Fig 1.
Response of VSMC to external pulling force.
(a) Schematic of a VSMC mechanically stimulated with a FN functionalized AFM probe and simultaneously imaged by spinning-disk confocal microscopy. (b-c) Fluorescence images of VSMC expressing mRFP1-actin-7 (left) and the 3D reconstructed image of the representative segmentation of actin filament bundles (right) for before (b) the AFM probe displacement at time 0 min, and after (c) the AFM probe displacement at time 68 min. Scale bar: 20 μm. Left panels used with permission from JOVE [21]. (d) The scheduled pulling force in three phases: small, intermediate and large forces. (e) The alignment index for the actin filament bundles increased rapidly as small force was applied, but slowed down as the force increased. (f) The normalized intensity for mRFP1-actin-7 increased steadily through all force ranges. Blue lines: piece-wise linear fit for forces below 0.5 nN and ≥0.5 nN.
Fig 2.
(a) A sketch of the simulation setup. The simulation box is 3 μm in x and y directions, and the initial height (z-direction) is 1.25 μm. The simulation box contains 300 free actin filaments, as well as diffusible G-actin, myosin, and α-actinin linkers. A semi-spherical AFM probe is located at the upper boundary, and 30 filaments are attached to the probe via stiff harmonic springs. At the beginning of simulations, all filaments are 0.108 μm long (containing 40 actin subunits). The input G-actin concentration is much higher than the equilibrium concentration, making actin filaments rapidly elongate. An average length of 0.8 μm is achieved and maintained after around 40s of simulation. (b) Simulated AFM-probe position, equivalent to the height of upper boundary, as a function of time for Cases i-iii. The control case (Case iv) is without AFM probe and without filament attachment, with only the upper boundary moving in the same way as in Case i to avoid potential boundary effects.
Fig 3.
(a-b) Representative snapshots of (a) bundle-like actin networks under Case i pulling condition at time t = 900 s, and (b) cluster-like actin networks without external force at time t = 900 s. Actin filaments, myosin motors, and crosslinkers are shown in red, blue, and green cylinders, respectively. The gray sphere in (a) represents the AFM probe. (c) Filament alignment index along the Z-axis for 500 nm step size (red, Case i), mixed step sizes (250 nm for the first three pulling events and 500 nm for the last two, blue), 250 nm step size (purple, Case ii) and no AFM-probe pulling (green, Case iii). α-actinin linker:actin is 0.1 and myosin:actin is 0.005 in all simulations. Error bars represent the standard deviation from the mean in 5–10 simulation replicates.
Fig 4.
(a) The F-actin fraction in the bundle region as a function of time for actin networks under 500 nm displacement steps (red, Case i) and actin networks free of external force (green, Case iv). The bundle region is defined as the volume under the simulated AFM probe, which is a cylindrical region of 500 nm in diameter and the height of the simulation box. The box size varies over time based on the position of the AFM probe. Insert shows a 2D illustration of the bundle region. (b) The rate of F-actin accumulation in the bundle region from simulations with the AFM probe pulling force (red, Case i) and without AFM probe pulling- force (green, control Case iv). The recruitment rates are calculated by linear-fitting of the data points every 50 seconds. Shaded colors and error bars are the standard deviations of 10 replica simulations for Case i and 5 replicas for Case iv, respectively.
Fig 5.
(a) F-actin distribution along the force direction (Z-axis) of the most representative trajectory before the 4th pulling at t = 600 s (blue), after the 4th pulling at t = 601 s (orange), and after the long-timescale chemical evolution at t = 750 s (yellow). (b-d) Corresponding simulation snapshots before pulling, after pulling, and after chemical evolution, respectively. (e) Standard deviations (σ) of the F-actin distribution along the force direction before pulling (blue), after pulling (orange), and after 150 s of chemical evolution (yellow) at the third, fourth, and fifth pulling events. σ are averaged over 10 duplicated trajectories, and error bars represent the standard errors. (f) The radius of gyration, Rg and (g) the rate of Rg change, , of actin networks in Case i with 500 nm pulling (red) and in Case iv without pulling (green). Shaded colors and error bars are the standard deviations of 10 duplicated trajectories for Case i and 5 duplicated trajectories for Case iv.
Fig 6.
Motor-driven chemical evolution generates contractility that induces the geometric collapse of the actin network.
In random networks without external forces, the geometric collapse would be isotropic, causing filaments to cluster into globular foci (a). However, the external tensile force induces filament directional alignment and favors anisotropic chemical evolution, resulting in filament bundling (b).
Table 1.
Parameters for the simulations.