Polarized Cell Motility Induces Hydrogen Peroxide to Inhibit Cofilin via Cysteine Oxidation

Mesenchymal cell motility is driven by polarized actin polymerization [1]. Signals at the leading edge recruit actin polymerization machinery to promote membrane protrusion, while matrix adhesion generates tractive force to propel forward movement. To work effectively, cell motility is regulated by a complex network of signaling events that affect protein activity and localization. H(2)O(2) has an important role as a diffusible second messenger [2], and mediates its effects through oxidation of cysteine thiols. One cell activity influenced by H(2)O(2) is motility [3]. However, a lack of sensitive and H(2)O(2)-specific probes for measurements in live cells has not allowed for direct observation of H(2)O(2) accumulation in migrating cells or protrusions. In addition, the identities of proteins oxidized by H(2)O(2) that contribute to actin dynamics and cell motility have not been characterized. We now show, as determined by fluorescence lifetime imaging microscopy, that motile cells generate H(2)O(2) at membranes and cell protrusions and that H(2)O(2) inhibits cofilin activity through oxidation of cysteines 139 (C139) and 147 (C147). Molecular modeling suggests that C139 oxidation would sterically hinder actin association, while the increased negative charge of oxidized C147 would lead to electrostatic repulsion of the opposite negatively charged surface. Expression of oxidation-resistant cofilin impairs cell spreading, adhesion, and directional migration. These findings indicate that H(2)O(2) production contributes to polarized cell motility through localized cofilin inhibition and that there are additional proteins oxidized during cell migration that might have similar roles.