Substrate engagement of integrins α5β1 and αvβ3 is necessary, but not sufficient, for high directional persistence in migration on fibronectin

Mesenchymal cell migration involves a complex, yet tightly regulated control over actin polymerization, adhesion dynamics and actomyosin contractility to enable cell translocation in its environment. Much of our understanding on how signals from the extracellular matrix (ECM) control cell migration stems from in vitro studies on flat substrates, on which both soluble and insoluble biochemical signals can be precisely manipulated1,2. Cell adhesion can be modulated by coating with ECM proteins, their fragments or small molecular ligands (e.g. peptides) and by employing engineering strategies to precisely vary ligand presentation, concentrations and mechanics3. Integrins are the major trans-membrane receptors cells employ to recognize, adhere and adapt to the chemical and mechanical properites of their ECM4. The 18 α and 8 β subunits assemble into 24 heterodimeric integrin complexes that exhibit varying affinity for ECM ligands and distinct signaling capabilities5,6. Interestingly, integrin expression profiles are often altered in pathological situations such as during wound healing, angiogenesis or tumor metastasis, presumably to promote efficient cell migration7,8. While integrins are probably not the sole receptor family responsible in regulating cell migration, understanding how cells respond to differential integrin engagement in respect to their motility, and in particular their directional persistence is a major open question9,10, and constitutes the underlying motivation of this study. Among integrins, particular attention has been placed on the “fibronectin receptor” α5β1 and “vitronectin receptor” αvβ3, and their impact on cell migration11. Previous work, based on exogenous integrin expression on cells that originally lack these integrins, has suggested that β1 promotes random cell migration, while β3 favor persistent migration12. More recently, pan-integrin-null fibroblasts were used to show that expression of αv integrins results in increased persistence compared to β1 integrin expression, and that there is substantial cross-talk between the two integrin classes13. Indeed, employing highly selective integrin peptidomimetics on spatially patterned surfaces, we recently provided further support of integrin cross-talk and demonstrated that integrin αvβ3 co-localizes with integrin α5β1 also in absence of αvβ3 ligand presentation14. The integrin dependence in directional migration was traced to the differential regulation of the family of RhoGTPases and the balance of actin polymerization mediators, including cofilin12,15. However, the aforementioned studies examining directional migration utilized exogenous control over integrin expression and tested migration only on fibronectin as the cell adhesive coating. Here, we presented fibroblasts with substrates coated with plasma fibronectin (FN) or vitronectin (VN), both ECM glycoproteins containing the integrin-binding RGD sequence16,17. In this manner, we studied how differential ECM receptor engagement affects single cell adhesion and migration avoiding genetic manipulation of cells. FN is a major constituent of provisional matrix during wound healing and is the most commonly-used cell adhesive coating for in vitro fibroblast migration studies. VN has received less attention, despite being an abundant serum protein, which is adsorbed readily on surfaces in vitro18, and having exhibited distinct behavior in initial cell motility studies19. FN and VN present binding sites for a range of different integrins and membrane receptors, including the heparan sulfate proteoglycan syndecan-4 for FN20, and the urokinase plasminogen receptor for VN21. Nevertheless, for the reasons outlined above, we focused on the role of α5β1 as the major integrin receptor for FN, which does not recognize VN, and αvβ3 that can bind both FN and VN5. Our findings revealed a pronounced effect of ECM protein coating on cell motility, with fibroblasts exhibiting directionally persistent migration on FN-coated substrates, as a function of FN surface density. Characterization of fibroblast adhesion and dynamics identified marked differences in adhesion plaque formation, cytoskeleton organization, focal adhesion dynamics and intracellular signaling between FN and VN. In order to examine the role of α5β1 or αvβ3 integrins, we used highly selective, antagonists against these integrins, either in soluble form to block their substrate engagement, or immobilized on a patterned substrate as cell adhesive ligands. Our results demonstrate that α5β1 and αvβ3 integrins are necessary but not sufficient for directional persistence in fibroblast migration.