Kinetics of cyclic AMP-dependent accumulation of novel intermediate filament proteins in cultured chick lens cells

Abstract

Purpose. To refine the parameters affecting the accumulation of cytoskeletal markers of lens fiber terminal differentiation. Methods. Primary cultures of chick lens annular pad cells were treated with a lipid soluble cyclic AMP analog under various culture conditions. The accumulation of beaded filament proteins, unique markers of lens fiber terminal differentiation, was quantified with an ELISA assay. The incorporation of beaded filament proteins into macromolecular structures was followed with immunofluorescence microscopy. Results. In a time- and dose-dependent manner, beaded filament protein levels were increased in cyclic nucleotide treated cells. The addition of serum to treated cells caused a further dose-dependent increase in beaded filament protein levels. The continuous presence of cyclic nucleotides for maximal beaded filament protein accumulation was also established. At the light microscopic level, cyclic nucleotide treatment produced much more extensive multilayering of cells and lentoid formation. Macromolecular structures containing beaded filament proteins also increased in both abundance and complexity after cyclic nucleotide treatment and were restricted to the multilayers/lentoids. Conclusions. These results indicate that multiple mechanisms (including cyclic AMP, serum factors, and the degree of cell–cell interactions) affect the accumulation of beaded filament proteins during the normal differentiation of lens fibers.