Myosin-I synergizes with Arp2/3 complex to enhance the pushing forces of branched actin networks

Abstract

Class I myosins (myosin-Is) colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by the Arp2/3 complex on the surface of beads coated with myosin-I and nucleation-promoting factor. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Myosin-I triggered symmetry breaking and comet tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations, suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.

Fig. 1.. Alteration of comet tail morphology by myosin-I.

(A) Schematic of (left) control- and (right) myosin-beads coated with NPF and neutravidin, where neutravidin is further conjugated with biotinylated CF640 fluorescent dye (control-bead) or biotinylated Drosophila Myo1d (myosin-bead). (B) Time-lapse sequence of a (red) myosin-bead walking on a (green) single actin filament track. Speed ~8 nm/s. See movie S1 for a full time series. Scale bar, 2 μm. (C) Time series of the growth of actin comet tail from symmetry breaking to generation of a polarized comet tail from (top) control-bead and (bottom) myosin-bead. Scale bar, 5 μm. (D) Phase map showing representative examples of 2-μm-diameter beads coated with a range of myosin densities in the presence of 30 to 200 nM CP, 4 μM (5% rhodamine-labeled) actin, and 200 nM Arp2/3 complex. For each myosin density condition, the left column (red) shows control-beads that were acquired in the same imaging field as the myosin-beads in the right column (blue). The myosin and NPF densities were determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel, where the NPF density ~6000 μm⁻² is the same for groups 0.28:1, 0.35:1, and 0.43:1, and ~4000 μm⁻² for group 0.80:1. Images were acquired 20 to 35 min after mixing. Brightness and contrast were linearly set to be the same for each control- and myosin-bead pair but different among panels at different conditions for better visualization of the bead position [see fig. S1 for panels using a common lookup table (LUT)]. Scale bar, 5 μm.

Fig. 2.. Disruption and fracturing of actin shells by myosin-I.

(A) Time series of (top) control-beads and (bottom) 0.43:1 myosin-beads acquired in the same imaging field in the presence of 200 nM CP showing the inability of myosin-beads to form a comet tail. (B) Time series of (top) control-beads and (bottom) 0.43:1 myosin-beads acquired in the same field in the presence of 25 nM CP showing fracturing of the actin shell and comet tail growth from a myosin-bead but not the control-bead. Conditions: 4 μM actin (5% rhodamine-labeled), 200 nM Arp2/3 complex, and 200 or 25 nM CP. Scale bars, 5 μm.

Fig. 3.. Myosin-I decreases the actin density of comet tails.

(A) Time series of (top) control and (bottom) 0.43:1 myosin-beads growing comet tails in the presence of 50 nM CP. The actin network growing from the myosin-bead is less dense but has a similar tail length as the control. Conditions: 4 μM actin (5% rhodamine-labeled), 200 nM Arp2/3 complex, and 50 nM CP. Scale bar, 5 μm. (B) Mean fluorescence intensities (Fluor. Int./area) of actin comet tails grown from control- and myosin-beads captured 20 min after mixing. The solid lines connect experimental pairs (N = 5 independent experiments, n = 11 pairs). (C) Actin comet tail length as a function of time and (D) tail growth rates derived from the slopes of the time courses in (C). (E) Comet tail fluorescence as a function of time. Control- and myosin-bead pairs are normalized to the average fluorescence level of the control-beads from 1100 to 1300 s. (F) Rate of fluorescent actin incorporation into comet tails derived from the slopes of the time courses in (E). (C and E) Large points show the averaged value at binned time intervals (every 100 s). Traces are from individual beads with each myosin-bead acquired with a control-bead in the same field of view (N = 5, n = 11). Error bars are SD. (G) Growth efficiency for control- and myosin-beads. Efficiency is defined as comet tail length per unit actin fluorescence intensity. Box plots (B, D, F, and G) show median (center line), interquartile range (box), and min-max values (whiskers). P values were calculated using a two-tailed paired t test. Each point represents a control- and myosin-bead pair acquired in the same field of view (N = 5, n = 11). See also fig. S2.

Fig. 4.. Comets grown from myosin-beads incorporate less Arp2/3 complex.

(A) Representative actin comet tails assembled from (top) control and (bottom) myosin-beads showing (magenta) actin and (green) SNAP-Arp2/3 complex distribution. Images were acquired approximately 25 to 35 min after mixing. Brightness and contrast were set differently for actin (invert LUT) and SNAP-Arp2/3 complex (invert LUT) panels for visualization. Conditions: 4 μM actin (5% rhodamine-labeled), 200 nM Arp2/3 complex (80% SNAP-Surface 488–labeled), and 40 nM CP. Scale bars, 5 μm. (B) Total actin fluorescence intensity, (C) total SNAP-Arp2/3 fluorescence intensity, and (D) actin to SNAP-Arp2/3 fluorescence ratio over the entire comet tail region. (E) Total SNAP-Arp2/3 fluorescence intensity on bead surface. The plot shows the median (center line), interquartile range (box), and min-max values (whiskers). P values were calculated using a two-tailed paired t test. Each point represents a pair of control- and myosin-beads (N = 2 independent experiments, n = 33 pairs), with intensity values normalized to the control-beads.

Fig. 5.. The myosin power stroke is required for altering network architectures.

(A) Time series of actin assembly around (top) control and (bottom) rigor myosin-beads at 50 nM CP. Rigor myosin heavily delayed the growth of actin comet tails. (B) Representative images of actin comet tails grown under different ATP concentrations (top: control-beads; bottom: myosin-beads). Adding ATP back rescued comet tail growth. Images were acquired 20 to 30 min after mixing. (C) Representative actin network patterns assembled on control, myosin, rigor myosin, and HaloABD-beads under three different CP concentrations, as indicated. Images were acquired 20 to 30 min after mixing. Brightness and contrast were set to the same values for each panel. (D) Representative actin comet tails generated by (top) control and (bottom) myosin-beads in the absence and presence of 100 μM free Ca²⁺ at 50 nM CP. Images were acquired approximately 15 to 20 min after mixing. (E) Length and (F) network density quantified by total fluorescence intensity per area for control- and myosin-beads in the absence and presence of 100 μM free Ca²⁺ (N = 1 independent experiments, n = 15 pairs). Control and myosin-bead experimental pairs are normalized to the average fluorescence level of the control-beads. Box plots (E and F) show median (center line), interquartile range (box), and min-max values (whiskers). P values were calculated using a two-tailed paired t test. Conditions: 4 μM actin (5% rhodamine-labeled), 200 nM Arp2/3 complex, 15 to 200 nM CP, and 0 to 1 mM ATP, as indicated. Scale bars, 5 μm.

Fig. 6.. The myosin power stroke can fracture the actin shell.

(A) Representative actin shells of control- and myosin-beads assembled under 50 nM CP, arrested by adding 20 μM (5 molar excess) of LatB and CK-666 before symmetry breaking, as well as myosin-beads assembled under the same conditions but arrested with the addition of 10 mM adenosine 5′-diphosphate (ADP) to inhibit myosin power stroke. Myosin-beads fractured and ejected from the actin shell, while control- and myosin-beads (with 10 mM ADP) remained enclosed in the shell. The control-bead assembled under 100 nM CP, showing similar network density as the myosin-bead, did not show shell fracture or bead ejection. The image was captured approximately 40 min after arrest. (B) The extents of shell breaking were classified by shell-breaking angle θ: θ = 0, no symmetry breaking; 0 < θ < 180, shell fracture; θ ≥180, bead ejected. Scale bar, 5 μm. (C) Percentage of populations with different extents of shell breaking for control (50 nM CP) (n = 182), myosin (n = 158), myosin (with 10 mM ADP) (n = 89), and control (100 nM CP) (n = 65); N = 2 independent experiments. Conditions: 4 μM actin (5% rhodamine-labeled), 200 nM Arp2/3, 50 or 100 nM CP. Twenty micromolar (5 molar excess) phalloidin and 2 mM ATP were also added to prevent actin depolymerization and preserve myosin motor activity. Actin assembly was arrested 100 s after mixing. Scale bars, 5 μm.

Fig. 7.. The filament-level model of actin comet tail recapitulates experimental results.

(A) Schematic of the model of actin polymerization around the nucleating bead. The model includes filament-level nucleation, branching, filament fragmentation, debranching, capping, and force exerted by implicit myosin. (B) Time-lapse of the symmetry-breaking event under intermediate capping conditions (branch length = 0.5 μm) with no myosin. The color scale indicates filament tension (red: tensile; blue: compressive). (C) Tension distributions within actin comet tails formed at either control (0.0 pN) or two myosin forces (0.2 or 0.4 pN) under intermediate capping conditions (branch length = 0.5 μm). The image shows a cut through the center of the comet tail. The color scale indicates filament tension (red: tensile; blue: compressive). (D) Simulated time-lapse of epifluorescence images under different myosin forces (branch length = 0.5 μm). (E) Elongation speed, (F) actin intensity, and (G) Arp2/3 complex intensity for simulated beads as a function of myosin force (branch length = 0.5 μm). (H) Forces acting on beads along the direction of bead propulsion due to actin polymerization (green) and myosin pushing (orange) as a function of the myosin force (branch length = 0.5 μm). Error bars are SD. (I) Orientation of filaments around the beads (within 0.15 μm of the bead surface) as a function of myosin force (branch length = 0.5 μm). (J) Simulated symmetry breaking after actin polymerization and branching arrest. Actin was allowed to polymerize around the bead for 42.7 s (0.2-pN myosin force with 0.5-μm filament length) to form a shell of intermediate thickness before being halted. The time of arrest was set as t = 0 s.

Fig. 8.. Schematic of how myosin-I modulates actin network structure through its power stroke.

(A) Schematic of branched actin assembly without the presence of myosin-I. (B) Schematic of branched actin assembly with the presence of myosin-I. Less dense actin networks in the presence of myosin-I result from the motor pushing actin filaments away from the NPF-coated surface through its force-generating power stroke.