Inhibition of Contractility and RhoA Deactivation Trigger Podosome Formation

Abstract

Development of integrin-mediated cell-matrix adhesions is important in the regulation of cell growth and differentiation. Activated integrin clusters are sites of actin polymerization, and the physical properties of extracellular matrix ligands play critical roles in the development of adhesion structures. Previously, we have utilized RGD-membranes, continuous supported bilayers with freely diffusing lipid-anchored RGD ligands, to activate integrins and investigate force dependence of initial integrin clustering during early adhesion formation (Yu et al., PNAS 2011). At later times (>45 minutes) on RGD-membranes, we found that fibroblasts natively form podosomes as adhesion structures when cell-matrix traction force cannot be stabilized. Podosome core assembly was initiated by Arp2/3-mediated actin polymerization locally within integrin clustering sites that were characterized by integrin-associated proteins, such as vinculin, paxillin, and talin. In addition, myosin-1E/1F and CARMIL1 were found as new markers at podosome cores. We utilized a FRET-based RhoA biosensor to measured differential RhoA activities in cells forming podosomes on RGD-membranes versus focal adhesions on RGD-glass. RhoA-GTP levels were indeed low in cells forming podosomes on RGD-membranes, whereas RhoA-GTP levels were high in the cells on RGD-glass. Constitutively active RhoA-Q63L mutant blocked podosome formation on RGD-membranes. Furthermore, fibroblasts on RGD-membranes with dense nano-partitions (1μm-pitch line partitions) in RGD-membranes provided mechanical barriers that enabled adhesion maturation that was not possible with wider line-partitions (4μm) in RGD-membrane. When a single cell adhered over both regions of high and low density, podosomes formed over low-density regions only. From our data, we suggest that development of podosomes as adhesion structures requires inhibition of traction forces between integrin receptors and matrix ligands. Activation of myosin contractility at integrin clusters activates focal adhesion formation and inhibits podosome formation at integrin clusters, giving rise to a mutually exclusive behavior of focal adhesions and podosomes.