A role for disulfide bonding in keratin intermediate filament organization and dynamics in skin keratinocytes

We recently reported that a trans-dimer, homotypic disulfide bond involving Cys367 in keratin 14 (K14) occurs in an atomic-resolution structure of the interacting K5/K14 2B domains and in keratinocyte cell lines. Here we show that a sizable fraction of the K14 and K5 protein pools participates in interkeratin disulfide bonding in primary cultures of mouse skin keratinocytes. By comparing the properties of wild-type K14 with a completely cysteine-free variant thereof, we found that K14-dependent disulfide bonding limited filament elongation during polymerization in vitro but was necessary for the genesis of a perinuclear-concentrated network of keratin filaments, normal keratin cycling, and the sessile behavior of the nucleus and whole cell in keratinocytes studied by live imaging. Many of these phenotypes were rescued when analyzing a K14 variant harboring a single Cys residue at position 367. These findings establish disulfide bonding as a novel and important mechanism regulating the assembly, intracellular organization, and dynamics of K14-containing intermediate filaments in skin keratinocytes.

Forced Expression of Keratin 16 Alters the Adhesion, Differentiation, and Migration of Mouse Skin Keratinocytes

Injury to the skin results in an induction of keratins K6, K16, and K17 concomitant with activation of keratinocytes for reepithelialization. Forced expression of human K16 in skin epithelia of transgenic mice causes a phenotype that mimics several aspects of keratinocyte activation. Two types of transgenic keratinocytes, with forced expression of either human K16 or a K16-C14 chimeric cDNA, were analyzed in primary culture to assess the impact of K16 expression at a cellular level. High K16-C14-expressing and low K16-expressing transgenic keratinocytes behave similar to wild type in all aspects tested. In contrast, high K16-expressing transgenic keratinocytes show alterations in plating efficiency and calcium-induced differentiation, but proliferate normally. Migration of keratinocytes is reduced in K16 transgenic skin explants compared with controls. Finally, a subset of high K16-expressing transgenic keratinocytes develops major changes in the organization of keratin filaments in a time- and calcium concentration-dependent manner. These changes coincide with alterations in keratin content while the steady-state levels of K16 protein remain stable. We conclude that forced expression of K16 in progenitor skin keratinocytes directly impacts properties such as adhesion, differentiation, and migration, and that these effects depend upon determinants contained within its carboxy terminus.