Assembly Dynamics of Epidermal Keratins K1 and K10 in Transfected Cells

Abstract

Keratins K1 and K10 are early markers of keratinization. Their expression starts when basal keratinocytes are committed to differentiate. To study keratin stability and intermediate filament (IF) assembly and dynamics, we have forced the expression of K1/K10 in epithelial and nonepithelial cell lines. It was observed that these keratins are unable to generate a normal cytoskeleton in fibroblasts, where they form, at most, abnormally short and twisted filaments. However, when transfected into epithelial cells they frequently cointegrate with the endogenous keratin into a well-developed cytoskeleton. These results suggest that the K1/K10 pair is unable to form a keratin network on its own and that, mimicking the in vivo situation, requires a preexisting cytoskeleton. Besides filaments, transfected K1/K10 also form fairly stable, regularly sized round aggregates with no evidence of organization into IF. Transfections with single genes demonstrated that these structures were generated by K10 alone. These aggregates interacted with most components of the cellular cytoskeleton, altering the distribution of the endogenous keratins, actin, vimentin, and tubulin. Kinetic experiments in transfected PtK2 cells showed that, contrary to expectations, K10 does not integrate directly into the preexisting cytoskeleton, but assembles into the stable nonfilamentous aggregates. From these structures, K10 evolves toward the formation of IF together with the endogenous keratins through a complex and highly dynamic process, which involves substantial rearrangement of the endogenous keratin cytoskeleton. These results demonstrate that K1 and K10 have properties different from those described for other keratins and that epithelial IF are surprisingly dynamic structures.