TIRF microscopy analysis of human Cof1, Cof2, and ADF effects on actin filament severing and turnover

J Mol Biol. 2016 Apr 24;428(8):1604-16. doi: 10.1016/j.jmb.2016.03.006. Epub 2016 Mar 17.

Abstract

Dynamic remodeling and turnover of cellular actin networks requires actin filament severing by actin-depolymerizing factor (ADF)/Cofilin proteins. Mammals express three different ADF/Cofilins (Cof1, Cof2, and ADF), and genetic studies suggest that in vivo they perform both overlapping and unique functions. To gain mechanistic insights into their different roles, we directly compared their G-actin and F-actin binding affinities, and quantified the actin filament severing activities of human Cof1, Cof2, and ADF using in vitro total internal reflection fluorescence microscopy. All three ADF/Cofilins had similar affinities for G-actin and F-actin. However, Cof2 and ADF severed filaments much more efficiently than Cof1 at both lower and higher concentrations and using either muscle or platelet actin. Furthermore, Cof2 and ADF were more effective than Cof1 in promoting "enhanced disassembly" when combined with actin disassembly co-factors Coronin-1B and actin-interacting protein 1 (AIP1), and these differences were observed on both preformed and actively growing filaments. To probe the mechanism underlying these differences, we used multi-wavelength total internal reflection fluorescence 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 much more rapidly than Cof1, decreasing the time interval between initial binding on a filament and severing at the same location. These differences in ADF/Cofilin activities and mechanisms may be used in cells to tune filament turnover rates, which can vary widely for different actin structures.

Keywords: ADF; AIP1; Cofilin; Coronin; actin disassembly.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actin Cytoskeleton / chemistry*
  • Actin Depolymerizing Factors / metabolism
  • Actins / chemistry
  • Animals
  • Cofilin 1 / chemistry*
  • Cofilin 2 / chemistry*
  • Destrin / chemistry*
  • Dose-Response Relationship, Drug
  • Escherichia coli / metabolism
  • Humans
  • Microscopy / methods*
  • Nucleotides / chemistry
  • Plasmids / metabolism
  • Protein Binding
  • Rabbits

Substances

  • Actin Depolymerizing Factors
  • Actins
  • CFL1 protein, human
  • CFL2 protein, human
  • Cofilin 1
  • Cofilin 2
  • DSTN protein, human
  • Destrin
  • Nucleotides