Loss of β-PIX inhibits focal adhesion disassembly and promotes keratinocyte motility via myosin light chain activation

Abstract

During healing of the skin, the cytoskeleton of keratinocytes and their matrix adhesions, including focal adhesions (FAs), undergo reorganization. These changes are coordinated by small GTPases and their regulators, including the guanine nucleotide exchange factor β-PIX. In fibroblasts, β-PIX activates small GTPases, thereby enhancing migration. In keratinocytes in vitro, β-PIX localizes to FAs. To study β-PIX functions, we generated β-PIX knockdown keratinocytes. During wound closure of β-PIX knockdown cell monolayers, disassembly of FA is impaired and their number and size are increased. In addition, in the β-PIX knockdown cells phosphorylated myosin light chain (MLC) localizes not only at the wound edge, but also in follower cells, while p21-activated kinase 2 (PAK2), a regulator of MLC kinase (MYLK), is mislocalized. Inhibition or depletion of MYLK restores FA distribution in β-PIX knockdown cells. Traction forces generated by β-PIX knockdown cells are increased relative to control cells, a result consistent with an unexpected enhancement in the migration of single β-PIX knockdown cells and monolayers of such cells. We propose that targeting β-PIX may be a means of promoting epithelialization of wounds in vivo.