Increased Number and Microtubule-Associated Dispersal of Acidic Intracellular Compartments Accompany Differentiation of Cultured Human Keratinocytes

Abstract

Intracellular acidic compartments serve several functions, including uptake of nutrients, processing and sorting of secreted and membrane-bound proteins, and even entry of viruses into cells. In this study, we examined the distribution of acidic compartments in normal human keratinocytes cultured in serum-free medium. Acridine orange was used to stain acidic organelles (red fluorescence), and adherent cells were evaluated by fluorescence microscopy and by interactive laser cytometry (ILC). Keratinocytes cultured in low [Ca++] (0.15 mM) exhibited morphologic characteristics associated with basal cells; red acidic vesicles in these cells were aggregated around the nucleus, sparing the peripheral cytoplasm. After 24 h of culture in high [Ca++] (1.5 mM) keratinocytes showed morphologic changes associated with differentiated cells, including increased number and dispersal of red vesicles to the periphery of the cytoplasm. Keratinocytes cultured in 0.15 mM [Ca++], but treated with phorbol 12-myristate 13-acetate (PMA, 5-100 ng/ml) to induce terminal differentiation, developed similar features. Incubation in media with either high [Ca++] or PMA also induced radial extension of the microtubule network, suggesting that the distribution of acidic organelles occurs along this network. Finally, crude keratinocyte membranes were evaluated by radioactive assay for the presence of three ion-translocating ATPase activities, plasma membrane Na/K ATPase, mitochondrial ATPase, and vacuolar H+ pump ATPase, the latter being the activity responsible for acidification of intracellular compartments. Both basaloid and differentiated keratinocytes exhibited similar vacuolar H+ pump ATPase activity, as measured by its sensitivity to bafilomycin.