Zyxin redistributes without upregulation in migrating human keratinocytes during wound healing

Cell migration, growth, and survival is modulated by focal adhesions linking extracellular matrix proteins, cell adhesion molecules, and the cytoskeleton. Zyxin is a focal adhesion phosphoprotein that shares homology with Listeria ActA protein in promoting actin filament assembly; it also has specialized protein-protein interface domains implicating an important role in cell growth and differentiation. We investigated the distribution of zyxin in normal and migrating human keratinocytes in wounds in vitro and in situ using confocal laser microscopy. Zyxin expression in high-density nonmigrating keratinocytes versus low-density migrating keratinocytes was determined by western immunoblotting and time lapse image analysis. In normal epidermis, zyxin exhibited a punctate staining pattern throughout the cytoplasm and was excluded from the intercellular spaces. In wounds, the punctate staining also localized in the edge of the migrating keratinocyte sheets; however, intercellular spaces were absent. Likewise, in vitro keratinocytes showed punctate staining throughout the cytoplasm. Migrating cultured keratinocytes next to wounds, however, had large focal contacts in the cell periphery where actin bundles converged at focal adhesions. Western immunoblots and confocal experiments with protein synthesis inhibition by cycloheximide confirmed that this difference in distribution of zyxin in migrating versus nonmigrating keratinocytes is due to the redistribution and not upregulation of zyxin. The abundance of zyxin and its relative change in distribution from normal to migrating keratinocytes in wounds is consistent with its role in cytoskeletal organization of actin bundles.