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.