In this study, we developed image analysis tools for the automated and quantitative analysis of cell spreading. For the first time, we have been able to make a detailed analysis of how activation of Rap1 modulates the kinetics and morphology of cells spreading over a fibronectin matrix. We observed that, under basal conditions, A549-Epac1 cells exhibited an anisotropic mode of spreading, where both an increase in cell spread area and a steady state of spread area was accompanied by extensive remodelling of the overall shape of the spread area of cells, mediated through continuous protrusion and retraction of the cell periphery. This anisotropic mode of spreading gave rise to the angular morphology of A549-Epac1 cells observed in fixed samples 
. Addition of 007, to activate endogenous Rap proteins specifically via the exchange factor Epac1, promoted a faster rate of cell spreading, induced cells to spread isotropically and reduced the remodelling of the spread area which occurred under basal conditions.
A factor that controls whether cells spread isotropically or anisotropically is the availability of ECM ligand sites to which cells can attach and make protrusions 
. Lower ECM concentrations promote anisotropic spreading, as cells have to “search” for ligand-dense sites which activate Src sufficiently to induce the formation of stable attachments 
. Our results show that Rap1 activation can induce isotropic spreading on an ECM substratum that otherwise drives anisotropic spreading. This indicates that Rap1 can promote and stabilise functional adhesion complexes at ligand sites that integrins may be able to attach to, but may not be sufficient to activate Src-driven protrusion. The ability of Rap1 to induce adhesion and spreading in the presence of PP2, even though outside-in signalling and the basal adhesion of A549-Epac1 cells were completely blocked, further supports this conclusion. Through the ability to induce adhesion, spreading and cell morphology changes by mechanisms that do not rely on the canonical ECM-derived signals, Rap1 may influence many stages of tissue development, including stem cell differentiation which is, in part, determined by the constraints induced by the extracellular matrix microenvironment 
One explanation for the rescue of spreading in the presence of PP2 is that, through the GEF, C3G, Rap1 functions downstream of Src, and, by stimulating Rap1, the next step in the Src pathway is being re-activated 
. However, our data show that the canonical phosphorylation sites in the FAK-Src-Paxillin module that were blocked by the inhibitors, PP2 and PF573228, were not rescued by Epac1-induced Rap1 activation. These data demonstrate that Rap1 does not regulate the FAK-Src signalling pathway and that the Src pathway is not truly reconstituted by Rap1 activation. Furthermore, as activation of Rap1 via C3G is reported to be mediated, in part, by actomyosin-induced force 
, our findings that Epac1-induced Rap1 activation can promote force-dependent FAs indicate that activating Rap1 is not simply reconstituting the Src pathway one-step down. Rather, our results place Rap1 regulation of adhesion and spreading in a parallel, or bypass, pathway from the FAK-Src-signalling module.
We have shown that Rap1 activation creates an ECM-integrin-actomyosin link, and focal adhesions that were responsive to changes in actomyosin contractility, in order to promote adhesion and spreading. It is, therefore, likely that the mechanisms by which Rap1 initiates spreading and FA formation in the presence of PP2 are analogous to the processes by which the FAK-Src module controls adhesion. It is well characterised that the FAK-Src signalling module induces a transient reduction of RhoA activity to promote cell spreading, followed by an induction of actomyosin-induced tension which induces the maturation of FAs 
. Therefore, as Rap1-initiated spreading in the presence of PP2, our data indicate that Rap1 itself could lower actomyosin-induced tension. Furthermore, as 007-induced focal adhesions were modulated by actomyosin contractility, it suggests that Rap may mediate a temporal and localised regulation of RhoA activity, similar to that which is instigated during Src-mediated spreading 
. Although Rap1 is implicated in modulating actomyosin tension, it is currently unclear which Rap effectors would regulate RhoA in our cell system, as we previously found that siRNA against Arap3, Krit1, or RA-RhoGAP/ARHGAP20 did not alter Rap-induced cell spreading of A549-Epac1 cells 
Our data implicate Rap1 activity as one of the mediators of force-dependent strengthening and maturation of FAs, a process that previously has largely been attributed to the FAK-Src-Paxillin cascade 
. Indeed, we found that the relative level of vinculin in Rap1-induced FAs was not altered upon the inhibition of the FAK-Src module although paxillin phosphorylation was strongly inhibited. In laser tweezer experiments using fibronectin-coated beads, mechanical force in the absence of Src kinase activity has been demonstrated to recruit vinculin to bead-cell focal complexes 
. Thus, in Rap1 activated and Src-inhibited cells, we created a similar situation at FAs, and demonstrated that Rap1 activation permitted the recruitment of vinculin to adhesions. As adhesions induced by Rap1 are mechanosensitive, the mechanism by which Rap1 activation recruits vinculin to adhesions may be primarily via stretch-dependent unfolding of Talin 
. This requires further investigation, however, our model system that combines inhibition of Src with active Rap1, will be a valuable tool for the characterisation of FAK-Src independent mechano-regulation of integrin adhesions.
Together, our data demonstrate that activation of Rap1 induces a functional ECM-integrin-actomyosin link that promotes adhesion and spreading, but which does not depend on the activity of the FAK-Src signalling module. Thus, Rap1 induced adhesion and spreading in A549-Epac1 cells shows similarities to Src-induced processes, but we propose that they are driven by parallel pathways. By regulating the ECM-integrin-actin link by distinct molecular mechanisms from the FAK-Src-paxillin cascade, a localised activation of Rap1 in cells within tissues may act to reinforce the contacts between cells and their extracellular environment. Indeed, the regulation of Rap1 activity has recently been implicated as being critical for preserving the attachment of neural stem cells to their niche 
. Therefore, activation of Rap1 may play a vital and significant role in modulating the physiological interaction between cells and their extracellular matrix environment to contribute to the structural maintenance and integrity of tissues.