The number and distribution of growing adhesions in the cell varies with substrate stiffness , (Extra cellular matrix) ECM chemistry (reviewed in ), topography (reviewed in ) and growth conditions (reviewed in ). These determine the force experienced by the adhesions and hence their molecular dynamics leading to mechanotransduction (see figure below).
Substrate rigidity has a great impact on the morphology of certain cell types such as fibroblasts, but has no effect on neutrophils . Rigidity sensing by focal adhesions (FA) can cause changes in FA dynamics and hence propagate signals that alter cell shape and cytoskeletal structures. For example, fibroblast polarization on rigid substrates is preceded by elongation, stabilization and uniform alignment of FAs along the elongation axis of the cell, whereas on compliant surfaces, small rounded and highly dynamic adhesions are formed and no proper polarization is observed .
Spacing of matrix ligands is also critical for establishment of stable adhesions. Cavalcanti-Adam et al. have shown that a spacing range of 50-70nm is required between ligand molecules, beyond which adhesions do not form and poor cell migration was observed . This distance seems to coincide with the length of talin, suggesting that talin is the principal mediator of integrin–actin linkage (reviewed in ). Independent of the global ligand density, it has been shown that nanoclustering of at least four integrin-binding sites in this threshold distance range is essential for effective integrin-mediated signaling . Ligand spacing has also been demonstrated to be important for integrin signaling in endothelial cells .
Cycling of integrins is a key aspect of mechanotransduction by focal adhesions. Integrins get activated, cluster, segregate and disassemble throughout the different stages of adhesions. This process facilitates both the transition of early complexes to mature and the ability to rapidly disassemble. All these steps are force-sensitive and dependent on other adhesion components (reviewed in ). Recently, it has been proven that integrins rapidly switch between active and inactive conformations within FAs . This influences their trafficking and hence FA dynamics .
Adhesion strengthening is required for nascent adhesions to survive shear forces exerted by actin retrograde flow. The friction between the adhesion components and hence the strengthening depends on the nature and organization of the receptor-ECM bonds (reviewed in ), and is known to generally correlate with increasing ligand density  and the ability of integrins to cluster laterally .
However, the adhesive bonds for β1 integrins are stronger than that of β3 integrins . In β1 integrins, the adhesive bond is further enhanced by applied forces that increase the catch bond behavior of the molecule , altogether with the engagement of a synergy site in fibronectin . Such catch bond behavior also fits a stochastic model that explains adhesion engagement state in relation to actin retrograde flow and corresponding traction stresses . In β3 integrins, the adhesion strength is increased by linkage to actin  and sustained by force-dependent recruitment of components such as talin, vinculin and paxillin , that reinforce the linkage  and initiate signaling cascades . Adhesion strength can modulate migratory behavior and shape in motile cells .
Flow of actin (i.e. actin retrograde flow)- The application of mechanical force that is generated by the actin system seems to be a prerequisite for the earliest stages of focal adhesion assembly and generation of traction forces . The coupling of the adhesion clutch to moving actin is mediated by the continuous association and dissociation of sliding molecular bonds referred to as ‘stick-slip behavior’ .Quantification of traction stresses exerted on the ECM by FAs using high-resolution traction force microscopy revealed that intermediate actin flow rates correspond to maximal traction . At the leading edge, where FAs form and flow is high, traction is minimal. However near the larger growing adhesion, the flow is slowed down  to ~8-10nm/s and is linearly proportional to traction stress  (reviewed in ).
Two stochastic models explain this biphasic relationship of friction at the moving interface between flowing actin and stationary adhesions . The competition between the elastic bonds at the interface and energy dissipation in the viscoelastic actin interior is proposed to modulate the stability of adhesions . The actin-receptor bonds are thought to reach a quasi-equilibrium state between bound and unbound forms. A catch bond model convincingly fits the experimental observations leading to clutch engagement and adhesion growth . These also clarify why traction on the substrate is maximum at intermediate flow .
Myosin contractile forces – FA growth, maturation and dynamics are highly dependent on myosin II contractility, that funnels the internal and applied forces to the adhesions as traction forces (, , reviewed in , see video below). MyosinIIA has been implicated in adhesion growth, remodeling of actin filaments into stress fibres and their maintenance between adhesions  while Myosin-IIB is responsible for the formation of FAs at the rear and hence serve to establish cell polarity . However it is to be noted that mature adhesions can withstand about six folds increase in traction stress without any effect on their size .
During cell spreading, myosin contraction gets activated at early stages at the end of initial rapid spreading phase, when the cell transforms to a flat morphology, due to the physical barrier posed by the plasma membrane . Thus increase in membrane tension seems to get converted into biochemical signal that activates actin assembly in the protruded area and causes local contraction that favors adhesion assembly . In an independent in vitro study, Yu et al. have shown that upon early actin polymerization from liganded integrin clusters, myosin II contraction of clusters activates protrusion with the actin providing outward forces and the myosin providing the local contraction and inward movement . This cyclic process known as the periodic lamellipodial contraction is mediated by myosin light chain kinase (MLCK) and the forces aid adhesion assembly and complete cell spreading . Hence, one cannot ignore the possibility that the two mechanisms could be interconnected.
The rate and extent of focal adhesion formation and maturation are regulated by factors such as synergistic integrin–syndecan signaling and alternating activation cycles of Rho (Rac1, Cdc42 and RhoA) GTPases.
Neither syndecan nor integrin is capable of independently supporting cell adhesion or spreading. Despite the cooperativity of integrin-syndecan pairs in various contexts (reviewed in ), recent studies have established synergistic signaling by integrin β1 and syndecan-4; they play cooperative yet distinct roles in cell spreading and maturation of adhesions as well as directional migration respectively . The receptors co-localize in early adhesion sites at the leading edge with ligand binding by both receptors (e.g fibronectin binds via cell binding domain [RGD to integrin and via HepII domain to syndecan) being necessary for downstream signaling . This is crucial as the cell polarity and migration is determined by differentially regulating signals at the leading and trailing edges.
Migration comprises of cycles of membrane protrusion, attachment, and cytoskeletal contraction, which causes forward movement. Immobilization of the integrin ligand is absolutely necessary to generate tension for adhesion formation and actin bundling while syndecan signaling primarily helps sense the environment for membrane protrusion. Localized signaling happens through alternating activation cycles of GTPases Rac1 (lamellipodium) and/or Cdc42 (filopodium) and RhoA, regulated by protein kinase pathways at the leading edge  (reviewed in ).
Stable adhesion induced by Rac1 may initially support tension, which allows RhoA-mediated contractility and pulling forces to impart stability on the bonds, thereby generating subsequent signals that are disseminated to the rest of the cell or axon ; these signals serve as feedback loops to restrict the direction of protrusion and reduce local activity of Rac1 , . It is to be noted that either receptor contributes to the regulation of both GTPases, however, Rac1 is primarily influenced by syndecan-4 . Coordination of such complex signaling is rendered by guidance signals.
Upon ligand binding, integrin signaling recruits GEFs for Rac1 and/or Cdc42 in lamellipodia and filopodia respectively . These initiate basal level Rac1 activity required for initial cell spreading , coordinated by Src–FAK signaling pathway  (reviewed in ). Further, liganded integrins retain active Rac1 at the leading edge by mediating a transient lipid redistribution which localizes GTP-Rac1 to the membrane .
During this relocation, the interaction of Rac1 with Rho-GDI is disrupted, allowing p21-activated kinase (PAK) coupling . In the lamellipodium, PAK promotes actin polymerization by inactivating cofilin and aids spreading by suppressing local myosin activity periodically . It also aids actin reorganization in the lamellae .
Upon engagement, syndecan-4 forms a ternary complex with PKCα and PIP2 ,  and its oligomerization leads to activation of PKCα . This is a critical step for further GTP-loading of Rac1, restricting Rac1 activity to the leading edge and environment sensing for directional migration . These facilitate downstream signaling for Rac1-mediated actin protrusion .
It is highly critical to suppress contractile signals during protrusion to aid forward movement and adhesion turnover. This is achieved again through convergent receptor signaling that retains RhoA inactive during Rac1 activity at the leading edge . On integrin β1 engagement, FAK phosphorylates and inactivates p190RhoGAP required for RhoA activation (essential role) . It then gets docked in the membrane fraction by binding p120RasGAP and FAK , and syndecan-4-mediated redistribution to membrane ruffles  (modulatory role). Further, p190RhoGAP phosphorylation triggers another wave of integrin-dependent lipid distribution that sustains RhoA suppression until the cell is fully spread.
The precise reciprocal feedback mechanism through which RhoA is activated in the lamellae during adhesion maturation/disassembly remained unclear until recently. However several lines of evidence exist for involvement of integrin– and syndecan-signaling pathways.
Besides reorganizing actin, Rac-activated PAK also phosphorylates the regulatory light chain (RLC) of myosin II, thus activating bundling of actin and the contractile mechanism. Myosin IIA/Myosin IIB-mediated actomysoin bundling generates stable adhesions, inhibit Rac-GEFs in the vicinity by modifying adhesion components that aid their recruitment and thus establish the cell rear . In a force-dependent manner, Rho-specific GEFs have been shown to get activated and recruited to focal adhesions through FAK and Fyn  (reviewed in ). Thus integrin-signaling pathway activates RhoA. Similarly, a syndecan-4 dependent pathway has been shown for the formation and maintenance of stress fibres, and focal adhesion maturation. Upon syndecan-4 clustering, GTP-loading of RhoA increases in a PKCα-dependent manner .
Activation of RhoA further enhances contractility and builds cellular tension through the Rho kinase, ROCK which sustains the myosin RLC phosphorylation (reviewed in ). Tension-dependent decrease of Rac activity has been demonstrated [ 16129884[/cite] and is believed to happen through stimulation of a Rac-GAP, ARHGAP22 by the Rho kinase, ROCK . Similarly CdGAP, has been shown to inhibit lamellipodial protrusion and is suggested to do so via its actions on both Rac and Cdc42 .