Early integrin binding to Arg-Gly-Asp peptide activates actin polymerization and contractile movement that stimulates outward translocation

Abstract

Integrin-mediated adhesions are critical for stem cell differentiation, cancer metastasis, and the immune response [Hynes RO (2009) Science 326:1216-1219. However, the mechanisms of early adhesion formation remain unclear, especially the effects of lateral clustering of integrins and the role of the Src family kinases. Using mobile Arg-Gly-Asp (RGD) peptide ligands on lipid bilayers with nano-fabricated physical barriers [Salaita K, et al. (2010) Science 327:1380-1385], we observe surprising long-range lateral movements of ligated integrins during the process of cell spreading. Initially, RGD-activated integrin clusters stimulate actin polymerization that radiates from the clusters. Myosin II contraction of actin from adjacent clusters produces contractile pairs that move toward each other against barriers. Force generated by myosin II stimulates a Src kinase-dependent lamellipodial extension and outward movement of clusters. Subsequent retraction by myosin II causes inward movement of clusters. The final cell spread area increases with the density of periodic barriers. Early integrin clustering recruits adhesion proteins, talin, paxillin, and FAK, irrespective of force generation. However, recruitment of vinculin is only observed upon contraction. Thus, we suggest that integrin activation and early clustering are independent of lateral forces. Clustering activates Src-dependent actin polymerization from clusters. Myosin contraction of clusters to lines stimulates active spreading with outward forces from actin polymerization followed by a second wave of contraction. Many of these early mechanical steps are not evident in cells spreading on immobilized matrices perhaps because of the low forces involved. These observations can provide new targets to control integrin-dependent adhesion and motility.cell adhesion | integrin reorganization | supported membranes T he integrins, a heterodimeric transmembrane receptor family, regulate many cellular processes including growth, differentiation, and death (1-3). Both chemical recognition and mechanical sensing are important factors in the outside-in integrin signaling pathway (4-6). Chemically, ligation of integrin receptors, such as fibronectin binding to α5β1 and αvβ3, results in conformational changes of the external domains and cytoplasmic tails causing the recruitment of various signaling molecules and adaptor proteins during focal adhesion formation (7,8). Mechanically, matrix rigidity and local matrix density regulate cell spreading, growth, and differentiation. Rigidity sensing depends upon local contractile force generation by the actin cytoskeleton on ligated integrin receptors (9-11). The spacing of Arg-Gly-Asp (RGD) peptide matrix ligands on 5 nm gold dots needs to be 60 nm or less for cell spreading (12). Recent findings with a constant overall density of RGD ligands, show that local clusters of four or more RGD ligands spaced by 60 nm are needed for sustained spreading, defining a minimal adhesion unit of four ligated integrins (13,14). Nevertheless, becau...

Cells test substrate rigidity by local contractions on submicrometer pillars

“…Once adhesions are formed, they couple to the rearward actin flow, which produces a centripetally directed force that increases with pillar diameter. The localization of myosin between 0.5-μm pillars suggests that the contractile complex consists of bipolar myosin filaments (with lengths of 0.3 to ∼0.4 μm) (21,22), attached to actin filaments that polymerize from adhesion sites as recently observed (23).…”

Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions

“…Here, αV-GFP-constrained and paxillin-mCherry formed mobile clusters with very low anisotropy, and cells did not spread (22) (Fig. 2A and Fig.…”

Large compact clusters and fast dynamics in coupled nonequilibrium systems

“…It is known that on the cell membrane, various proteins and lipids like integrins, T-cell receptors are present in the form of nanoclusters [21,22]. This clustering is shown to be induced by the cortical actin cytoskeleton [2,23], and within a recent theoretical model of the process, an FDPO state has been observed [24].…”

“…This clustering is shown to be induced by the cortical actin cytoskeleton [2,23], and within a recent theoretical model of the process, an FDPO state has been observed [24]. There is now experimental evidence that the actin cytoskeleton also gets reorganized by these membrane components [22,25]. This raises the interesting possibility of new phases arising if the treatment of [24] is extended to account for the back action of membrane components on the cytoskeleton.…”