ROLES OF DISULFIDE BONDING IN THE ASSEMBLY, ORGANIZATION, AND DYNAMICS OF KERATIN INTERMEDIATE FILAMENTS IN EPIDERMAL KERATINOCYTES
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Our laboratory recently reported on the crystal structure of the interacting 2B regions from the rod domain of keratins K5 and K14. In addition to providing much needed atomic-resolution insight into the heteromeric character of K5-K14 coiled-coil dimers, this effort revealed the occurrence of a trans-dimer, homotypic disulfide bond bridging two K14 polypeptides via a Cys367 in both crystal as well as in epidermal keratinocytes in vivo (Lee et al., NSMB 19:707, 2012). This disulfide linkage appears poised to promote the formation/maintenance of a perinuclear network of keratin filaments that contributes to determining the size and shape of the nucleus. In this thesis, we report that a sizable fraction of the K14 and K5 protein pools is engaged in inter-keratin disulfide bonding in wild-type mouse keratinocytes in primary culture, under both basal and calcium-promoted differentiation conditions. Like its wild-type counterpart, a cysteine-free variant of K14 forms normal 10-nm filaments when using standard assembly conditions in vitro and following transfection in K14-/- mouse keratinocytes in primary culture. However, allowing for the formation of disulfide bonding by using oxidizing conditions in vitro significantly limits filament elongation, consistent with previous studies. Using live-imaging, we further show that total loss of K14-dependent disulfide bonding impairs the normal keratin filament cycle, interferes with the formation and/or maintenance of perinuclear networks, and is associated with enhanced movement of the nucleus and whole cell. These striking phenotypes can be partially rescued by transfection of a K14 variant harboring a single Cys367. Albeit important, C367 does not account for all of disulfide bonding-dependent characteristics of K14 in vitro and in vivo. In follow-up studies, we showed that two other cysteine residues located in K14’s head domain, C4 and C40, are also involved in inter-keratin disulfide bonding and play a functional role in filament assembly in vitro, their influence being dominant over C367. In live mouse keratinocytes, C4 and C40 are also required for the execution of normal keratin filament cycle, formation of a perinuclear-concentrated network of keratin filaments, and the motility of the nucleus. These results establish that disulfide bonding plays a physiologically important role in the assembly, intracellular organization, and dynamics of keratin IFs, with an impact on the location, shape and motility of the nucleus in skin keratinocytes.