Transient expression of mouse hair keratins in transfected HeLa cells: interactions between "hard" and "soft"...

Although it has been shown previously that an acidic (type I) "soft" keratin can interact with many basic (type II) "soft" keratins to form 10-nm intermediate filaments, it has been unclear whether "soft" keratins are compatible with the "hard" keratins typically found in hair and nail. To address this issue and to generate more structural information about hard keratins, we have isolated and sequenced a cDNA clone that encodes a mouse hair basic keratin (b4). Our sequence data revealed new information regarding the structural conservation of hard keratins as a group, being significantly different from soft keratins. Using expression vectors containing appropriate cDNA inserts, we studied the expression of this basic (b4) as well as an acidic (a1) mouse hair keratin in HeLa cells. The expression of these alien hair keratins in the transfected cells was surveyed using a panel of monoclonal and polyclonal antibodies. Our results indicated that the basic and acidic hair keratin readily incorporated into the existing endogenous soft keratin network of HeLa cells. Overproduction of hair keratin, however, occasionally led to the formation of cytoplasmic aggregates containing both hard and soft keratins. These data suggest that although small amounts of newly synthesized hair keratins can incorporate into the "scaffolding" of the preformed soft keratin filament network, possibly through dynamic subunit exchange, overproduction of hard keratins can lead to the partial collapse of the soft keratin network. These observations, along with the deduced amino acid sequence data, support and extend the concept that hard and soft keratins, although closely related, are divergent enough to justify their being divided into two separate subgroups.