The mechanism of actin polymerization by FH1FH2-formins and profilin

Background. Formin proteins assemble linear actin filament structures for a number of cellular processes in eukaryotes. The formin-homology (FH) 2 domain, the signature domain of formins, speeds actin polymerization by promoting the formation of actin filament nuclei. Single dimers of FH2 also bind and faithfully track the growing barbed ends of actin filaments through several round of actin subunit addition---a property known as processive association. The flexible FH1 domain lies N-terminal to the FH2 domain and binds to the actin-monomer binding protein profilin through individual tracks of polyproline. The bulk of polymerization-competent actin in cells is bound to profilin. Processive association with FH1FH2-formins can speed elongation of barbed ends growing in profilin-actin to rates exceeding the diffusion-limited rate of growth of free barbed ends.;Results. Here, I measured the kinetics of actin polymerization in the presence of FH1FH2-formins and profilin to study the mechanism by which formins nucleate actin filament ends and processively elongate barbed ends in physiological conditions. The major findings are: (i) to achieve fast barbed end elongation with profilin, FH1FH2-formins use individual FH1 polyproline tracks to bind dimers of profilin-actin in solution and rapidly deliver these actin subunits onto the FH2-associated barbed end; (ii) the addition of actin subunits onto formin-associated barbed ends, but not gamma-phosphate release from polymerizing ATP-actin filaments, provides the energy for processive elongation; (iii) the addition of actin subunits occasionally stimulates dissociation of formin from the barbed end, suggesting that formins are most vulnerable to dissociation during translocation along the growing end; (iv) FH2 domains from different formin homologs are variably susceptible to dissociation; (v) the flexible linker of the FH2 domain influences the rate of formin dissociation form barbed ends, perhaps by modulating the duration of the weakly-attached state of formin during translocation; (vi) profilin profoundly inhibits nucleation of filament ends by FH2 and FH1FH2-formins, but profilin-actin bound to FH1 may contribute weakly to nucleation.