Research Project
10212353
Project Summary The goal of this proposal is to understand how two different members of the Actin Depolymerizing Factor Homology (ADF-H) family of actin-regulatory proteins, Cofilin and Abp1, influence each other?s interactions with and functional effects on actin. Cofilin and Abp1 are highly abundant and ubiquitous in vivo, and they both bind directly to actin filaments using structurally related ADF-H domains, yet these two proteins have strikingly different effects on actin filament dynamics. Cofilin promotes severing, depolymerization, and debranching of filaments, whereas Abp1 instead promotes assembly of filaments by Arp2/3 complex and protects (rather than destabilizes) filament branch junctions. This raises a key question: how do Cofilin and Abp1 co-exist on the same filamentous actin structures in cells, and how do they affect each other?s functions at these locations? Based on preliminary data that I have gathered, my working hypothesis is that cells express a level of Abp1 that allows it to carry out its functions in promoting actin assembly without interfering with Cofilin?s functions in actin disassembly. Further, I hypothesize that this balance is achieved in part because of key differences in the binding kinetics of Cofilin and Abp1 on actin filaments. However, my preliminary data also suggest that the same physiological concentrations of Abp1 strongly interfere with Cofilin-mediated debranching of filaments. I hypothesize that this is because Abp1 stably binds to branch junctions and competitively blocks Cofilin binding to Arp2/3 complex. To test these models, I will use an integrated approach combining biochemistry and in vitro single molecule imaging with genetics and live-cell imaging in S. cerevisiae. The aims of the proposal are: (1) In vitro analysis of Abp1 effects on Cof1-mediated actin disassembly and debranching, and (2) In vivo analysis of the relationship between Abp1 and Cofilin in regulating actin dynamics.
