Cell stiffening in response to external stress is correlated to actin recruitment

Abstract

We designed a micromanipulation device that allows the local application of a constant force on living cells, and the measurement of their stiffness. The force is applied through an Arg-Gly-Asp-coated bead adhering on the cell and trapped in optical tweezers controlled by a feedback loop. Epifluorescence observations of green fluorescent protein-actin in the cells are made during force application. We observe a stiffening of cells submitted to a constant force within a few minutes, coupled to actin recruitment both at the bead-cell contact and up to several micrometers from the stress application zone. Moreover, kinetics of stiffening and actin recruitment exhibit a strong correlation. This work presents the first quantification of the dynamics of cell mechanical reinforcement under stress, which is a novel insight into the elucidation of the more general phenomenon of cell adaptation to stress.

FIGURE 1

Mechanical measurements on single cells. (A) Creep function of a single C2C12 cell as a function of force application time, fitted by a power law J(t) = 5.9.10⁻³ t^0.18 Pa⁻¹ (in log-log scale). (B) Successive creep functions on a single A549 cell: a series of step forces is applied. For each step, the creep function is measured. For better readability, J(t) is displayed for only four of the nine-step force applications: number 2, 4, 5, and 7. (C) The corresponding viscoelastic modulus G₀ versus time, fitted by a sigmoid with a rising time τ_G = 75 s. Mechanical saturation is reached in this example.

FIGURE 2

Distribution histograms and cumulative probability functions of the power-law parameters: exponent-α (A) and viscoelastic modulus G₀ (B), measured on 39 different C2C12 cells.

FIGURE 3

A C2C12 cell during force application: transmission image with an indication of the force direction (A′); a zoom around the bead (A); fluorescence images of GFP-actin at times t₁ = 420 s (B), t₃ = 1080 s (C), t₅ = 1440 s (D), t₇ = 1920 s (E), t₉ = 2400 s (F), and t₁₀ = 2640 s (G). Arrowheads in images D and E point at adhesion patches. A circle representing the bead in size and position is superimposed on image D. (Bars: 5 μm.)

FIGURE 4

(A) Graphic of the actin recruited in FA around the bead during one experiment. The arrow gives the force direction. The ellipses are the best fits for the actin patches detected (crosses indicate their centers), and are labeled according to the image number on which they are measured. For patches present on step forces number-8 and more, only the centers are displayed. (B) Distribution of the actin patches orientations. Angles are calculated relatively to the direction of force application, as displayed on the top panel.

FIGURE 5

Plot of the actin density δQ′_i in successive rings around the bead (of radii δR₁ = 2.5 to δR₈ = 6.0 μm), at different step force application times t_k, k = 2 (open down-triangles), 4 (open up-triangles), 6 (open circles), 8 (shaded down-triangles), 10 (shaded up-triangles), and 12 (solid circles). During force application, the actin quantity increases even far from the bead. An exponential decay fit δQ′(r) = A + B exp(– r/R_c) yields a cutoff radius R_c of ∼3.5 μm (1.4, 5.2, 3.25, and 4.55 μm, respectively, for t₄, t₆, t₁₀, and t₁₂).

FIGURE 6

Normalized cell viscoelastic modulus g(t) (shaded dots), actin quantities q(t) (solid diamonds), and q′(t) (open diamonds) versus time, during a series of step-force applications. Fitting this experiment by a sigmoid yields the parameters {q₀ = 1.9.10⁻⁴, q_f = 1.01, τ_Q = 300 s}; {q′₀ = 6.2.10⁻³, q′_f = 1.03, τ′_Q = 471 s}; and {g₀ = 1.6.10⁻³, g_f = 1.2, τ_G = 612 s}.