Tensional homeostasis in single fibroblasts

Abstract

Adherent cells generate forces through acto-myosin contraction to move, change shape, and sense the mechanical properties of their environment. They are thought to maintain defined levels of tension with their surroundings despite mechanical perturbations that could change tension, a concept known as tensional homeostasis. Misregulation of tensional homeostasis has been proposed to drive disorganization of tissues and promote progression of diseases such as cancer. However, whether tensional homeostasis operates at the single cell level is unclear. Here, we directly test the ability of single fibroblast cells to regulate tension when subjected to mechanical displacements in the absence of changes to spread area or substrate elasticity. We use a feedback-controlled atomic force microscope to measure and modulate forces and displacements of individual contracting cells as they spread on a fibronectin-patterned atomic-force microscope cantilever and coverslip. We find that the cells reach a steady-state contraction force and height that is insensitive to stiffness changes as they fill the micropatterned areas. Rather than maintaining a constant tension, the fibroblasts altered their contraction force in response to mechanical displacement in a strain-rate-dependent manner, leading to a new and stable steady-state force and height. This response is influenced by overexpression of the actin crosslinker α-actinin, and rheology measurements reveal that changes in cell elasticity are also strain- rate-dependent. Our finding of tensional buffering, rather than homeostasis, allows cells to transition between different tensional states depending on how they are displaced, permitting distinct responses to slow deformations during tissue growth and rapid deformations associated with injury.

Figure 1

Single cells spread between two surfaces reach a steady-state height, spread area, and tension. (A) Schematic diagram of the contraction-force microscopy setup. Cartoon depicts a cell undergoing morphological changes as it spreads between an AFM cantilever and a glass substrate. The two surfaces are patterned with fibronectin to constrain cell adhesion and spreading. The cell spreads and contracts against the cantilever, which enables nanoNewton-level measurements of traction force in the vertical direction. The cell eventually fills up the micropatterned substrates to form an hourglass shape. Note that the deflection of the cantilever is exaggerated to illustrate changes in force measurements. (B) TIRF images of the spreading process on the bottom surface. The cell was visualized using a membrane dye (scale bar: 10 μm). (C) A side projection of an NIH3T3 fibroblast expressing mCherry-LifeAct at steady state taken with confocal microscopy showing a columnar shape and cortical actin underneath the membrane. (D) Example trace of cell spread area increasing over time and eventually reaching a steady state when the cell has filled up the patterned area. (E) Average height (N = 39) and spread area (N = 19) during steady state. Error bars indicate standard error. (F) Example trace of cell tension also increasing during spreading and reaching a plateau when spreading ceased. Note: Area and force traces from panels E and F are taken from different cells. (G) Average steady-state force (N = 42). Error bars indicate standard error. To see this figure in color, go online.

Figure 2

Steady-state tension of single cells is altered by cell displacement in a rate-dependent manner, but not by changes in extracellular stiffness. (A) Cartoon depicting the loading perturbation applied by displacing the cantilever by 1 μm either toward the bottom substrate or away from the substrate at rates of 0.1 μm/min, 1 μm/min, or with a step motion, after a cell has reached steady state. (B) Application of a 1-μm step displacement induced a jump in contractile force, followed by a partial viscous dissipation to a smaller but still significantly higher value compared to before loading. (C) Contractile force increased slightly when a cell was slowly strained at 0.1 μm/min by 1 μm. (D) Force was increased when a cell was quickly strained at 1 μm/min by 1 μm and the cell remained at higher tension even after the ramp displacement ended. (E) Force changes for each loading condition were calculated as the difference between average force before and after ramp or step displacement and were normalized to initial steady-state force. Normalized force change after a fast ramp or step displacement was significantly larger than force change after a slow ramp. Error bars represent standard error. Paired t-tests indicate that changes in steady-state force before and after mechanical perturbations were statistically significant for all three strain rates (N = 16, 13, and 9, for the slow ramp, fast ramp, and step conditions, respectively; ^∗p < 0.05). (F) After a cell has reached steady-state force, the apparent cantilever stiffness was cycled from 2000 nN/μm to 20 nN/μm, and back to 2000 nN/μm at 2-min intervals. (G) Normalized force change after a 100× decrease in stiffness and a 100× increase in stiffness. There was no significant change in steady-state force during step changes in the apparent cantilever stiffness. Error bars represent standard error. (Number of step changes, N_step = 9, 11 for decreasing and increasing stiffnesses, respectively.) To see this figure in color, go online.

Figure 3

Mechanical properties of single cells reach a steady state after spreading but are altered after cell displacement. (A) Contractile force increased during spreading and reached a plateau when spreading ceased. (B) The storage modulus and (C) loss modulus of a cell increased during spreading. Both moduli reached a plateau when the cell was no longer increasing its tension. (D) Normalized change in storage modulus after ramp and step displacements of the cell. (E) Normalized change in loss modulus after ramp and step displacements of the cell. (N = 9, 10, and 9, for the slow ramp, fast ramp, and step conditions, respectively; ^∗∗p < 0.1). Error bars represent standard error. To see this figure in color, go online.

Figure 4

Rate-dependent changes in steady-state tension are dependent on cytoskeletal crosslinking but do not involve changes in adhesions. (A) A pseudo-colored image of vinculin after a cell has reached steady-state spread area (scale bar: 10 μm). (Open square) Size of the subset region. (B) Vinculin intensity was tracked before, during, and after ramp displacements in height. Images show the intensity of a subset region of different cells over time subjected to a slow ramp, fast ramp, or a large step strain (see panel A for subset area depiction). Vinculin intensity change was observed only after a large strain (consisting of multiple step displacements) was applied (scale bar: 1 μm). (C) The average intensity of adhesions remained unchanged after slow and fast ramp displacements. However, significant reinforcement was observed when a very large strain was applied to the cell. Error bars represent standard error (N_slow = 2, N_fast = 4, N_step = 3; ^∗p < 0.05). (D) Cells overexpressing α-actinin-1 were significantly stiffer than normal cells (N_wt = 18, N_actn = 9; ^∗p < 0.05). (E) Cells overexpressing α-actinin 1 showed a large normalized force change when they were displaced by 1 μm at a slow (0.1 μm/min) and fast (1 μm/min) rate. The contractile responses of wild-type cells after slow, fast, and step strains were presented again for easier comparison. Error bars represent standard error (N_slow = 6, N_fast = 6; ^∗p < 0.05). (F) Normalized changes in storage modulus of cells overexpressing α-actinin-1 after slow and fast ramp displacements. The normalized changes in storage modulus of wild-type cells after slow, fast, and step strains were presented again for easier comparison. No significant difference in storage modulus change was observed for the two loading rate conditions. Error bars represent standard error (N_slow = 6, N_fast = 6; ^∗∗p < 0.1). To see this figure in color, go online.