Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY 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 in Aim 1. Neural Wiskott-Aldritch syndrome protein will be used as a nucleation promoting factor, because our lab has previously shown 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. In doing so, we will determine how the interplay between branch stabilization and destabilization affects the ability of actin networks to provide pushing forces. Many debranchers have also been implicated in bundling actin, so cryo-electron tomography will identify whether actin branches dissociate or remodel as bundles. Then, in Aim 2, we will assess how these proteins affect the recycling of monomeric actin from branched actin networks. By incorporating the filamentous actin disassembly protein, cofilin, into the bead motility assay, we will assess whether the interplay between cortactin and debranching proteins on sustaining bead motility over longer periods of time, which will be verified by an actin co-precipitation assay and cryo-electron tomography. Finally, in Aim 3, we will characterize the activity of a more enigmatic debrancher, coronin 7. In addition to the bead motility assay to identify the role of coronin 7 on force production by branched actin networks, we will utilize total internal reflection fluorescence microscopy to observe the frequency of debranching in the presence of coronin 7 with or without cortactin. We will then observe binding and dissociation of fluorescently labeled coronin 7 to actin filaments made from ATP (newer branches) or ADP (older branches) to assess nucleotide preference.
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