Persistence of fan-shaped keratocytes is a matrix-rigidity-dependent mechanism that requires α₅β₁ integrin engagement

Despite the importance of matrix rigidity on cell functions, many aspects of the mechanosensing process in highly migratory cells remain elusive. Here, we studied the migration of highly motile keratocytes on culture substrates with similar biochemical properties and rigidities spanning the range between soft tissues (~kPa) and stiff culture substrates (~GPa). We show that morphology, polarization and persistence of motile keratocytes are regulated by the matrix stiffness over seven orders of magnitude, without changing the cell spreading area. Increasing the matrix rigidity leads to more F-actin in the lamellipodia and to the formation of mature contractile actomyosin fibers that control the cell rear retraction. Keratocytes remain rounded and form nascent adhesions on compliant substrates, whereas large and uniformly distributed focal adhesions are formed on fan-shaped keratocytes migrating on rigid surfaces. By combining poly-L-lysine, fibronectin and vitronectin coatings with selective blocking of α_vβ₃ or α₅β₁ integrins, we show that α_Vβ₃ integrins permit the spreading of keratocytes but are not sufficient for polarization and rigidity sensing that require the engagement of α₅β₁ integrins. Our study demonstrates a matrix rigidity-dependent regulation of the directional persistence in motile keratocytes and refines the role of α_vβ₃ and α₅β₁ integrins in the molecular clutch model.