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

Abstract

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.

Abl2:cortactin interactions regulate dendritic spine stability via control of a stable filamentous actin pool

“…We show here that Abl2 and cortactin are essential to maintain the stable actin pool in dendritic spines. Although Abl2 and cortactin can each independently bind and stabilize actin filaments, they synergize at much lower binding stoichiometries in vitro (Courtemanche et al, 2015; Scherer et al, 2018). The Abl2 N-terminal half, which neither binds actin nor promotes cortactin binding to actin, is sufficient to support the stable actin pool in Abl2-deficient neurons.…”

“…Abl2 binds actin filaments cooperatively (Wang, Miller, Mooseker, & Koleske, 2001) and increases the binding stoichiometry of cortactin for actin filaments (MacGrath & Koleske, 2012a). These interactions significantly impact actin filament stability – at saturated binding, Abl2 and cortactin each stabilize actin filaments (Courtemanche, Gifford, Simpson, Pollard, & Koleske, 2015; Scherer, Anand, & Koleske, 2018), but mixtures of Abl2 and cortactin at concentrations far below saturated binding also stabilize actin filaments (Courtemanche et al, 2015). While Abl2 and cortactin synergize to stabilize actin filaments in vitro , it is unclear whether or how they contribute to actin stability in neurons or how this mechanism might impact dendritic spine shape or stability.…”

Primary immunodeficiencies caused by mutations in actin regulatory proteins

“…15,110 Profilin regulates actin dynamics in part by depolymerizing filaments, 111 while Cortactin localizes with the ARP2/3 complex at sites of F-actin polymerization contributing to ARP2/3 complex activation and actin filament stabilization. 112,113 WIP can serve as a bridge between Profilin and the adaptor protein NCK. 17 Likewise, WIP facilitates the interaction between G-actin monomers and the Cortactin-ARP2/3 complex.…”

“…WIP also associates with Profilin and Cortactin . Profilin regulates actin dynamics in part by depolymerizing filaments, while Cortactin localizes with the ARP2/3 complex at sites of F‐actin polymerization contributing to ARP2/3 complex activation and actin filament stabilization . WIP can serve as a bridge between Profilin and the adaptor protein NCK .…”

Hematopoietic cell-specific lyn substrate (HCLS1 or HS1): A versatile actin-binding protein in leukocytes

“…However, HS1 is a weak activator of Arp2/3dependent actin polymerization, 6 and rather connects the Arp2/3 complex and actin filaments, thus stabilizing newly formed branched actin networks, as it has been described for its homologue cortactin. 11 HS1 can also interact with signaling molecules such as different kinases and the guanylate exchange factor (GEF) Vav1, 12 thus serving as scaffolding molecule for many cellular processes. Accordingly, and similar to what has been described for cortactin, 13 HS1 is important for proper activation of GTPases such as Rac1 and Rap1.…”