Dissecting Regional Variations in Stress Fiber Mechanics in Living Cells with Laser Nanosurgery

Abstract

The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.

Geometry and network connectivity govern the mechanics of stress fibers

“…By contrast, peripherally located SFs such as the ones severed in our experiments are primarily regulated by myosin light chain kinase and are thus expected to be minimally perturbed by ROCK manipulation (17,18). To suppress ROCK activity and dissipate central connections, we treated cells with 5 μM Y-27632.…”

“…We then applied SLA on SFs along the FN-free edge and observed their retraction kinetics. As in our previous studies, we fit each retraction curve to a Kelvin-Voigt viscoelastic cable model described by a time constant (τ), which reflects the SF's effective viscosity/ elasticity ratio, and a plateau retraction distance (L o ), which correlates to the elastic energy dissipated by half of the severed SF (3,17,18) (Fig. 1D) (see SI Text and Fig.…”

“…We and others have applied SLA to determine the viscoelastic contraction of sarcomeric structures within SFs (14-16) and tension distribution after SF ablation to focal adhesions (FAs) throughout the cell (9). Moreover, SLA has been used to spatially map SF viscoelastic properties within the cell and associate activities of specific NMMII activators and isoforms to these subpopulations (17,18).…”

14-3-3σ stabilizes a complex of soluble actin and intermediate filament to enable breast tumor invasion

“…2 A and B), suggesting the dynamics of actin turnover and/or assembly are perturbed. To test this, we performed fluorescence recovery after photobleaching (FRAP) experiments using an F-actin live-cell probe (30) and observed a ∼twofold increase in the rate of F-actin fluorescence recovery in sh-14-3-3σ cells (Fig. 2C), consistent with an increase in actin-polymerization kinetics rather than a suppression of filament turnover (31).…”

Biophysical Tools for Cellular and Subcellular Mechanical Actuation of Cell Signaling

“…Notably, Kumar et al (34) observed viscoelastic retraction of single actin stress fiber in living cells using nanosurgery and demonstrated that tensional prestress exists in stress fiber bundles. Leveraging the precision of subcellular control of nanosurgery, the spatial tensional variation was mapped by measuring the viscoelastic recoil of contractile stress fibers (38). A similar study has been performed in Caenorhabditis elegans zygotes in which actomyosin meshwork was ablated and the initial outward velocity of adjacent cortex was proportional to cortical tension (39).…”