NIH Reporter #5F32GM156029-03

The role of cortactin-mediated actin branch stabilization in force-producing actin networks

This award funds research to understand the role of cortactin in stabilizing actin branches that produce cellular forces. The study will use biochemical assays and microscopy to determine how cortactin prevents premature branch removal and supports force production in actin networks.

Description

Networks of branched actin filaments produce forces necessary for cellular processes ranging from cell migration to endocytosis. These branches are formed by proteins actin-related protein 2/3 (Arp2/3) complex, along with proteins that promote Arp2/3 activation. Cortactin is a multi-functional protein that can activate Arp2/3 complex on its own at high concentrations, synergize with certain nucleation promoting factors, or stabilize branches after they are formed. This multifunctionality has made isolating the role of cortactin as a branch stabilizer difficult. Actin branches are intrinsically stable, so why cortactin-mediated branch stabilization is important for regulating force-producing actin networks is unknown. One possibility is that cortactin protects branches from being removed by a class of debranching proteins, such as coronin 1B or glia maturation factor-γ. Additionally, actin networks are age-segregated, with newer branches near the surface being thought to be more important in the ability of the network to provide pushing forces, so the importance of cortactin may be related to whether debranchers target the younger or older regions of the network. In this proposal, we will utilize a reconstituted assay to isolate cortactin's role as a branch stabilizer, and determine whether it is important for force production by preventing branches from being removed prematurely. Neural Wiskott-Aldrich syndrome protein will be used as a nucleation promoting factor, because our lab has previously shown it does not synergize with cortactin. Lower concentrations of cortactin will minimize cortactin-mediated branch nucleation. Bead motility assays will allow for observing force production by age-segregated actin networks that push against beads in the presence of cortactin alone and with differentially targeted debranching proteins. Cryo-electron tomography will identify whether actin branches dissociate or remodel as bundles. In Aim 2, we will assess how these proteins affect the recycling of monomeric actin from branched actin networks, incorporating cofilin into the bead motility assay. In Aim 3, we will characterize the activity of the debrancher, coronin 7, using bead motility assays and total internal reflection fluorescence microscopy to observe debranching frequency and binding/dissociation to actin filaments made from ATP or ADP.

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