Absence of filamin A prevents cells from responding to stiffness gradients on gels coated with collagen but not fibronectin

Abstract

Cell types from many tissues respond to changes in substrate stiffness by actively remodeling their cytoskeletons to alter spread area or adhesion strength, and in some cases changing their own stiffness to match that of their substrate. These cell responses to substrate stiffness are linked to substrate-induced changes in the state, localization, and amount of numerous proteins, but detailed evidence for the requirement of specific proteins in these distinct forms of mechanical response are scarce. Here we use microfluidics techniques to produce gels with a gradient of stiffness to show the essential function of filamin A in cell responses to mechanical stimuli and dissociate cell spreading and stiffening by contrasting responses of a pair of human melanoma-derived cell lines that differ in expression of this actin cross-linking protein. M2 melanoma cells null for filamin A do not alter their adherent area in response to increased substrate stiffness when they link to the substrate only through collagen receptors, but change adherent area normally when bound through fibronectin receptors. In contrast, filamin A-replete A7 cells change adherent area on both substrates and respond more strongly to collagen I-coated gels than to fibronectin-coated gels. Strikingly, A7 cells alter their stiffness, as measured by atomic force microscopy, to match the elastic modulus of the substrate immediately adjacent to them on the gradient. M2 cells, in contrast, maintain a constant stiffness on all substrates that is as low as that of A7 cells on the softest gels examined (1000 Pa). Comparison of cell spreading and cell stiffening on the same gradient substrates shows that cell spreading is uncoupled from stiffening. At saturating collagen and fibronectin concentrations, adhesion of M2 cells is reduced compared to that of A7 cells to an extent approximately equal to the difference in adherent area. Filamin A appears to be essential for cell stiffening on collagen, but not for cell spreading on fibronectin. These results have implications for different models of cell protrusion and adhesion and identify a key role for filamin A in altering cellular stiffness that cannot be compensated for by other actin cross-linkers in vivo.

Figure 1

(a) Gradient generator, which has three inlets supplying the relative amounts of bisacrylamide or fluorescent dye shown and one wide outlet. (b)The chemical gradient produced at the outflow from the mixing apparatus of the microfluidic system visualized by doping the bisacrylamide solution with a fluorescent dye. Higher fluorescence intensities represent lower bisacrylamide concentration, and hence lower gel stiffness. (c) Local elastic modulus (solid circles) measured by AFM across a 2 mm wide gradient gel. Fluorescence intensity profile across a gradient gel coated with rhodamine-labeled fibronectin (solid squares) and a gradient gel coated with nonfluorescent fibronectin (open squares) to correct for gel autofluorescence.

Figure 2

Morphological change of melanoma cells on stiffness gradient gels. Shapes of A7 (a) and M2 (b) cells bound to a PA gradient gel coated with collagen I and fibronectin, taken 24 h after plating. Gel stiffness increases from left to right. Scale bar is 40 μm. The full width of the gel is 1.8 mm, and its stiffness ranges from 1 to 30 kPa. Higher magnification images show examples of A7 (c and f) and M2 (d and g) cells on soft (c and d, 0.5 kPa) and stiff (f and g, 15 kPa) gels. Scale bar for these images is 10 μm Western blot (e) shows equal levels of talin and actin expression in M2 and A7 cells, but no filamin A expression in M2 cells. Molecular mass bars on left of image are 200 kDa for filamin and talin, and 40 kDa for actin.

Figure 3

Cellular stiffness measured by AFM (a and b) and adherent area (c and d) of A7 (a, c) and M2 (b and d) melanoma cells cultured for 24 h on polyacrylamide gels laminated with collagen I (circles), fibronectin (triangles), or mixture of collagen I and fibronectin (diamonds). Both proteins were added at saturating concentrations to the gels using methods described in (47). Error bars representing standard errors (n = 3) for stiffness measurements of individual cells and the adjacent gel shown in panel a are representative for data in panels a and b. Error bars in panels c and d represent larger populations of cells (n = 30–50) within gels of the average stiffness shown on the abscissa.

Figure 4

Adhesion strength of M2 and A7 cells to surfaces coated with different densities of fibronectin and collagen. Relative cell adhesion strength is calculated as the intensity of cell staining on surfaces after nonadherent cells are removed by washing with medium.