Control of actin stability and branching by Arg and cortactin Abl family kinases act downstream of cell surface receptors to coordinate changes in actin cytoskeletal structure. These signaling roles make Abl kinases essential in fundamental processes that depend on actin regulation, such as cell migration and cell and tissue morphogenesis. Aberrant Abl family kinase activation also causes leukemia and contributes to the progression of other cancers. These biological processes have been largely ascribed to kinase activation, but our lab has shown that the Abl2/Arg kinase can also directly bind and stabilize actin filaments. In addition, Arg synergizes with its substrate cortactin to activate Arp/3 complex- mediated actin branching. Our lab has established that Arg regulates actin-rich structures that are essential for fibroblast migration, breast cancer invasion, and neuronal morphogenesis. Elucidating the direct effects of Arg- actin binding is critical for understanding how Abl family kinases regulate motility and morphogenesis in these diverse cell types. In this proposal, I will test the hypothesis that Arg-mediated actin filament stabilization and stimulation of filament branching is critical for the formation and turnover of actin-based cell edge protrusions.
My first aim i s to determine which domains of Arg and cortactin mediate actin stabilization and branching. I will use total internal reflection fluorescence (TIRF) microscopy to measure the effects of Arg and cortactin mutants on actin stability and branch formation. Arg phosphorylates cortactin, but our lab has shown that this is not required for Arg and cortactin synergy in actin regulation. I will utilize a panel of Arg and cortactin fragments and deletion mutants to determine the minimal functional domains of Arg and cortactin necessary for their synergy in stabilizing actin and promoting Arp2/3 complex-mediated branching.
My second aim i s to elucidate how Arg-actin interactions activate cortactin- and Arp2/3 complex- mediated filament branching. Arg binds actin cooperatively and changes the helical twist of filaments, which increases cortactin binding to actin. I will use cosedimentation assays to test the hypothesis that Arg increases actin branching by recruiting the Arp2/3 complex to filaments. I will also use two-color TIRF microscopy to determine whether branching is increased on Arg-actin filaments relative to naked actin filaments.
My third aim i s to determine how Arg-actin interactions drive dynamic cell edge protrusions in vivo. The two Arg actin-binding domains (ABDs) have distinct effects on actin stability and branching, however, the role of Arg-actin binding in regulating the formation of actin-based structures in cells is not well understood. Arg is required for adhesion-dependent cell edge protrusions in fibroblasts. To determine which Arg residues are necessary for protrusions, I will express mutants with deletions and mutations in each ABD in arg-/- cells. Arg may have cortactin-independent effects on actin branching modulated by one of its ABDs, AB2. I will express Arg mutants in cortactin knockdown fibroblasts to elucidate the cortactin-independent effects of Arg in cells.
Precise control of cell shape and movement is necessary for normal development of human tissues, and disruption of normal cytoskeletal control is a hallmark of cancer progression and neurodegenerative diseases. This project will investigate a common pathway by which all of these cell types form actin-based protrusive structures. Determining the fundamental means by which all cells regulate their structure is foundational to understanding these disorders and could lead to new methods to diagnose and treat them.
| Scherer, Alexander N; Anand, Neha S; Koleske, Anthony J (2018) Cortactin stabilization of actin requires actin-binding repeats and linker, is disrupted by specific substitutions, and is independent of nucleotide state. J Biol Chem 293:13022-13032 |
