Zyxin redistributes without upregulation in migrating human keratinocytes during wound healing

Cell migration, growth, and survival is modulated by focal adhesions linkin g extracellular matrix proteins, cell adhesion molecules, and the cytoskele ton, Zyxin is a focal adhesion phosphoprotein that shares homology with Lis teria ActA protein in promoting actin filament assembly; it also has specia lized protein-protein interface domains implicating an important role in ce ll growth and differentiation. We investigated the distribution of zyxin in normal and migrating human keratinocytes in wounds in vitro and in sib usi ng 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 w as 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 p unctate staining throughout the cytoplasm, Migrating cultured keratinocytes next to wounds, however, had large focal contacts in the cell periphery wh ere actin bundles converged at focal adhesions. Western immunoblots and con focal experiments with protein synthesis inhibition by cycloheximide confir med that this difference in distribution of zyxin in migrating versus nonmi grating 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.