During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

Cell adhesion, spreading and migration require the dynamic formation and dispersal of contacts with the extracellular matrix (ECM). In vivo, the number, availability and distribution of ECM binding sites dictate the shape of a cell and determine its mobility. To analyse the geometrical limits of ECM binding sites required for cell attachment and spreading, we used microcontact printing to produce regular patterns of ECM protein dots of defined size separated by nonadhesive regions. Cells cultured on these substrata adhere to and spread on ECM regions as small as 0.1 microm2, when spacing between dots is less than 5 microm. Spacing of 5-25 microm induces a cell to adapt its shape to the ECM pattern. The ability to spread and migrate on dots > or =1 microm2 ceases when the dot separation is > or =30 microm. The extent of cell spreading is directly correlated to the total substratum coverage with ECM-proteins, but irrespective of the geometrical pattern. An optimal spreading extent is reached at a surface coating above 15%. Knowledge of these geometrical limits is essential for an understanding of cell adhesion and migration, and for the design of artificial surfaces that optimally interact with cells in a living tissue

A talin intermolecular interaction autoinhibits its own activation and regulates β3-integrin binding. When bound, β3-integrin undergoes structural alterations that prevent its β and α subunits from associating, maintaining β3-integrin's clustering capability.

Cultured myofibroblasts are characterized by stress fibers, containing alpha-smooth muscle actin (alpha-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of alpha-SMA-negative fibroblasts. We have investigated the role of alpha-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of alpha-SMA-negative fibroblasts. Then, myofibroblasts incorporate alpha-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When alpha-SMA expression is induced in 5 d culture by TGFbeta or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with alpha-SMA fusion peptide (SMA-FP), which inhibits alpha-SMA-mediated contractile activity, reduces their adhesion to the level of alpha-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and beta3 integrin-containing FAs; alpha5 integrin distribution is not affected. FRAP of beta3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high alpha-SMA-mediated contractile activity of myofibroblast stress fibers.

Mechanisms regulating thrombus stabilization remain largely unknown. Here, we report that loss of any 1 of the Gas6 receptors (Gas6-Rs), i.e., Tyro3, Axl, or Mer, or delivery of a soluble extracellular domain of Axl that traps Gas6 protects mice against life-threatening thrombosis. Loss of a Gas6-R does not prevent initial platelet aggregation but impairs subsequent stabilization of platelet aggregates, at least in part by reducing "outsidein" signaling and platelet granule secretion. Gas6, through its receptors, activates PI3K and Akt and stimulates tyrosine phosphorylation of the β 3 integrin, thereby amplifying outside-in signaling via α IIb β 3 . Blocking the Gas6-R-α IIb β 3 integrin cross-talk might be a novel approach to the reduction of thrombosis.