Cell size and actin architecture determine force generation in optogenetically activated cells

Abstract

Adherent cells use actomyosin contractility to generate mechanical force and to sense the physical properties of their environment, with dramatic consequences for migration, division, differentiation, and fate. However, the organization of the actomyosin system within cells is highly variable, with its assembly and function being controlled by small GTPases from the Rho family. To understand better how activation of these regulators translates into cell-scale force generation in the context of different physical environments, here we combine recent advances in non-neuronal optogenetics with micropatterning and traction force microscopy on soft elastic substrates. We find that, after whole-cell RhoA activation by the CRY2/CIBN optogenetic system with a short pulse of 100 ms, single cells contract on a minute timescale in proportion to their original traction force, before returning to their original tension setpoint with near perfect precision, on a longer timescale of several minutes. To decouple the biochemical and mechanical elements of this response, we introduce a mathematical model that is parametrized by fits to the dynamics of the substrate deformation energy. We find that the RhoA response builds up quickly on a timescale of 20 s, but decays slowly on a timescale of 50 s. The larger the cells and the more polarized their actin cytoskeleton, the more substrate deformation energy is generated. RhoA activation starts to saturate if optogenetic pulse length exceeds 50 ms, revealing the intrinsic limits of biochemical activation. Together our results suggest that adherent cells establish tensional homeostasis by the RhoA system, but that the setpoint and the dynamics around it are strongly determined by cell size and the architecture of the actin cytoskeleton, which both are controlled by the extracellular environment.

Figure 1

Cell size is the major determinant of strain energy and strain energy gain during photoactivation. (a) From left to right: (i) disc-shaped fibronectin micropatterns on polyacrylamide hydrogels with increasing surface area. The patterns cover an area of 500–1000–1500 μm². (ii) Individual actin-labelled cells. (iii) Quantification of the actin orientation by orientation map. The color-coded maps show the angle of oriented features in the image from ${-90}^{\circ }$ to ${90}^{\circ }$ (see materials and methods). Larger cells are more polarized. (iv) Adhesion pattern from vinculin staining. The contrast of the vinculin images is enhanced to facilitate visualization of small and thin focal adhesions. (v) Results for traction force microscopy. Traction forces are localized at the cell contour. (b) Exemplary substrate deformation map and bright-field images. Cells show dipolar traction patterns. Substrate deformation is larger for larger cells. (c) Substrate displacement measured with respect to distance from the cell edge along the lines in (b). Vertical lines indicate the mean values of the decay lengths l_d defined by the half-decay of the displacement. (d) Decay length l_d for cells on different pattern sizes. (e) Global cellular actin fiber alignment for cells spread on each disc size. This is represented by the actin order parameter. (f) Static strain energy for cells spread on the three different disc sizes. Using a one-way ANOVA test, significant difference is found between cells spread on 500 μm² pattern and the other two bigger sizes. (g) Quantification of the mean strain energy over time for cells on the different disc sizes subjected to one light pulse of 100 ms. (h) Strain energy increase for every activated cell on the three different disc sizes. Calculation is made by subtracting the strain energy value before activation to the highest strain energy value obtained after light activation. To see this figure in color, go online.

Figure 2

A mathematical model decouples activation and force generation. (a) The active Kelvin-Voigt model describes a viscoelastic solid with active stresses, which here are controlled by optogenetics. The cell on a soft substrate is modeled as a thin contractile sheet coupled to an elastic foundation. (b) Finite element modeling is used to implement the model for anisotropic cell organization like a polarized cell on a disc pattern. The resulting traction patterns resemble the experimentally observed ones. (c) The model predicts the variation of displacements, strain energies, localization lengths and background stresses as a function of cell size in very good agreement with experimental observations. (d) Photoactivation is modeled by a double sigmoid. (e) The model predicts the internal dynamics of the active stresses that cannot be measured directly. (f) Predicted values for strain energy gain, gain in active stress, time constants, and sigmoid centers. The model suggests a strong asymmetry between activation (fast) and relaxation (slow). In addition, it reveals peaked values for intermediate cell size. To see this figure in color, go online.

Figure 3

Actin architecture modulates magnitude and variability of strain energy gain during activation. (a) From left to right: (i) 1000 μm² disc shaped and hazard shaped fibronectin micropatterns on polyacrylamide hydrogels. (ii) Actin staining. (iii) Actin orientation map. (iv) Adhesion pattern from vinculin staining. The contrast of the vinculin images is enhanced to facilitate visualization of small and thin focal adhesions. (v) Traction stress map. (b) Actin order parameter and strain energy for cells spread on disc or hazard micropatterns. Despite the differences in actin organization, the static strain energy for cells spread on the disc and the hazard shapes is very similar. Using a one-way ANOVA test, significant difference is not found between the two cases. (c) Normalized quantification of the mean strain energy over time for cells on both shapes subjected to one light pulse of 100 ms. (d) The model reveals that internal stresses are very different during activation. (e) Model parameters reveal large differences despite similar strain energies. To see this figure in color, go online.

Figure 4

The dynamics of photoactivation strongly depend on actin architecture. (a) Strain energy during a series of photoactivation pulses of increased duration (represented by stripe width). Dotted lines show mean values, shaded regions correspond to standard deviations and full lines show model fits. The curves represent averages of seven disc and hazard patterns. For each pulse we specify pulse duration and injected energy input. (b) Active stresses extracted from the model. (c) Maximal active stresses extracted from the model for the two different patterns reveal saturation at 25 ms pulse duration. Solid lines are exponential fits. (d) Sigmoid centers t and time constants τ for activation and relaxation for disc and hazard. There is a strong difference between the two actin architectures, reflecting the different internal organization of the cell. To see this figure in color, go online.