Focal adhesion proteins associated with apical stress fibers of human fibroblasts.

Human fibroblasts stained with fluorescently labeled phalloidin revealed many stress fibers within the apical cytoplasm in addition to those located along the basal plasma membrane and associated with focal adhesions. The staining patterns of these apical stress fibers with fluorescent phalloidin, anti-alpha-actinin, and antimyosin were identical to those of the basal stress fibers, suggesting the same macromolecular organization for both types of stress fibers. There were two types of apical stress fibers that clearly interacted with the apical plasma membrane, those extending between the basal and the apical plasma membrane and those having both ends on the basal membrane forming arches whose top interacted with the apical plasma membrane. By electron microscopy, we observed that apical stress fibers were associated with the apical plasma membrane via electron-dense plaques reminiscent of the focal adhesion. Since several proteins have been specifically localized to the focal adhesion site, we examined whether they were also present at the apical stress fiber-membrane association site by using immunocytochemical methods and image reconstruction techniques. We found that vinculin, talin, paxillin, a fibronectin receptor protein, several integrin subunits including beta 1, fibronectin, and proteins with phosphorylated tyrosine were also components of the apical plaque. These observations indicate that apical stress fibers are attached to the plasma membrane by using principally the same molecular assembly as the focal adhesion associated with the basal stress fiber. We suggest that the complex molecular organization of the focal adhesion is not demanded by cell adhesion, but rather it is needed for anchoring stress fibers to the plasma membrane. Apical plaques did not stain with the anti-integrin alpha v subunit or anti-focal adhesion associated kinase (FAK), although these antibodies stained focal adhesions. These results suggest that the apical stress fiber-membrane contact has some important functions different from those of the focal adhesion.

Effects of phalloidin on the epithelial regeneration in the mouse gastric mucosa.

Effects of phalloidin on the early (5-60 min) processes of wound repair were examined in the ethanol-injured mouse gastric mucosa. Within 5 min after ethanol was washed out from the stomach, survived epithelial cells changed their shape and became flat extending both lamellipodia and filopodia. The denuded basal lamina was re-covered by these stretched cells within 30 min. When a low dose of phalloidin (5 micrograms) was applied to the wounded stomach, it took 60 min or more to cover the exposed basal lamina. Epithelial cells at the wound edge of the phalloidin-treated stomach often contained unusually thick layer of microfilaments in their subapical region. This observation suggests that phalloidin induces active microfilament assembly mainly in the apical region of epithelial cell processes.

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.

[Cell adhesion and the cytoskeleton].

Most cells have macromolecules on their outer surface that are specialized for adhesion. Cells can attach to another cell and/or various extracellular matrix components. When tissue culture cells attach to the substrate, they form a specialized structure called adhesion plaque. At the cytoplasmic side of the adhesion plaque, stress fibers terminate, forming an electron-dense plasma membrane undercoat structure. Integrin is localized to the adhesion plaque and this is a transmembrane protein that connects the cytoskeleton to the extracellular matrix. Endothelial cells in vivo have stress fibers, and we have recently found that the ends of these stress fibers also terminate at a structure similar to the adhesion plaque of cultured cells. It appears, therefore, that endothelial cells in vivo employ similar, if not identical, mechanism for adhesion as the one used by tissue culture cells.

An osmium-ferricyanide staining method for the intercellular space in the small intestinal epithelium.

The post-fixation with osmium tetroxide and potassium ferricyanide (OsFeCN) produced dense deposits on the outer surface of the lateral plasma membrane in the mouse small intestinal epithelium. The deposits also filled up the intercellular space. No deposits were found on the surface of apical and basal plasma membranes. This staining pattern was highly reproducible when pH of the OsFeCN solution was adjusted to 7.0 by cacodylate buffer without calcium ion. Thus, our modified OsFeCN method is useful to selectively stain the intercellular space in epithelial tissues.

Cytotoxic effect of the thermostable direct hemolysin produced by Vibrio parahaemolyticus on FL cells.

The thermostable direct hemolysin produced by Vibrio parahaemolyticus showed cytotoxic activity on FL cells derived from human amniotic membrane. Scanning electron micrographs of the whole cells showed that the microvilli on the cell surface decreased in number and changed in shape on treatment with the hemolysin. Most of the microvilli disappeared before death of the cells, as judged from the results of staining the cells with trypan blue and measuring release of alkaline phosphatase from the cells. Electron micrographs of thin sections ofthe cells showed that the cytoplasm of the cells was not significantly affected by treatment with sublethal amounts of hemolysin, even when the microvilli on the cell surface were significantly affected. Lethal amounts of hemolysin affected the cytoplasm and caused disarovilli on the cell surface are affected by treatment with the hemolysin before cytoxic effects develop.

Interaction of thermostable direct hemolysin of Vibrio parahaemolyticus with human erythrocytes.

The interaction between thermostable direct hemolysin produced by Vibrio parahaemolyticus WP-1 and human erythrocytes was studied. The lysis of human erythrocytes by the hemolysin was dependent of temperature and no hemolysis occurred at low temperature (0-4 C), but the hemolysin was adsorbed on human erythrocytes even at low temperature. No hemolysis was observed when antihemolysin antiserum was mixed with the hemolysin and human erythrocytes at zero time. On the other hand, lysis of the cells by hemolysin was not completely inhibited when the antiserum was added during the lag time and the inhibitory effect decreased with delay in the time of addition of antiserum. The inhibitory effect of the antiserum decreased with increase in the incubation temperature, increase in the concentration of divalent cations, and decrease in pH. These results suggest that lysis of human erythrocytes by the hemolysin is at least a two-step process consisting of adsorption of the hemolysin to human erythrocytes and the step(s) following adsorption.