Cortactin stabilization of actin requires actin-binding repeats and linker, is disrupted by specific substitutions, and is independent of nucleotide state.

The actin-binding protein cortactin promotes the formation and maintenance of actin-rich structures, including lamellipodial protrusions in fibroblasts and neuronal dendritic spines. Cortactin cellular functions have been attributed to its activation of the Arp2/3 complex, which stimulates actin branch nucleation, and to its recruitment of Rho family GTPase regulators. Cortactin also binds actin filaments and significantly slows filament depolymerization, but the mechanism by which it does so and the relationship between actin binding and stabilization are unclear. Here we elucidated the cortactin regions that are necessary and sufficient for actin filament binding and stabilization. Using actin cosedimentation assays, we found that the cortactin repeat region binds actin but that the adjacent linker region is required for binding with the same affinity as full-length cortactin. Using total internal reflection fluorescence microscopy to measure the rates of single filament actin depolymerization, we observed that cortactin-actin interactions are sufficient to stabilize actin filaments. Moreover, conserved charged residues in repeat 4 were necessary for high-affinity actin binding, and substitution of these residues significantly impaired cortactin-mediated actin stabilization. Cortactin bound actin with higher affinity than did its paralog, hematopoietic cell-specific Lyn substrate 1 (HS1), and the effects on actin stability were specific to cortactin. Finally, cortactin stabilized ADP-actin filaments, indicating that the stabilization mechanism does not depend on the actin nucleotide state. Together, these results indicate that cortactin binding to actin is necessary and sufficient to stabilize filaments in a concentration-dependent manner, specific to conserved residues in the cortactin repeats, and independent of the actin nucleotide state.