Volume 9, Issue 6, Pages 900-910 (June 2016) A Processive Arabidopsis Formin Modulates Actin Filament Dynamics in Association with Profilin  Sha Zhang, Chang Liu, Jiaojiao Wang, Zhanhong Ren, Christopher J. Staiger, Haiyun Ren  Molecular Plant  Volume 9, Issue 6, Pages 900-910 (June 2016) DOI: 10.1016/j.molp.2016.03.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 Single-Molecule Analysis of AtFH14 FH1-FH2-Mediated Actin Assembly Processes. (A and C) Two-color TIRFM micrographs (time given in minutes) of actin (0.75 μM, 15% Oregon green labeled, green) assembled with 50 nM FH1-FH2 (A) and 10 nM FH1-FH2 (C) Arrowheads indicate SNAP-549-FH1-FH2-associated (red) barbed ends. Scale bar, 1 μm. Merged kymographs of filament length (x axis; scale bar, 1 μm) over time (y axis; scale bar, 2 min). The corresponding concentrations of FH2 gave similar results which are not shown. (B) Average elongation rates of filaments with or without FH1-FH2 or mDia1 on the barbed end at different concentrations. Error bars represent SE, n ≥ 30, N ≥ 3. (D) The number of observed FH1-FH2 dissociation events at different concentrations. The numbers 0–5, and >5 represent the times that FH1-FH2 dissociates from the barbed end. (E) The fraction of barbed end attached time to the total growing time at different concentrations. Error bars represent SE, n ≥ 10, N ≥ 3. (F) The observed overall elongation rates at different concentrations of FH1-FH2 (1 μM, 15% Oregon green labeled actin). The data were fit with Equation 1 in the Supplemental Information to determine the predicted dissociation equilibrium constant. (G) Distribution of the number of photobleaching steps per barbed end with a processively attached SNAP-549-FH1-FH2 spot (bars ±SE; n = 60). Predicted distributions for oligomeric states (symbols) were calculated based on a binomial distribution model where the measured labeling stoichiometry is 0.65 dye per monomer. Molecular Plant 2016 9, 900-910DOI: (10.1016/j.molp.2016.03.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 ABP29 Blocks Elongation at the Filament Barbed End and Competes with AtFH14 FH1-FH2. (A) Preformed F-actin (0.5 μM) seeds were incubated with ABP29 and 1 μM G-actin (10% pyrene labeled) was added to initiate actin elongation at the barbed end in the presence of 200 μM free Ca2+. (B) The initial elongation rates at different concentrations of ABP29. (C) Preformed F-actin (0.5 μM) seeds were incubated with ABP29, SNAP-649-ABP29, and FH1-FH2 at different concentrations, then 1 μM G-actin (10% pyrene labeled) was added to initiate actin elongation at the barbed end in the presence of 200 μM free Ca2+. (D) Dependence of the percent of 20 nM SNAP-549-FH1-FH2 (○) or 50 nM ABP29 (●) bound filaments on time. (E) Two-color TIRFM (time given in minutes) showing the barbed end of the actin filament (0.5 μM, 15% Oregon green labeled, green) capped by ABP29 (marked by yellow arrowheads). Scale bar, 0.5 μm. (F) Three-color TIRFM of the assembly of actin (0.5 μM, 15% Oregon green labeled, green) in the presence of 50 nM SNAP-549-FH1-FH2 (red), 30 nM SNAP-649-ABP29 (blue), and 200 μM Ca2+. Images (left; time in minutes) and corresponding profiles of fluorescence intensity along the length of the filament (right). Arrowheads mark the barbed end (green), FH1-FH2 (red), and ABP29 (blue). Scale bar, 0.5 μm. (G and H) Steric clash between the predicted three-dimensional structure of ABP29 and FH2. The 167°-twisted F-actin barbed end is depicted as a surface representation (4A7N), while the predicted structural models of ABP29 and FH2 are illustrated in a ribbon diagram. The FH2 hemidimers (FH2-1 and FH2-2) position on the barbed end of actin filaments (B1–B5). The G1 domain of ABP29, ABP29-G1 (red), binds to actin B2 subdomains 1 and 3, while the flexible G1-G2 linker (aquamarine) allows the G2 domain of ABP29, ABP29-G2 (yellow), to contact actin B4 subdomain 2. As illustrated in the figure, there is a steric clash between ABP29-G1 and the knob region of FH2-2. Molecular Plant 2016 9, 900-910DOI: (10.1016/j.molp.2016.03.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 AtFH14 FH1-FH2-Mediated Actin Assembly in the Presence of Profilin. (A) Actin (2 μM, 10% pyrene labeled) was assembled in the presence of the indicated concentrations of FH1-FH2 with 6 μM Arabidopsis profilin4, AtPRF4. (B) Pyrene-actin polymerization assays containing 2 μM monomers (10% pyrene). Actin alone (blue), actin plus 20 nM FH1-FH2 (brown), actin plus 6 μM AtPRF4 (red), actin plus 20 nM FH1-FH2 and AtPRF4 (green). (C) Processive actin-filament elongation by SNAP-549-FH1-FH2 in the presence of AtPRF4. Two-color TIRF micrographs (in seconds) showing that at the beginning, SNAP-549-FH1-FH2 processively attaches to the barbed end forming a dim, fast-growing actin filament (50% Oregon green labeled) and then SNAP-549-FH1-FH2 dissociates from the filament end, resulting in an increase in filament brightness and a decrease in the elongation rate of the free barbed end. Yellow arrowheads indicate SNAP-549-FH1-FH2-associated barbed ends. Scale bar, 1 μm. Merged kymographs of filament length (x axis; scale bar, 1 μm) over time (y axis; scale bar, 40 seconds). (D) Average elongation rates of actin alone (0.5 μM, 15% Oregon green labeled) and SNAP-549-FH1-FH2–assembled filaments with or without different profilins (2.5 μM). Error bars represent SE, n ≥ 30, N = 3. (E) Histogram showing the average fluorescence intensity of spontaneously assembled control filaments and SNAP-549-FH1-FH2-elongated filaments with different profilins. (F) Processive SNAP-biotin-FH1-FH2 adsorbed to the cover glass; filament barbed end (yellow arrowhead), buckle (white arrow). Actin filaments (0.5 μM, 50% Oregon green labeled, with 2.5 μM AtPRF4) grow out of a fixed point, which is considered to be an immobilized SNAP-biotin-FH1-FH2 (35 nM), and buckle when another part of filament is anchored to the slide (time given in seconds). Scale bar, 0.5 μm. Molecular Plant 2016 9, 900-910DOI: (10.1016/j.molp.2016.03.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 Actin Bundles Generated by AtFH14 with or without Plant Profilin. (A–C) Time-lapse micrographs of the spontaneous assembly of actin (0.5 μM, 15–30% Oregon green labeled) with 100 nM FH1-FH2 (A), 200 nM FH1-FH2 (B), 2.5 μM AtPRF4 and 100 nM FH1-FH2 (C) visualized by TIRFM (time given in minutes). Scale bar, 5 μm. (D) Actin filaments (0.125 μM, 15% Oregon green labeled, with 0.625 μM LlPRF1) extend in opposite directions in the bundle (time in seconds). White arrows indicate the growing barbed ends of actin filaments. Scale bar, 1 μm. (E) Low magnification view of two-color TIRF micrographs of actin bundles (0.125 μM, 15% Oregon green labeled, with 0.625 μM LlPRF1) annealing and zippering mediated by 100 nM SNAP-549-FH1-FH2 (time in seconds). Scale bar, 5 μm. (F) Two-color TIRF micrographs of actin bundles (0.125 μM, 15% Oregon green labeled, with 0.625 μM LlPRF1) mediated by 100 nM SNAP-549-FH1-FH2. Boxes, white and red, show the process of zippering (time in seconds). Scale bar, 0.5 μm. Molecular Plant 2016 9, 900-910DOI: (10.1016/j.molp.2016.03.006) Copyright © 2016 The Author Terms and Conditions