Abstract
Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by Actin-Depolymerizing Factor (ADF)/Cofilin proteins. Mammals express three ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest they perform both overlapping and unique functions. First, I directly and quantitatively compared the actin filament severing activities of human Cof1, Cof2, and ADF using TIRF microscopy, which revealed Cof2 and ADF severed filaments more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Further, Cof2 and ADF were more effective than Cof1 in promoting 'enhanced disassembly' when combined with actin disassembly cofactors Coronin1B and Actin-Interacting Protein 1 (AIP1), both on preformed and actively growing filaments. To probe the mechanism underlying these differences, I used multi-wavelength TIRF microscopy to directly observe Cy3-Cof1 and Cy3-Cof2 interacting with actin filaments in real time during severing. Cof1 and Cof2 each bound to filaments with similar kinetics, yet Cof2 induced severing more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing. Additionally, a pairwise combinatorial analysis revealed novel relationships, where specific pairs have additive severing effects while other pairs show neutral or inhibitory effects. Furthermore, I characterized a Cof2 mutant implicated in human disease and identified defects in regulating actin dynamics which likely contribute to the condition. Lastly, I contributed to work characterizing the three-component pathway by which Cof1, Coronin1B, and AIP1 promote rapid disassembly. Taken together, these data establish key quantitative differences in the activities of the three human ADF/Cofilins with important implications for how these proteins are utilized for tuning rates of actin network turnover.