Filamin A is essential for active cell stiffening but not passive stiffening under external force - PubMed (original) (raw)

Filamin A is essential for active cell stiffening but not passive stiffening under external force

K E Kasza et al. Biophys J. 2009.

Abstract

The material properties of a cell determine how mechanical forces are transmitted through and sensed by that cell. Some types of cells stiffen passively under large external forces, but they can also alter their own stiffness in response to the local mechanical environment or biochemical cues. Here we show that the actin-binding protein filamin A is essential for the active stiffening of cells plated on collagen-coated substrates. This appears to be due to a diminished capability to build up large internal contractile stresses in the absence of filamin A. To show this, we compare the material properties and contractility of two human melanoma cell lines that differ in filamin A expression. The filamin A-deficient M2 cells are softer than the filamin A-replete A7 cells, and exert much smaller contractile stresses on the substratum, even though the M2 cells have similar levels of phosphorylated myosin II light chain and only somewhat diminished adhesion strength. In contrast to A7 cells, the stiffness and contractility of M2 cells are insensitive to either myosin-inhibiting drugs or the stiffness of the substratum. Surprisingly, however, filamin A is not required for passive stiffening under large external forces.

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Figures

Figure 1

Figure 1

Viscoelastic material properties of M2 and A7 melanoma cells cultured on rigid glass substrates. (A) Elastic, _G_′(ω) (closed), and viscous, _G_″(ω) (open), moduli for A7 (squares) and M2 (circles) cells cultured for 24 h, as measured by MTC. (B) Confocal images of actin cytoskeleton at the basal surface of cells fixed at various times after plating. Bar = 10 _μ_m. (C) Cell stiffness (geometric mean ± SE) measured at ω = 5 rad/s as a function of time after plating; similar time evolution observed for all ω (top). Ratio of viscous to elastic moduli (mean ± SE) of cells as a function of time after plating (bottom). Error bars, if not visible, are smaller than the points in this plot.

Figure 2

Figure 2

Contractile stresses exerted by M2 and A7 cells on collagen I-coated E = 1.3 kPa polyacrylamide substrates. (A) Mean contractile moment M. Examples of contractile deformations induced in the substrate by typical M2 (B) and A7 (C) cells. Image size, 100 _μ_m.

Figure 3

Figure 3

Contractility, stiffness, and spreading of M2 and A7 cells grown on collagen I-coated polyacrylamide substrates of increasing stiffness. (A) Cell contractile moment (mean ± SE). (B) Cell stiffness (geometric mean ± SE). (C) Projected cell area (mean ± SE). (D–I) Examples of cells grown on E = 0.1, 1.3, and 24 kPa substrates.

Figure 4

Figure 4

(A) Schematic showing various roles for filamin A in cell mechanics. Filamin A cross-links actin filaments into orthogonal networks, which support contractile stresses generated by myosin II, and also binds some integrins, linking the actin cytoskeleton to the cell membrane. (B) Effect of myosin II inhibition on material properties of A7 and M2 cells. Cell stiffness (geometric mean ± SE) and the ratio of viscous to elastic moduli (mean ± SE) for control cells and cells treated with 50 _μ_M blebbistatin for 20 min. ∗ indicates that p < 0.05.

Figure 5

Figure 5

(A) Western blots showing similar levels of pMLC and MLC in M2 (lanes 1–3) and A7 (lanes 4–6) cells plated on collagen I. pMLC levels are decreased in a dose-dependent manner by treatment with 0 _μ_M (lanes 1 and 4), 10 _μ_M (lanes 2 and 5), or 50 _μ_M (lanes 3 and 6) of the Rho-associated kinase inhibitor Y-27632. (B) Contractile moment, M, of a single M2 (circles) or A7 (squares) cell after addition of 10 _μ_M Y-27632 to the media at t = 0. (Inset) Contractile moment (mean ± SE) for cells before (white) and after (black) 20 min of treatment with either 10 _μ_M Y-27632 or control media.

Figure 6

Figure 6

Adhesion strength of M2 (circles) and A7 (squares) cells to collagen I-coated substrates, measured by the spinning disk method (34). After cells are exposed to fluid shear stress, adherent fraction is measured as a function of distance r from the center of the coverslip. Shear stresses on apical surface of cells increase linearly with r. (Inset) Relative adhesion strength determined from radius at which 50% of cells have detached.

Figure 7

Figure 7

Stiffening of A7 and M2 cells subjected to large nanonewton-scale external forces, applied by magnetic tweezers through a fibronectin-coated magnetic bead. (A) Cell stiffness and estimated modulus (geometric mean ± SE) as a function of force for A7 (squares, n = 30) and M2 (circles, n = 19) cells. (Inset) Percentage of beads disrupted from cells as a function of force. (B) Cell stiffness as a function of force for A7 (n = 9) and M2 (n = 7) cells treated with 5 _μ_M cytochalasin-D for 15 min.

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References

    1. Bausch A.R., Ziemann F., Boulbitch A.A., Jacobson K., Sackmann E. Local measurements of viscoelastic parameters of adherent cell surfaces by magnetic bead microrheometry. Biophys. J. 1998;75:2038–2049. - PMC - PubMed
    1. Fabry B., Maksym G.N., Butler J.P., Glogauer M., Navajas D. Scaling the microrheology of living cells. Phys. Rev. Lett. 2001;87:148102. - PubMed
    1. Hoffman B.D., Massiera G., Van Citters K.M., Crocker J.C. The consensus mechanics of cultured mammalian cells. Proc. Natl. Acad. Sci. USA. 2006;103:10259–10264. - PMC - PubMed
    1. Radmacher M., Fritz M., Kacher C.M., Cleveland J.P., Hansma P.K. Measuring the viscoelastic properties of human platelets with the atomic force microscope. Biophys. J. 1996;70:556–567. - PMC - PubMed
    1. Na S., Collin O., Chowdhury F., Tay B., Ouyang M. Rapid signal transduction in living cells is a unique feature of mechanotransduction. Proc. Natl. Acad. Sci. USA. 2008;105:6626–6631. - PMC - PubMed

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