Serhan Karvar

Phosphorylation of radixin regulates cell polarity and Mrp-2 distribution in hepatocytes

Radixin, the dominant ezrin-radixin-moesin (ERM) protein in hepatocytes, has two important binding domains: an NH(2)-terminal region that binds to plasma membrane and a COOH-terminal region that binds to F-actin after a conformational activation by phosphorylation at Thr564. The present studies were undertaken to investigate the cellular changes in expression of radixin in WIF-B cells and to assess radixin distribution and its influence on cell polarity. We used a recombinant adenoviral expression system encoding radixin wild-type and Thr564 mutants fused to cyan fluorescent protein (CFP), as well as conventional immunostaining procedures. Functional analyses were characterized quantitatively. Similar to endogenous radixin, adenovirus-infected radixin-CFP-wild type and nonphosphorylatable radixin-CFP-T564A were found to be expressed heavily in the compartment of canalicular membrane vacuoles, typically colocalizing with multidrug resistance-associated protein 2 (Mrp-2). Expression of radixin-CFP-T564D, which mimics constant phosphorylation, was quite different, being rarely associated with canalicular membranes. The WIF-B cells were devoid of a secretory response, T567D radixin became predominantly redistributed to the basolateral membrane, usually in the form of dense, long spikes and fingerlike projections, and the altered cell polarity involved changes in apical membrane markers. Differences in polar distribution of radixin suggest a role for the linker protein in promoting formation and plasticity of membrane surface projections and also suggest that radixin might be an organizer and regulator of Mrp-2 and cell polarity in hepatocytes.

A new approach for high-pressure freezing of primary culture cells: the fine structure and stimulation-associated transformation of cultured rabbit gastric parietal cells

A newly designated procedure for high‐pressure freezing of primary culture cells provided excellent ultrastructure of rabbit gastric parietal cells. The isolated parietal cells were cultivated on Matrigel‐coated aluminium plates for conventional subsequential cryoimmobilization by high‐pressure freezing. The ultrastructure of different organelles (Golgi apparatus, mitochondria, multivesicular bodies, etc.) was well preserved compared to conventional chemical fixation. In detail, actin filaments were clearly shown within the microvilli and the subapical cytoplasm. Another striking finding on the cytoskeleton system is the abundance of microtubules among the tubulovesicles. Interestingly, some microtubules appeared to be associating with tubulovesicles. A large number of electron‐dense coated pits and vesicles were observed around the apical membrane vacuoles in cimetidine‐treated resting parietal cells, consistent with an active membrane uptake in the resting state. Immunogold labelling of H+/K+‐ATPase was seen on the tubulovesicular membranes. When stimulated with histamine, the cultured parietal cells undergo morphological transformation, resulting in great expansion of apical membrane vacuoles. Immunogold labelling of H+/K+‐ATPase was present not only on the microvilli of expanded apical plasma membrane vacuoles but also in the electron‐dense coated pits. The present findings provide a clue to vesicular membrane trafficking in cultured gastric parietal cells, and assure the utility of the new procedure for high‐pressure freezing of primary culture cells.

Cellular Localization and Stimulation‐Associated Distribution Dynamics of Syntaxin‐1 and Syntaxin‐3 in Gastric Parietal Cells

Syntaxins are differentially localized in polarized cells and play an important role in vesicle trafficking and membrane fusion. These soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) proteins are believed to be involved in tubulovesicle trafficking and membrane fusion during the secretory cycle of the gastric parietal cell. We examined the cellular localization and distribution of syntaxin‐1 and syntaxin‐3 in rabbit parietal cells. Fractionation of gastric epithelial cell membranes showed that syntaxin‐1 was more abundant in a fraction enriched in apical plasma membranes, whereas syntaxin‐3 was found predominantly in the H,K‐ATPase‐rich tubulovesicle fraction. We also examined the cellular localization of syntaxins in cultured parietal cells. Parietal cells were infected with CFP‐syntaxin‐1 and CFP‐syntaxin‐3 adenoviral constructs. Fluorescence microscopy of live and fixed cells demonstrated that syntaxin‐1 was primarily on the apical membrane vacuoles of infected cells, but there was also the expression of syntaxin‐1 in a subadjacent cytoplasmic compartment. In resting, non‐secreting parietal cells, syntaxin‐3 was distributed throughout the cytoplasmic compartment; after stimulation, syntaxin‐3 translocated to the apical membrane vacuoles, there co‐localizing with H,K‐ATPase, syntaxin‐1 and F‐actin. The differential location of these syntaxin isoforms in gastric parietal cells suggests that these proteins may be critical for maintaining membrane compartment identity and that they may play important, but somewhat different, roles in the membrane recruitment processes associated with secretory activation.

The Cell Biology of Gastric Acid Secretion

Abstract The regulation of gastric HCl secretion by the parietal cell occurs primarily through the regulated recycling of H,K-ATPase. Secretagogue stimulation of the parietal cell initiates HCl secretion by effecting an intracellular signaling and biochemical pathway that culminates with the membrane fusion of intracellular H,K-ATPase-rich tubulovesicles with, and thus, the delivery of the H,K-ATPase to the secretory canaliculus (apical membrane). On withdrawal of the secretagogue, cessation of secretion occurs concomitantly with the retrieval of the H,K-ATPase from the secretory canaliculus, and the reformation of the tubulovesicular membrane compartment. The H,K-ATPase resides quiescently in this compartment until another round of secretion is initiated. Intracellular signaling pathways, cytoskeletal elements, and vesicular trafficking machinery must be coordinately regulated to effect this membrane-recycling pathway. This chapter summarizes key findings from experimental approaches taken toward the elucidation of the mechanism of gastric HCl secretion, through the use of electron microscopy, electrophysiology, membrane biochemistry, molecular biology, cell biology, and atomic structure, in the characterization of the regulation of activity of the H,K-ATPase itself, to that of associated ion channels and membrane transporters, as well as of the regulation of H,K-ATPase trafficking.