Fig. 1: Mical-oxidized actin binds and inhibits profilin-assisted actin polymerization.

a Profilin facilitates polymerization by fueling F-actin assembly by elongation-promoting factors (formins, Ena/VASP). In particular, most cellular monomeric/G-actin is complexed with G-actin binding proteins such as profilin (pink). These profilin–actin complexes associate with elongation-promoting factors such as formins (black) and Ena/VASP (blue) to drive monomers’ incorporation (thin green arrows) leading to F-actin elongation (thick green arrows) in a tightly controlled manner. Based on current models, formins trigger actin nucleation by stabilizing transient actin dimers. Formins are shown here as dimers, such that each FH1 domain binds profilin–actin and delivers it to FH2 domains, which are bound to actin’s barbed-end. For simplicity, the cell membrane is neither illustrated, nor formins’ or Ena/VASP’s association with it. b Mical post-translationally and specifically oxidizes actin to induce F-actin disassembly. In particular, based on current models, Mical with its Redox region (orange, left model and inset) directly associates with F-actin. F-actin triggers Mical’s Redox enzymatic activity (inset)—such that with its co-enzyme NADPH, Mical stereospecifically (in the R-isomer conformation) oxidizes Met44 and Met47 residues (inset) in the D-loop at actin’s pointed end. This generates Mical-oxidized actin (Mox-actin) (red, model and inset). Since this oxidation occurs on residues along the interface of actin filament subunits, it weakens the interactions between individual filament subunits and promotes F-actin disassembly (thin red arrows) and shortening (thick red arrow). c Profilin binds to Mical-oxidized actin to a similar extent as to unoxidized actin. Binding of human profilin-1 to actin was measured by changes in tryptophan fluorescence of actin. Unoxidized actin (closed circles). Mox-actin (open circles). n = 2 (two different preps of both actins) with each data point an average of two independent experiments. Dissociation constants (Kd) for unoxidized and Mox-actin under G-buffer conditions are estimated as 0.5 and 0.8 µM, respectively. [Actin] = 0.15 µM; excitation wavelength (295 nm), emission (330 nm). F₃₃₀ = normalized fluorescence at 330 nm. d Profilin complexes with unoxidized actin (blue, green traces), but not with Mical-oxidized actin (red, black traces), polymerize (with or without F-actin seeds). [Actin] = 3 µM; [profilin] = 9 µM; [F-actin-phalloidin seeds] = 0.25 µM (stabilized with phalloidin (Ph) at 1:50 Ph:actin molar ratio). A.U. arbitrary units. n = 2 separate experiments with similar results. Source data for Fig. 1 are provided as a Source Data file.