Resolving the role of actoymyosin contractility in cell microrheology

Abstract

Einstein's original description of Brownian motion established a direct relationship between thermally-excited random forces and the transport properties of a submicron particle in a viscous liquid. Recent work based on reconstituted actin filament networks suggests that nonthermal forces driven by the motor protein myosin II can induce large non-equilibrium fluctuations that dominate the motion of particles in cytoskeletal networks. Here, using high-resolution particle tracking, we find that thermal forces, not myosin-induced fluctuating forces, drive the motion of submicron particles embedded in the cytoskeleton of living cells. These results resolve the roles of myosin II and contractile actomyosin structures in the motion of nanoparticles lodged in the cytoplasm, reveal the biphasic mechanical architecture of adherent cells-stiff contractile stress fibers interdigitating in a network at the cell cortex and a soft actin meshwork in the body of the cell, validate the method of particle tracking-microrheology, and reconcile seemingly disparate atomic force microscopy (AFM) and particle-tracking microrheology measurements of living cells.

Figure 1. Multiple-particle tracking in Swiss 3T3 fibroblasts.

A and B. Typical trajectories of 100 nm-diameter fluorescent beads that were bombarded in the cytoplasm of 3T3 fibroblasts. After overnight incubation, the beads were tracked with ∼10 nm spatial resolution and 33 ms temporal resolution for 20 s. Each slope discontinuity in (B) represents a 33 ms timestep. Scale bars, 20 µm (A) and 100 nm (B). C. Mean squared displacements (MSDs) of the beads 1–3 showed in panel A. The time lag-dependent MSDs showed a slope smaller than 1, indicative of sub-diffusive displacements within the cytoplasm.

Figure 2. Cell morphology, actin, myosin, and phosphorylated-myosin organization following myosin inhibition.

A–C. Typical morphology, actin filament organization (red), distribution of myosin heavy chain IIA (green), and nuclear DNA (blue) in control cells (A), cells treated with 25 µM myosin II inhibitor blebbistatin (B), and cells treated with 20 µM myosin light chain kinase inhibitor ML-7 (C). D–F. Typical morphology, actin filament organization (red), distribution of phosphorylated myosin light chain 2 (green), and nuclear DNA (blue) in control cells (D), cells treated with 25 µM myosin II inhibitor blebbistatin (E), and cells treated with 20 µM myosin light chain kinase inhibitor ML-7 (F). Scale bars, 20 µm.

Figure 3. Role of myosin activity on the displacements of beads in the cytoplasm.

A–C. Typical (light color) and ensemble-averaged MSDs (dark color) of beads embedded in the cytoplasm of control cells (A), cells treated with 25 µM blebbistatin (B), and cells treated with 20 µM ML-7 (C). D. Ensemble-averaged MSDs that are color-coded according to panels A–C. The time lag-dependent MSDs showed a slope smaller than unity, indicative of sub-diffusive displacements within the cytoplasm. At least 10 cells were probed per condition for a total of at least 30 cells (n = 3) and >200 particles.

Figure 4. Cross-correlation magnitudes of simulated Brownian particles and particles in treated and untreated fibroblasts.

A–B. Cross-correlation magnitudes of simulated Brownian particle pairs whose average diffusion coefficient is on the same order of magnitude as in untreated and drug-treated fibroblasts. Traces consistently above the x-axis represent positively correlated particle pairs, whereas traces consistently below the x-axis represent negatively correlated particle pairs. Since Brownian particle pairs were generated randomly and should not maintain any meaningful correlational data, these traces are the established “noise” threshold—magnitudes greater than Brownian pair traces are considered significant, while magnitudes lower than these tracers are considered uncorrelated over the frequency range presented. The data is redisplayed showing only the frequency range of interest, 1 to 10 Hz (B). C. Cross-correlation magnitudes of particle pairs in untreated fibroblasts. D. Cross-correlation magnitudes of particle pairs in blebbistatin-treated fibroblasts. E. Cross-correlation magnitudes of particle pairs in ML-7-treated fibroblasts. At least 10 different, representative particle pair traces are shown for each condition.

Figure 5. Role of myosin activity in intracellular microrheology.

A. Averaged frequency-dependent elastic modulus of the cytoplasm of control cells (black), cells treated with blebbistatin (red), and cells treated with ML-7 (blue). B and C. Averaged cytoplasmic elastic moduli at frequencies of 1 Hz (B) and 30 Hz (C). At least 10 cells were probed per condition for a total of at least 30 cells (n = 3) and >200 particles.

Figure 6. Mechanical architecture of adherent cells.

A. The cytoskeleton of adherent cells is composed of stiff contractile actomyosin structures located in the cortex of the cell, including the basal and apical surfaces of the lamella and the top of the nucleus, which envelop a soft actin filament network devoid of stress fibers. B. Simple model describing how cell mechanics is measured differently by AFM and particle tracking microrheology. AFM measures the combined response of stiff contractile stress fibers and the soft actin network weaved within it. This is equivalent to a soft and a stiff spring in parallel. Particle tracking microrheology only measures the soft actin filament network in the body of the cell that surrounds each bead, which is devoid of stress fibers. This is equivalent to a soft and a stiff spring in series.