Intracellular distribution of beta-actin mRNA is polarized in embryonic corneal epithelia.
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abstract
The intracellular distribution of filamentous actin (F-actin), all actin isoforms and beta-actin mRNA were analyzed in whole-mount preparations of freshly isolated corneal epithelia. Filamentous actin distribution was analyzed with fluorescently tagged phalloidin. An antibody that recognizes an epitope on both globular (G-actin) and F-actin was used in an immunohistochemical analysis of actin protein distribution. Whole-mount epithelial tissues were examined with a confocal laser scanning microscope (CLSM). Biotinylated oligonucleotide probes specific for the beta-actin mRNA were used, and visualized with avidin-FITC. The intracellular localization of the beta-actin mRNA was similar to the F-actin protein distribution. In the most apical optical sections of embryonic cornea, actin staining delineated the cell borders and microvilli of the periderm cells. The actin is also detected as an organized network at the interface between the basal and periderm cells. At the level of the basal cell nucleus, F-actin is sparse, associating only with the lateral cell membranes. However, at the optical plane below the nuclei, the actin forms an elaborate actin cortical mat. Actin mRNA staining was visualized as discrete punctate areas. The beta-actin mRNA was positive at the optical plane just below the periderm cell apical membrane surface, similar to actin in microvilli. These cells also contained punctate staining near the cell membranes and in the periderm-basal cell junction area. At the level of the basal cell nucleus the actin mRNA was present in a punctate pattern along the cell membranes. Below the basal cell nuclei the actin mRNA staining increased at the level of the actin cortical mat. These experiments are the first demonstration that actin mRNA is polarized in embryonic corneal epithelia and co-localized with actin protein in an intact tissue.
