property of cells, at least in the context of the lung. In support to this interpretation, a previous study reported examples of force versus relative length curves measured on a single fibroblast sandwiched between two microplates for several minutes and subjected to low frequency cycles of either compression or extension [11]. Although the effect of force directionality was not analyzed in that study, qualitative reexamination of their cyclic measurements suggests that the fibroblast exhibited smaller apparent stiffness in extension than in compression, in agreement with our observations. Furthermore, it is worth noting that the asymmetric resistance to force directionality observed in lung cells is not physiologically surprising, since asymmetric elasticity is a hallmark of a variety of non-homogenous inert materials as well as biological materials such as bones, ligaments and soft tissues [34,35]. Further comparisons between EC and ET data revealed that there was a consistent positive correlation between them, which was linear for nearly all experimental conditions as indicated by the constant asymmetric ratio EC/ET for each cell type. In agreement with these findings, previous studies reported a positive linear correlation between traction forces and cell stiffness in human airway smooth muscle cells [2,36] and in human lung endothelial cells [37]. However, we found that this linearity is not universal, since EC/ET was dramatically reduced in all cell types specifically upon inhibition of actin polymerization (Figure 4 and Figure S5). Role of actin in lung cell asymmetric resistance to compression and extension A straightforward mechanism underlying cell asymmetric resistance to compression and extension is that, as observed in tissues, different structures are involved in cell mechanoresponse to force directionality. This interpretation is further supported by the observation that EC was ,10-fold smaller upon treatment with the actin polymerization inhibitor latA compared to vehicle, whereas ET was only ,4-fold smaller in both CCD19-Lu and A549 cells (Figure 4 and Figure S5). These data confirm previous observations that an intact actin CSK is required to resist both compression [29,37,38] as well as extension applied at ECM adhesion sites [2,5,17]. In addition, these findings provide new insights in the role of actin in cell asymmetric mechanoresponse. First, they highlight a prominent role of actin polymerization in response to compressive loading. Second, they reveal that different processes underlie the assembly of the actin networks involved in resisting compression and extension. These latter observations were further expanded by our finding that both ET and F* reached a plateau after ,30 s of tip-cell contact, whereas EC continued rising after 5 min of tip-cell compressive contact, in agreement with previous studies showing actin recruitment in response to an exogenous force [31]. Finally, these results indicate that the raise in EC after 5 min of tip-cell contact did not contribute to ET, thereby suggesting that the actin network assembled to resist compression is, at least in part, distinct from the actin network assembled to resist extension and provide adhesion strength. Integrin-specific mechanoresponses of lung cells to compression and extension Local attachment to the ECM in cultured cells begins with a dot-like initial adhesion that progressively matures into ,1 mm2 focal complex rich in integrins, actin, vinculin and few other FA proteins in a timescale of few seconds, and eventually forms larger and more tightly bound FAs [7,27,28,39,40]. Likewise, recent simulation studies of integrin dynamics on a rigid ECM substrate Lung Cell Mechanoresponse to Force Directionality reported that 35 s was enough time to induce integrin bond formation and clustering close to maximum values [18]. Accordingly, we held an RGD-coated FE-AFM tip of ,1 mm2 of cross-section area in contact with the cell surface for 30 s prior to examining its mechanoresponse to compression and extension. In these conditions, we expected cell mechanoresponses to be largely orchestrated by integrins in the context of FA precursors. To check the integrin-specificity of the RGD-induced mechan- oresponses further, experiments were repeated with either bare tips or tips coated with non-integrin specific molecules (RGE/ BSA). Using this approach, we identified two mechanoresponses with RGD-coated tips only regardless the cell type: (i) the largest resistance to compression and extension (Figure 2), and (ii) the highest cell-tip adhesion (Figure 3). Both mechanoresponses were inhibited in the presence of EGTA (Figure 4), which is consistent with the Ca2+ dependency of integrin adhesion [41]. On the other hand, the efficiency of integrins in probing ECM rigidity has been largely associated with their ability to physically link ECM to the actin CSK through adaptor proteins that are typically accumu- lated at FA precursors. We provided three pieces of evidence supporting this interpretation. First, integrin-specific mechanor- esponses required an intact actin CSK, as revealed by latA experiments (Figure 4). Second, F* measured with RGD-coated tips was comparable to previous data including detachment forces reported using RGD coated microbeads on fibroblasts [12], and traction forces developed at focal complexes, whereas they were smaller than traction forces measured in FAs [27]. Third, a positive correlation was found between ET and F* (Figure 3C), consistent with a positive correlation between traction force and vinculin accumulation at adhesion sites reported elsewhere [27]. Conversely, compromising the contractility generated within the actin CSK increased vinculin dissociation rate in FAs [42]. These latter positive correlations data suggest that the largest F* at integrin adhesion sites may arise from direct stiffening of the integrin linkage to the actin CSK through vinculin and/or other integrin-adaptor proteins present at FA precursors. However, this stiffening could also be supported by an increase in the number of integrins linked to the CSK [10,27]. In addition, high F* could arise from the activity of mechanosensitive ion channels physically connected to the CSK [4,17,31]. Whatever the detailed mecha- nism might be, our data reveal that FA precursors are sufficient to orchestrate rapid (,30 s) mechanoresponses that elicit strength- ening or reinforcement of lung cell adhesions. Mechanochemical signals underlying integrin-specific mechanoresponses In addition to cell adhesion to RGD, integrins and actin polymerization, we identified other mechanochemical cues required to elicit integrin-specific mechanoresponses in lung cells: (i) sustained compressive loading for ,30 s, and (ii) tyrosine phosphatases and Ca2+ signaling. Conversely, we demonstrated that compression without RGD adhesion was not sufficient to induce these mechanoresponses. Likewise, exogenous cytokines and other soluble factors present in serum were not required. In this context, stiffening in a timescale of minutes is well documented in a variety of cell types in response to either shear or tensile stresses applied by ECM coated microneedles or microbeads [17,31,33,43,44]. Consistently, stiffening was observed when extension was applied to lung cells with RGD-coated tips only (Figure 2C, E). This stiffening has been associated with integrin activation through extension enhanced outside-in signaling [6,44]. However, to our knowledge, this is the first study showing directly RGD-induced stiffening in compression, therefore suggesting that compression can also modulate integrin-specific mechanore- PLoS ONE | www.plosone.org 9 February 2012 | Volume 7 | Issue 2 | e32261