INTRACELLULAR RELATIONSHIP BETWEEN ACTIN AND ALPHA-ACTININ IN A WHOLECORNEAL EPITHELIAL TISSUE

Alpha-actinin is an actin crosslinking protein that may be one of the proteins involved in the attachment of the actin cytoskeletal framewor k to the plasma membrane. We investigated the distribution of alpha-ac tinin in whole-mount embryonic chick corneal epithelia using confocal laser scanning analysis. The intracellular alpha-actinin distribution was compared with F-actin using phalloidin, or total actin using an an ti-actin antibody. Corneal epithelial tissues were isolated with or wi thout the basal lamina (+ or -BL), and fixed immediately. In addition, epithelia isolated -BL were cultured for 2 hours with either control medium, laminin-supplemented medium or laminin and cytochalasin D (CD) -containing medium. The single- and double-labeled epithelia showed th at alpha-actinin delineated the cell borders and microvilli of the per iderm cells in the most apical optical sections of control and laminin -treated epithelia. At the optical plane through the basal cell nuclei , the alpha-actinin was distributed diffusely throughout the cytoplasm , whereas the actin was sparse, only associated with the lateral cell membranes. Epithelia (-BL) cultured in control medium had cytoplasmic protrusions or blebs on the basal cell surface. The blebs contained bo th actin and alpha-actinin. In epithelia cultured with laminin, the ba sal cell surface was flat. The actin cortical mat became reorganized w ithin two hours. Actin and alpha-actinin were colocalized in the re-fo rmed basal cytoskeletal network. In cells cultured with cytochalasin D (CD) and laminin the actin cortical mat was not reorganized. Actin ne tworks from both cell layers were eliminated and replaced by aggregate s scattered throughout the cytoplasm. The alpha-actinin remained diffu sely distributed in the cytoplasm and failed to colocalize with the ac tin aggregates. The alpha-actinin appeared closer to the basal cell me mbrane than the actin in cross-sectional views of the tissue. Results from these double-labeling experiments confirmed the intimate associat ion of alpha-actinin and actin in the laminin-stimulated actin cortica l mat reorganization. This study is the first to demonstrate that CD-a ggregated F-actin does not capture the alpha-actinin. The alpha-actini n appeared to remain diffuse throughout the cytoplasm and separate fro m F-actin; however, there was some overlap with G-actin.