G. Forte

Department of Cell & Molecular Biology,  University of California, Berkeley
 
 

The parietal cell canalicular network, visualized by fluorescent labeling of F-actin and ezrin in isolated rabbit gastric glands

  
Ezrin is concentrated at canalicular (apical) membrane of gastric parietal cell, showing extent, and complex branching, of apical membrane invaginated from small apical pore at lower right.  (Probed with mouse anti-ezrin & Texas Red-labeled secondary) F-actin is concentrated along canalicular membrane and extending through cell's apical pore into gland lumen, part of which is seen at lower right.  (Probed with FITC-labeled phalloidin) Lower-power view of gastric gland, with ezrin and F-actin images merged.  Colocalization is seen as yellow in canalicular networks of several parietal cells.  F-actin staining continues into lining of gland lumen. 
  Blank spaces between narrow apical poles of parietal cells are not empty, but are occupied by other cells that secrete pepsinogen or mucus.  They have too little ezrin or F-actin (except for F-actin at their apical membranes, which form most of the lining of the gland lumen) to be visible at the exposure settings used for parietal cells.

 

Taken from the following publication:

 

  THE EMBO JOURNAL, Vol. 10, No. 9, 2363-2373, Sept. 1991
 

The secretion-stimulated 80K phosphoprotein of parietal cells is ezrin, and has properties of a membrane cytoskeletal linker in the induced apical microvilli
  D. Hanzel, H. Reggio, A. Bretscher, J. G. Forte  and P. Mangeat
Department of Molecular and Cell Biology, University of California, Berkeley, CA  94720 		 
Abstract: Stimulation of gastric acid secretion in parietal cells involves the translocation of the proton pump (H,K-ATPase) from cytoplasmic tubulovesicles to the apical membrane to form long, F-actin-containing microvilli. Following secretion, the pump is endocytosed back into tubulovesicles. The parietal cell therefore offers a system for the study of regulated membrane recycling, with temporally separated endocytic and exocytic steps. During cAMP-mediated stimulation, an 80 kDa peripheral membrane protein becomes phosphorylated on serine residues. This protein is a major component, together with actin and the pump, of the isolated apical membrane from stimulated cells, but not the resting tubulovesicular membrane. Here we show that the gastric 80 kDa phosphoprotein is closely related or identical to ezrin, a protein whose phosphorylation on serine and tyrosine residues was recently implicated in the induction by growth factors of cell surface structures on cultured cells [Bretscher, A. (1989) J. Cell Biol., 108, 921-930]. Light and electron microscopy reveal that ezrin is associated with the actin filaments of the microvilli of stimulated cells, but not with the filaments in the terminal web. In addition, a significant amount of ezrin is present in the basolateral membrane infoldings of both resting and stimulated cells. Extraction studies show that ezrin is a cytoskeletal protein in unstimulated and stimulated cells, and its association with the cytoskeleton is more stable in stimulated cells. These studies indicate that ezrin is a membrane cytoskeletal linker that may play a key role in the control of the assembly of secretory apical microvilli in parietal cells and ultimately in the regulation of acid secretion. Taken together with the earlier studies, we suggest that ezrin might be a general substrate for kinases involved in the regulation of actin-containing cell surface structures.

 

 

 

From Duman, et al., J Cell Sci. 2002:

 
TEM of canalicular cross-sections in gastric parietal cell.  Many mitochondria (m) and some areas of tubulovesicles (arrowheads) surround the canaliculi.  A few examples of endocytosis from the canalicular membrane (arrows) are also seen.  Many microvilli extend into the canalicular lumina, most seen in cross-section, but a few lying in the plane of the thin section.  Bar = 1μm.
(Click to see large image in new window.) 		3-dimensional reconstruction of microvilli
in portion of canaliculus, from serial
ultra-thin sections of rabbit gastric glands

 

 

 

Cultured parietal cells:

When parietal cells are isolated and cultured (method of Chew, et al., 1989 PubMed link), the apical pore closes and the previously basolateral membrane becomes the complete plasma membrane.  The canaliculi pinch off from this plasma membrane to form several intracellular vacuoles that retain apical membrane characterisics.  Stimulation causes tubulovesicles to fuse with these engulfed apical membranes.  The accompanying graphic is a 3-D reconstruction from serial-section confocal microscopy of a cultured, resting parietal cell. It was probed with green-labeled anti-H,K-ATPase, identifying the tubulovesicles, and with red-labeled phalloidin, which binds to F-actin, concentrated at the vacuolar (apical) membranes and, more diffusely, near the plasma membrane.  In this cell, small amounts of H,K-ATPase remaining at the apical membrane cause the vacuolar outlines to appear slightly orange compared to the pure red seen at the periphery of the cell.

Such cultured cells are being used in our lab for expression of certain proteins altered by site mutation (e.g., of phosphorylation sites) and by linkage to Green Fluorescent Protein (GFP) to study their function and localization in parietal cell activation and determination of cell polarity.  (See Am J Physiol Cell link, May 2010)