Fig. 8. CNF-1 induces polarized restructuring of F-actin and actin-binding proteins in epithelial monolayers. Rhodamine phalloidin was used to highlight F-actin organization in the apical and basal poles of T84 epithelial cells following polarized incubation with CNF-1 or vehicle control. While control monolayers (a) and those incubated apically with CNF-1 (b) revealed identical organization of F-actin in en face confocal micrographs, basolateral exposure to CNF-1 (c) induced dramatic polarized F-actin restructuring. Slight increases in staining intensity were observed in the perijunctional F-actin ring, but the normal pattern of punctate microvillous staining was virtually abolished in the apical pole of these monolayers (arrow) relative to controls. These changes were accompanied by altered immunolocalization of the brush border actin-binding protein villin in CNF-treated monolayers (f) relative to control cells (d) and those treated apically with toxin (e). F-actin distribution at the basal epithelial pole was similar in both control (g) and apically-treated monolayers (h), and consisted of diffuse meshworks of short stress fibers. By contrast, basolateral exposure to CNF-1 (i) stimulated the aggregation of stress fibers into thicker, `cabled' bundles. Parallel changes were detected in immunolocalization of the actin-binding protein paxillin at the basal epithelial pole. Control monolayers (j) displayed `plaque-like' paxillin immunoreactivity, similar to that in monolayers exposed apically to toxin (k). Basolateral treatment with CNF-1 (l) was associated with a slight increase in plaque number and size. CNF-induced changes in F-actin, villin and paxillin (m, n, o, respectively) were not accounted for by changes in the total levels of these proteins as assessed by western blot analysis of lysates from control (lanes 1-3) and CNF-treated monolayers (lanes 4-6) for the times indicated. Slight increases in the molecular mass of paxillin upon CNF incubation reflected tyrosine phosphorylation of the protein, as shown by the increased level of phospho-specific paxillin detected in lysates from CNF-treated cells relative to controls (p). Bar10 µm.