Abstract
Cells sense and respond to the mechanical properties of their microenvironment. We investigated whether these properties affect the ability of cells to assemble a fibrillar fibronectin (FN) matrix. Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates. Stimulating integrins with Mn(2+) treatment increased FN assembly on softer gels, suggesting that integrin binding is deficient on soft substrates. Guanidine hydrochloride-induced extension of the substrate-bound FN rescued assembly on soft substrates to a degree similar to that of Mn(2+) treatment and increased activation of FAK along with the initiation of assembly at FN matrix assembly sites. In contrast, increasing actin-mediated cell contractility did not rescue FN matrix assembly on soft substrates. Thus, rigidity-dependent FN matrix assembly is determined by extracellular events, namely the engagement of FN by cells and the induction of FN conformational changes. Extensibility of FN in response to substrate stiffness may serve as a mechanosensing mechanism whereby cells use pericellular FN to probe the stiffness of their environment.
Highlights
Cell behavior is affected by changes in extracellular matrix stiffness during disease progression
Analysis of matrix assembled by cells grown on FN-coated polyacrylamide gels of varying stiffnesses showed that rigid substrates stimulate FN matrix assembly and activation of focal adhesion kinase (FAK) compared with the level of assembly and FAK signaling on softer substrates
No DOC-insoluble material was detected on the soft substrate, and the level on the intermediate substrate was very low. These low levels precluded determination of band intensities at the 6-h time point. These results show that substrate stiffness affects cellmediated FN matrix assembly with rigid substrates enhancing assembly and softer substrates limiting the amount of FN matrix assembled
Summary
Cell behavior is affected by changes in extracellular matrix stiffness during disease progression. Whether cells increase FN fibril formation in response to sensing a rigid pericellular environment is an important question especially because FN assembly precedes and often seeds assembly of other ECM proteins such as collagen [20]. We observed that FN matrix assembly is up-regulated on rigid substrates and propose that this is primarily due to a deficiency in cell-mediated FN conformational changes on softer substrates. These findings establish an extracellular mechanism for stiffness-dependent regulation of FN matrix assembly
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