mDia1 senses both force and torque during F-actin filament polymerization

Fig. 1. Schematics of experimental set-up and force calibration. a Actin filaments are tethered in four different designs using two differently tagged mDia1ΔN3. The GST-mDia1ΔN3-bound barbed end of the filament is either attached to an anti-GST–Protein A coated coverslip surface (top-left) or to an anti-GST–Protein A coated superparamagnetic bead (top right). The biotin-mDia1ΔN3-bound barbed end is attached to a streptavidin-coated superparamagnetic bead either directly (bottom left) or indirectly (bottom right) through a torsionally unconstrained DNA linker. The actin filament is tethered to an immobilized NEM-HMM-coated bead on a coverslip through a point on the actin filament somewhere near the tipped end. The actin filament was stretched at 6–10° above the surface using transverse magnetic tweezers. The rotationally unconstrained or constrained tethers are indicated. b Fluorescence imaging revealed sparsely distributed seeding filaments on the surface under our preparation conditions. Scale bar represent 10 μm. c The calibrated force-distance profiles for 2.8-μm-diameter M270 beads and 1.0-μm-diameter MyOne beads. 12 beads for M270 and 10 beads for MyOne

Fig. 2. Actin elongation speed is force-dependent. a Elongation trajectories of a single-F-actin filament in 500 nM G-actin solution under different forces. b The elongation of the F-actin filament stopped after free G-actin was washed off. The number of replication of the force-dependent actin polymerization at each force is indicated in Fig. 4

Fig. 3. The F-actin elongation rate varies depending on tether. The elongation trajectories of F-actin in 500 nM G-actin solution at 1.9 ± 0.4 pN obtained from 17 different tethers show a wide variation in speed. The inset shows the histogram of the elongation rates based on the 17 tethers

Fig. 4. Force-dependent F-actin elongation rate varies depending on tether configuration. The data show the actin elongation speed as a function of force for cases where GST-mDia1ΔN3 was attached to coverslip surface (red solid and hollow squares represent the fast and slow elongating species, respectively), or to the superparamagnetic bead surface (blue), or when biotin-mDia1ΔN3 was directly attached to streptavidin-coated superparamagnetic bead surface (purple) or to an anti-DIG coated superparamagnetic bead surface through a torsionally unconstrained DNA linker (brown). Vertical error bars represent standard errors of mean (s.e.m) obtained from multiple data points obtained at the force. Horizontal error bars represent the 20% uncertainty in force calibration. The value labeled on each data point indicates the number of independent experiments carried out at the corresponding force. Inset shows the zoom-in of the slow elongating species. All of the data were collected in the presence of 500 nM G-actin. The solid black line is the predicted polymerization rate based on Eq. (1) with best fitting parameters Δ ≈ 5.36 nm, \documentclass[12pt]{minimal} \usepackage{amsmath}…

Fig. 5. Kinetic model of FH2 dependent actin polymerization. a Schematics of the closed-open transition of FH2 associated at the barbed end and recruitment of actin monomers at the open state of the FH2 ring. b The corresponding transition diagram of actin elongation. c The corresponding transition diagram of recruiting one actin monomer, from which the polymerization speed can be derived