dependent features were observed in A549 cells (Figure S5), confirming that the role of actin CSK is cell type-independent. In contrast, nocodazole dramatically enhanced the RGD-specific stiffening and adhesion strengthening in A549 cells, indicating that the role of microtubules is cell type dependent. Moreover, because a large body of evidence indicates that extracellular forces trigger mechanoresponses through molecular mechanisms that are often mediated by alterations in tyrosine phosphorylation and/or Ca2+ signaling [2,30,31], we probed RGD-induced mechanoresponses in CCD-19Lu cells upon treatment with either the broad spectrum tyrosine phosphatase inhibitor PAO, or with EGTA or BAPTA/ AM to chelate the extracellular and intracellular Ca2+, respec- tively. We found that all these three compounds inhibited both the integrin-specific stiffening in compression and in extension (Figure 4D), and the highest F* probed with RGD-coated tips (Figure 4F). Conversely, none of the compounds compromised the EC/ET ratio (Figure 4E). Among these inhibitors, EGTA elicited the lowest E and F* values, underscoring the role of extracellular Ca2+ in integrin-specific mechanoresponses. Discussion Understanding how lung parenchymal and stromal cells respond to bidirectional forces is important for both normal physiology and diseased conditions, since many prevalent pathologies including asthma, fibrosis and acute lung injury are associated with a loss of the normal balance of forces within the lung [1,3]. Because force transmission occurs at discrete adhesion sites rather than continuously throughout the cell surface, we studied lung cell mechanoresponse to exogenous forces applied locally at the cell surface using FE-AFM tips in conditions that enabled formation of FA precursors [7]. Suitability of FE-AFM cylindrical tips to probe cell-ECM mechanical interactions The identification of cell mechanoresponses to force direction- ality relied on the nanofabrication of cylindrical FE-AFM tips and the validation of the experimental protocol. A cylindrical geometry was chosen instead of a parallelepiped because it avoids applying unwanted high mechanical stresses to the cell surface due to sharp edges. The protocol validation was accomplished by reporting several hallmarks of linear elastic materials in PAA+ gels, as expected based on previous findings [19]. This validation provided also a proof of principle that FE-AFM tips can be used to quantify E in extension, which may be a useful tool in future studies in material sciences and cell biophysics. FE-AFM tips also enabled quantifying the tip-sample adhesion strength. Because the cross-section of the tips was much larger than either the width of PAA+ filaments or the size of ECM receptors [12,18], it is conceivable that tip-sample adhesion was mediated through multiple parallel bonds in both cells and PAA+ samples. Nonetheless, the shape of the unloading curves beyond zc were dramatically different in gel and cell samples (Figure 1F, G), revealing the presence of distinct physicochemical processes at the tip-sample interphase. Because PAA+ gels contain a single monomer type, tip-PAA+ gel bonds are expected to exhibit similar affinities and rupture forces. Accordingly, once the extensional force is slightly higher than the rupture force of a single bond, all bonds should detach very rapidly. Consistently, we observed a sudden decay in force during unloading in PAA+ gels (Figure 1F), similar to single-bond rupture experiments [32]. In contrast, the cell surface is highly heterogeneous and dynamic, and contains a non-homogeneous receptor distribution which typically exhibit distinct affinities and rupture forces [12,16,17]. Therefore, it is Lung Cell Mechanoresponse to Force Directionality expected that cell-tip bonds will detach at different times, pulling forces and locations. Accordingly, we observed a progressive and nonlinear tip detachment from the cell surface (Figure 1G). These findings revealed that sudden or progressive tip-sample detach- ments are indicative of tip-sample adhesion mediated by either multiple homogeneous or heterogeneous parallel bonds, respec- tively, and highlighted the suitability of FE-AFM tips to study quantitatively different mechanical aspects of local cell-ECM interactions. Lung cells respond asymmetrically to local compression and extension To our knowledge, this is the first quantitative study of local cell mechanoresponse to force directionality. Although ET probed with RGD-coated tips was higher than with bare or RGE coating, all ET data were smaller than EC in all cell types, in marked contrast with the equal EC and ET reported in PAA+ gels. Accordingly, EC/ ET.1 in all conditions, and largely independent of the specific coating of the tip, whereas it was cell type dependent (Figure 2D, F). These findings indicate that integrin binding to RGD is not necessary to induce the asymmetric mechanoresponse to com- pression and extension. We also observed that the asymmetric resistance to force directionality remained and even increased after holding an RGD-coated tip in contact with the cell for 5 minutes using a force control protocol, which is a time window that enables forming mature FAs in culture [6,27,28]. The mechanical interpretation of E data obtained with the force control protocol is less straightforward than with the protocol used in all other experiments (Figure 1B,E), since measurements performed with the former protocol may be subjected to unwanted variability sources. These sources include force variability due to the effects of mechanical drift of the AFM setup on zc that are not corrected with the force control protocol, as well as the uncontrolled stress relaxations induced specifically on each cell by the strain steps applied when adjusting the piezotranslator position during the force control. Although these limitations could introduce variabil- ity in the calculation of E, several pieces of evidence support that the conclusions drawn from E data measured with the force control protocol are not compromised by these variability sources. First, these sources are likely to affect to a similar extend EC and ET, thereby cancelling each other out when computing the ratio EC/ET. Second, the slopes in the extension region of the F versus t curve recorded with the force control protocol (Figure S4), which are proportional to E according to the F(d) equation, were consistently lower than the slopes observed during compression for all cells examined, thereby indicating qualitatively that EC/ET.1. Third, the observed three-fold increase in EC after 5 min compared to EC after 30 s of tip-cell contact falls within the same range of cell stiffening due to mechanical stimuli reported elsewhere. Thus, a five-fold increase in EC measured with AFM was observed in fibroblasts upon sustained mechanical stimulation by pulling during 30 min collagen-coated magnetic beads attached to the cell surface [31]. Likewise, ,three-fold stiffening was measured in airway smooth muscle cells upon mechanical stimulation by cyclically twisting during 60 min magnetic beads bound to the cell surface [33]. Finally, unlike EC, we observed little variations in ET after 5 min of tip-cell adhesion compared to 30 s, in agreement with the weak (,1–1.5 fold) increase in traction force observed in airway smooth muscle cells subjected to either intermittent or transient stretch for 5 min [2]. Our force control measurements confirmed that the elastic asymmetry observed after 30 s of tip-cell contact was not due to insufficient contact time, and strongly suggests that asymmetric resistance to compression and extension may be an intrinsic PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32261