Cell culture and reagents

The mouse mammary epithelial cell line NMuMG (ATCC, Manassas, VA), was maintained in Dulbecco's modified Eagles medium with 4.5 g/l glucose (Lonza, Köln, Germany), 10% fetal calf serum (PAA Laboratories GmbH, Cölbe, Germany) and 4 mM L-glutamine (Biochrom, Berlin, Germany). Additionally, 10 µg/ml insulin (Sigma-Aldrich, Munich, Germany) was added as stated in the cultivation protocol by ATCC (American Type Culture Collection). During all measurements, the regular culture medium supplemented with 15 mM Hepes, Amphotericin and Penicillin/Streptomycin (all PAA Laboratories GmbH) was used. For all of the measurements mentioned below NMuMG cells were seeded to 70% confluency on Petri dishes without any substrate coating providing sufficient area for morphological changes during EMT. Fibroblasts obtained from 2-d-old rats were kindly provided by Marco Tarantola, Katharina Schneider and Marion Kunze (Max Planck Institute for Dynamics and Self-Organization, Göttingen). During the measurement fibroblasts were kept in Dulbecco's modified Eagles medium (Invitrogen, Karlsruhe, Germany) supplemented with 1% L-glutamine (Invitrogen), 10% FCS (Invitrogen) and 1% penicillin/streptomycin (Life Technologies). All preparations were done in regard to the guidelines of the German Animal Protection Act (TierSchG) and were reported to the responsible animal welfare representative. All animals were killed before preparations by decapitation, which conforms with the ‘Guidelines for Euthanasia of Rodent Fetuses and Neonates’ of the NIH, and were reported referred to §4 para. 3 TierSchG. Since animal testing was not conducted the protocol did not require approval by the Committee on the Ethics of Animal Experiments. Necessary certificates of exemption for technical staff members were obtained from the Lower Saxony State Office for Consumer Protection and Food Safety (Laves) with respect to §9 para. 1 sentence 4 (TierSchG). Tissue was only used to prepare cultures of cardiomyocytes and fibroblasts. TGF-β1 was obtained from Life Technologies GmbH (Darmstadt, Germany) and used in a final concentration of 10 ng/mL diluted in culture medium including Hepes, Amphotericin and Penicillin/Streptomycin. EDTA was received from Sigma-Aldrich and used in a final concentration of 2 mM diluted in culture medium with Hepes, Amphotericin and Penicillin/Streptomycin.

Cytochalasin D was obtained from Life Technologies and used in a final concentration of 10 µg/mL diluted in culture medium including Hepes, Amphotericin and Penicillin/Streptomycin. For single molecule experiments control measurements an E-cadherin antibody (Life Technologies GmbH) diluted in serum-free medium to a final concentration of 200 µg/mL was employed. Prior to measuring, cells were incubated with the antibody for 1 h. Thereafter, the serum-free medium was removed and fresh culture medium supplemented with 15 mM Hepes was added. The inhibitors rapamycin, cycloheximide, blebbistation and Y-27632 were purchased from Sigma-Aldrich and used in concentrations as indicated. Generally, application of rapamycin leads to a decline in both cell motility and de novo protein synthesis via inhibition of the mTOR signaling pathway in TGF-β1-stimulated NMuMG cells [23]. Cycloheximide is a well-known inhibitor for protein synthesis by blocking translational elongation [24]. Blebbistatin prevents rebinding of the myosin head to the actin cytoskeleton, thereby decreasing acto-myosin contractility within treated cells [25]. Finally, Y-27632 is a Rho kinase inhibitor interfering with the formation of actin stress fibers [26]. The impact of these compounds on cellular dynamics and presence of cell-cell contacts during EMT of NMuMG cells has recently been investigated by us in a separate study [27].

Fluorescence microscopy

Immunostaining combined with fluorescence microscopy was carried out using an upright microscope (Olympus BX51, 20× or 40× water immersion). Cells were grown on Ibidi™ petri dishes (Ibidi, Martinsried, Germany), treated with 10 ng/ml of TGF-β1 for either 48 h or 10 d and compared to untreated reference samples. Fixation of the cells was carried out using a 4% paraformaldehyde solution (Sigma-Aldrich), and an incubation time of 15 min. This was followed by washing the cells twice with 1× PBS (PAA) without Ca²⁺ and Mg²⁺ denoted PBS⁻ throughout the manuscript and permeabelizing them with a 1× PBS⁻ solution containing 0.4% Triton-X (Sigma-Aldrich) and 5% BSA (Sigma-Aldrich) to block unspecific binding sites. For E-cadherin staining cells were incubated for 1 h with a monoclonal Alexa488-conjugated IgG2a antibody (BD Bioscience) diluted to a final concentration of 5 µg/mL in 1× PBS⁻ supplemented with 1% BSA and 0.3% Triton-X. For DNA staining 4′-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) was used and an incubation time of 15 min was chosen.

Indentation experiments with conical indenter

Force indentation curves on cell layers were recorded using a Nanowizard II setup (JPK Instruments, Berlin, Germany) placed on an inverted Olympus IX81 microscope (Hamburg, Germany). Silicon nitride cantilevers (MLCT, Bruker Daltonics, Camarillo, USA) with a nominal spring constant of 0.01 N/m were employed. A petri dish heater (JPK Instruments) set to 37°C as well as Hepes-buffered culture medium supplemented with Amphotericin and Penicillin/Streptomycin were used to assure physiological conditions throughout all experiments.

During indentation experiments, the indentation force of either 500 or 1000 pN, and the velocity of cantilever approach and retraction (1 µm/s) were kept constant. Analysis of the recorded force-distance curves was conducted using a tension based model considering the properties of adhering cells in contrast to non-adhering cells according to the following equations:(1)(2)The complete theory is described precisely in literature [28], [29], [30] and a detailed derivation given in the supporting information (Supplementary Text S1). In brief, the force T acting against cantilever indentation during deformation of the cellular structures strongly depends on indentation depth. Whereas apical tension T₀ arising from membrane tension and cortical tension dominates the elastic response at lower values, stretching of the cellular membrane expressed in the area compressibility modulus K_A occurs at higher indentation depth. The influence of both parameters on the shape of the recorded force-distance curves is visualized in Fig. S1 and Fig. S2. Certainly stretching of the membrane during indentation strongly depends on the amount of free accessible membrane material. Therefore, ΔA and A₀ describe the area change due to indentation of the conical indenter and the apical side of the spread cell excluding folds or invaginations, respectively. Due to the fact that the excess area of cellular membranes (A_Ex), bearing mainly membrane folds and invaginations, is unknown, only an apparent area compressibility modulus K_A^′ can be determined (equation 2).

Within this study, we focused on pre-tension T₀ as this parameter is directly accessible from the recorded force curves even without knowledge of the buried membrane area. Bending and shearing of the membrane are neglected. According to our model, cells are treated as spherical caps with characteristic radii and contact angles for each cell type before and after the transition. This information was obtained from AFM images (Fig. 1 D/E). The images were recorded using a closed loop feedback circuit and the piezo movement was corrected with the help of capacitive sensors implemented into the scanner of the AFM (JPK instruments). The mean relative height and mean radius were calculated by analyzing at least 3 cells per category. For NMuMG cells treated with either EDTA or cytochalasin D and NMuMG cells before the transition, a mean radius of 10.1 µm and a mean relative height of 2.2 µm for the spherical cap are computed. After 24 h of TGF-β1 incubation we found a radius of 10.3 µm and a relative height of 1.8 µm, whereas upon reaching the final mesenchymal-like state a radius of 10.3 µm and a relative height of 1.4 µm for the cells were used as input parameters for the indentation model. Similar values were used for mesenchymal-like NMuMG cells incubated with rapamycin, cycloheximide, blebbistatin, Y-27632 or cytochalasin D. Finally, for single untreated NMuMG cells parameters of 12.3 µm and 2.0 µm are determined. Adapting the input parameters in dependence of the cellular state during EMT is necessary as the morphological changes influence the elastic response upon cantilever indentation as inferred from simulation studies (Fig. S3 and S4). Although we performed indentation experiments with pyramidal shaped MLCT cantilevers, we used a conical shape as input parameter for tension model. Due to the asymmetry of the MLCT cantilevers a calculation of the cellular deformation would not be tractable analytically. However, considering a conformal contact our model is valid as the contact area of both shapes differs only by a factor of 1.3 comparing a conical indenter with a pyramidal one. For calculation an mean opening angle of 17.5 was assumed for the conical/pyramidal tip.

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Figure 1. Structural alterations during epithelial-to-mesenchymal transition. (A–C) AFM deflection images of fixed untreated and fixed TGF-β1 treated NMuMG cells (24 h or 48 h incubation time, B and C, respectively).

Fixation was carried out using glutardialdehyde (10 min incubation time) leading to cross-linking of proteins. Subsequent loss of cell-cell junctions after 24 h of incubation is marked by white arrows. Zoom-ins of the corresponding AFM height images show a loss of cellular protrusions during EMT. White boxes in the deflection images mark the chosen regions for the zooms. Size of the height images is 9.3×9.3 µm². (D) AFM height images of living untreated and living treated (48 h incubation time) NMuMG cells. (E) The obtained cross-sections from these images provide information about the contact angle, the radius and the height of the cells before (black) and after the transition (grey). All AFM images were recorded in closed-loop contact mode using MLCT cantilevers and a scan rate of 0.2 Hz. Scale bars: 20 µm.

https://doi.org/10.1371/journal.pone.0080068.g001

Indentation experiments with colloidal probe

A similar setup was used here as for the indentation experiments with a conical indenter (vide supra). Spherical glass beads (Duke borosilicate glass 9015, Duke Scientific Corporation, Palo Alto, CA, USA) with a diameter of 15 µm were glued onto tip-less silicon nitride cantilevers for indentation experiments (tipless MLCT-O10, Bruker Daltonics) using an epoxy resin cured at a temperature of about 90°C (Epikote 1004, Brenntag GmbH, Mühlheim, Germany). The bead was placed onto the cantilever with the help of a nanomanipulator (MM3A-LS, Kleindiek Nanotechnik GmbH, Reutlingen, Germany). Cantilever retraction and approach were kept constant at 1 µm/s. Cells were indented in the center.

In order to extract apical tension T₀ of NMuMG cells in the absence and presence of TGF-β1 from colloidal probe force measurements, we employed a theoretical approach devised by Lomakina and coworkers [31]. Essentially, the approach is based on determining the pressure change P of a bead (radius R_b), the colloidal probe, in conformal contact with the cell of radius R_c using Laplace's law:(3)providing the force F acting on the cell. The contact radius R_con can be used to compute the indentation depth z:(4)With equations (3) and (4) the force-indentation curve can be modeled to estimate tension values from colloidal probe measurements.

Tether pulling experiments

For tether pulling experiments the same setup as for indentation experiments with pyramidal indenters was used. Tips of MLCT cantilevers were first cleaned with argon plasma for 1 min (Plasma Cleaner, Harrick, NY, USA) followed by rinsing with isopropanol (VWR International GmbH, Darmstadt, Germany). Finally, the cantilevers were incubated with concanavalin A (Sigma-Aldrich) diluted in 1× PBS⁻ to a final concentration of 2.5 mg/mL for 30 min at room temperature. Concanavalin A is known for strong binding to carbohydrates on the cellular surface leading to tether formation upon cantilever retraction [32]. Measurements were performed in Hepes-buffered cell medium with Amphotericin and Penicillin/Streptomycin. We chose an approach/retraction velocity of 1 µm/s, a setpoint of 500 pN and a contact time of 1 s between the tip and the cells. Using the following equation, membrane tension from tether pulling experiments before and after the transition is computed [33].(5)Besides static membrane tension T_t membrane viscosity η has to be taken into consideration. v denotes the velocity of tether pulling. The bending modulus of the cellular membrane was assumed to be κ = 2.7e⁻¹⁹ as obtained by Dai and Sheetz (1999) for renal epithelial cells [34]. C is a correction factor with a value of 1.6 [35].

Single-cell force spectroscopy (SCFS)

Measurements were accomplished using a Cellhesion200 setup (JPK Instruments) combined with an Olympus IX81 microscope. Again we assured physiological conditions using a Petri dish heater (JPK Instruments) set to 37°C. Tipless cantilevers (Arrow TL-2, Bruker Daltonics) with a nominal spring constant of 0.03 N/m were employed for the experiment and, prior cell picking, they were washed twice in isopropanol and ultra-pure water. Additionally, cantilevers were sterilized using argon plasma for 1 min. The cleaned cantilevers were then incubated for 15 min in a 30 mM CellTak solution (BD Biosciences) diluted in 1× PBS⁻ for 15 min at room temperature. NMuMG cells were seeded onto Petri dishes in Hepes-buffered cell culture medium at least 48 h before starting the measurement ensuring formation of proper cell-substrate and cell-cell adhesion sites. In case of the mesenchymal-like cells, incubation times of at least 8 d in presence of the cytokine TGFβ-1 were applied to guarantee a complete EMT and down-regulation of E-cadherin. Thereafter, freshly suspended cells, either epithelial ones or mesenchymal-like ones, were added to the Petri dish and a single cell was captured using the Celltak-coated cantilever. Using the cell as a probe, it is brought into contact with either a spread epithelial or mesenchymal-like cell. A contact force of 500 pN and an approach/retraction velocity of 10 µm/s were employed for all measurements. Furthermore, we chose a contact time of 1 s between the cells in either state since at longer times of up to 10 s the variances of detachment forces increase considerably, which in turn reduces significance of the obtained results (data not shown). All adhesion measurements were again conducted in cell medium supplemented with Hepes, Amphotericin and Penicillin/Streptomycin.

Single-molecule force spectroscopy (SMFS)

SMFS was carried out with a Nanowizard II setup (JPK Instruments) mounted on an inverted Olympus IX81 microscope. We used gold-coated cantilevers (Bio-lever, BL-RC150VB, Olympus, Japan) with a nominal spring constant of 0.06 N/m. First, cantilevers were cleaned in argon plasma for 30 s and subsequently functionalized by incubation for 3 h in a 1 mM benzylguaninthiol : matrix-thiol (MeO) (1∶100) solution dissolved in isopropanol. This was followed by rinsing the cantilevers with pure solvent and finally with buffer (10 mM HEPES, 100 mM NaCl, 1 mM EDTA). Thereafter, cantilevers were incubated with protein solution (2 µM) to couple Ecad15 (E-cadherin including all 5 ectodomains) to the tip. This coupling is induced via a Snap-Tag C-terminally fused to Ecad15. The coupling procedure is described in detail by Wedlich and coworkers [36]. Force-distance measurements were performed using a pulling speed of 1 µm/s and a contact force of 100 pN. The contact time between the functionalized cantilever and either the epithelial or mesenchymal-like cell was kept constant (1 s). The matrix-thiol, Methoxy-capped tri(ethyleneglycol)undecanthiol, was synthesized at the Fraunhofer Institute for Manufacturing Technology and Applied Materials Research (IFAM). Benzylguanine-terminated disulfide (BGT) was obtained from New England Biolabs (Ipswich, USA). The E-cadherin construct is expressed in stably transfected HEK293 cells and purified afterwards.

AFM Imaging and roughness analysis

AFM imaging of NMuMG cells was accomplished using a Nanowizard II setup. Due to lateral mobility of the cellular membrane, cells were fixed using a 2.5% glutardialdehyd solution (Fisher Scientific GmbH, Nidderau, Germany) diluted in 1× PBS⁻ (15 min incubation time) prior to scanning. MLCT cantilevers with a nominal spring constant of 0.01 N/m were employed. A scan rate of 0.2 Hz was used for contact-mode imaging.

Roughness of the cells within the different stages during EMT was analyzed as follows. In a first step, a user-defined area of 9.7×9.7 µm² on any cellular body was chosen for analysis. Subsequently, a general trend was subtracted from this section to correct for differences in cellular height. Therefore a 2-D sliding window of 11×11 points was adopted. In order to avoid boundary problems when applying the moving average box, the images were mirrored in both x and y direction. The algorithm computes variances for each row perpendicular to the scan direction of the AFM cantilever. Finally, the mean variances for each category were computed. In case of NMuMG cells within the epithelial state as well as for NMuMG cells from the mesenchymal-like state three different areas were chosen for analysis, whereas for the transitional state one area was selected (Fig. S5).

AFM rheology measurements and data acquisition

A MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA, USA) placed on an inverted IX51 Olympus microscope was used for rheological measurements. The cantilever (MLCT, Bruker Daltonics) was excited to a sinusoidal oscillation (5 Hz to 200 Hz) at a defined indentation depth δ₀ within the samples. According to a model introduced by Alcaraz and coworkers [37], we were able to obtain quantitative information about the elastic and viscous properties of our samples from the recorded datasets. Although Hertzian contact mechanics forms the base, the model has to be extended by an additional frequency-independent tension term as already described [38]. Considering a correction for the hydrodynamic drag [39] and a linearization for small amplitude oscillations [21], the complex shear modulus G*, as well as the storage G′ and loss modulus G″ were obtained. According to the Hertz model only force-distance curves with low indentation depths of 50–550 nm were analyzed in order to avoid influence of the underlying substrate or stiff intracellular structures like the nucleus and to stay in the limits of the model [40]. Similarly to the indentation and adhesion measurements cells were investigated under physiological conditions using Hepes-buffered medium and a Petri dish heater (JPK Instruments) adjusted to 37°C.

Cantilever calibration

Spring constants of all cantilevers were determined after cleaning (Ar plasma, 1 min) but before the individual incubation steps using the thermal noise method [41]. Although cells are mostly grown to confluent monolayers, we always found uncovered spots on the Petri dishes to calibrate the cantilever. All of the force measurements mentioned above are then performed on cells which are several mm away from the free area.

Statistics

To evaluate the statistics of the obtained data, a non-parametric statistical hypothesis test, Wilcoxon rank-sum test, was employed as indicated accounting for non-normal data. The alpha level was either set to 0.1 or 0.01.

Structural alterations of NMuMG cells during EMT

Figure 1 shows AFM deflection, as well as relative height images of untreated (Fig. 1A) and treated NMuMG cells incubated for either 24 h (Fig. 1B) or 48 h (Fig. 1C) with the cytokine TGF-β1. During EMT, we observe a dramatic change of the cellular shape including an increased formation of stress fibers and a loss of cell-cell contacts. Furthermore, a decrease in the number of membrane protrusions, as well as a decline in the cellular height of the cellular cap during EMT (Fig. 1D,E) are clearly visible in the recorded AFM images underscoring the considerable structural alterations during the transformation. The surface structure of epithelial and mesenchymal-like cells shows huge differences in corrugation supporting the idea of severe changes in membrane area and structure during transition. Whereas in the epithelial state membrane folds and protrusions are numerous, the surface during EMT adopts a much smoother topography already after 24 h of incubation with TGF-β1. To quantify these observations, we performed a roughness analysis of the AFM height images as described in the Materials and Methods section (Fig. S5). By doing so, we could unequivocally show that the calculated variances of the cellular surface heights decrease significantly from 480 nm²±190 nm² (n = 300) within the epithelial state to 370 nm²±110 nm² (n = 71, p-value<0.01, Wilcoxon rank sum test) during the transitional state down to 350 nm²±180 nm² (n = 209, p-value<0.01, Wilcoxon rank sum test) after completion of EMT. However, due to the decline in cellular height and the concomitant rearrangement of the actin cytoskeleton during the transition, stress fibers become visible upon AFM imaging. These putative corrugations are also captured by our roughness analysis leading to a decline in significance of the calculated variances. Consequently, the amount of free accessible membrane material allocated by membrane protrusions is even smaller than expected from roughness analysis, particularly in the latter stages of development. However, these observations are in good agreement with results from literature describing a strong influence of TGF-β1 on this epithelial cell line [42].

Alterations of cellular mechanics during EMT

Understanding the interplay between structural transformation of cells and their response to mechanical challenges provides deeper mechanistic insight into mechanoregulation during EMT on all time scales. The large-scale structural changes during EMT are expected to cause substantial changes in cellular mechanics. Particularly, dissociation of cell-cell contacts representing a tension-generating boundary, as well as the degradation of cortical actin accompanied by stress fiber formation and an increased cell-substrate adhesion should have a strong impact on the cell's mechanical behavior. To quantify these time-dependent alterations we performed force indentation experiments (Fig. 2A) as well as tether pulling experiments (Fig. 2B) on living untreated and treated NMuMG cells using atomic force microscopy. We assume that the restoring force in response to indentation of the cells is predominantly generated by a constant isotropic tension T comprising the sum of cortical and overall tension T₀ and a term describing the stretching or area dilatation of the bilayer (eq. 1). The associated area compressibility modulus K_A of the plasma membrane is usually in the range of 0.1 N/m emphasizing the inextensibility of the lipid bilayer, while that of the underlying actin mesh is expected to be substantially smaller. Generally, bending and tension due to pre-stress prevails at low penetration depths, larger deformation resulting in stretching of the surface is governed by plasma membrane mechanics. Since the membrane is largely inextensible allowing only 2–3% of area dilatation, stretching of the bilayer leads in first order to a contribution described by a 2-D Hookean behavior that adds to membrane's overall tension (eq. 1). Therefore, the restoring force F generated by the isotropic tension T increases nonlinearly with indentation depth with a dominant contribution from area dilatation of the plasma-membrane at large strains. The tension T₀ is dominated by the cortical tension generated by the active actomyosin cortex, the attachment of the plasma membrane to the cytoskeleton, and adhesion due to cell-cell or cell-substrate contacts. The theoretical procedure to parameterize the cell in order to obtain force indentation curves to describe experimental data is given in the supporting information (Supplementary Text S1) following the publication of Discher and coworkers [28]. The shape of the cell prior to indentation is essentially a spherical cap described by the two topographical parameters R₁ and the contact angle φ.

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Figure 2. Scheme showing (A) AFM indentation and (B) tether pulling experiments before and after the epithelial-to-mesenchymal transition (EMT).

The dashed arrows indicate the direction of force exerted to the membrane cortex upon either indentation or pulling. (A) Force indentation curves are analyzed using a tension model model [28], [30], [43]. Exemplarily chosen force-distance curve (red) and the corresponding fit by applying the extended indentation model (blue). Whereas T₀ dominates the elastic response upon low indentation depths, the influence of K_A increases at higher indentations a lateral stretching of the cellular shell occurs. A typical AFM force-distance curve of an untreated NMuMG cell including cantilever approach is shown (red). (B) During cantilever retraction (blue) the characteristic formation of a tether at constant force is observable at about 1.5 µm away from the surfaces.

https://doi.org/10.1371/journal.pone.0080068.g002

In case of tether pulling experiments only tension within the membrane is addressed, whereas upon indentation the cellular membrane and the underlying actin cortex are deformed (Fig. 2A, left panel). Consequently, application of the two techniques enables us to address the contribution of both membrane tension T_t and cortical tension T_C to the overall apical tension or pretension T₀ of the cells (eq. 6).(6)Pretension or in-plane tension T₀ of the cortical shell including the cellular membrane and the actin cytoskeleton as well as the apparent area compressibility modulus K_A^′ (Fig. 2A, right panel) were extracted from experimental force indentation curves by fitting the tension model to the data [28], [30], [43]. A detailed description of the procedure is given in the supplementary part of the manuscript. Notably the shape of the cells plays an important role to accurately determine the area compressibility modulus since the surface area is an important input parameter.

Mainly a broadening of the distribution of T₀ including a slight shift of the maximum towards larger values was detectable 24 h after cytokine addition (transitional state, Fig. 3B). Prolongation of the incubation time up to 48 h (mesenchymal-like state) results in a strong increase of the tension values approaching those obtained for single untreated NMuMG cells (Fig. 3C). In contrast to confluent NMuMG cells bearing a cubical shape, solitary NMuMG cells show a more elongated morphology and an increased spreading behavior due to the absence of cell-cell contacts and a stronger cell-substrate adhesion including a high tendency for stress fiber formation (Fig. S6). Furthermore, the migratory behavior of cells organized in a confluent monolayer is much lower than that of single isolated NMuMG cells mirrored in lamellipodium formation in the latter case. Consequently, as solitary NMuMG cells bear nearly the same cellular morphology as NMuMG cells within the mesenchymal-like state, it is not surprising that also their mechanical behaviors are comparable. However, the broad distribution of the calculated pre-tension T₀, still comprising values found for untreated cells, reflects a somewhat transitional state of the cells. To exclude that local effects during indentation determine the obtained results, colloidal probe indentation experiments were performed (Fig. S7) confirming that apical tension significantly rises from about 0.04 mN/m±0.01 mN/m (n = 10) before the transition to 0.11 mN/m±0.09 mN/m (n = 10) 48 h after EMT induction (Wilcoxon rank sum test, p-value<0.01). As stated above mainly structural changes during EMT are responsible for alterations of the mechanical properties of the cells. In order to evaluate the impact of the most relevant endogenous alterations taking place during the transition, we used a set of agents influencing distinct cellular process necessary for a proper proceeding of EMT (Fig. S8). Interestingly, inhibition of the translational elongation during protein synthesis by application of cycloheximide as well as administration of the Rho-kinase inhibitor Y-27632 does not alter membrane tension of the mesenchymal-like state. However, either pre-incubation of the cells with rapamycin, an inhibitor of cellular motility and de novo protein synthesis, or addition of blebbistatin lead to a significant decline of apical tension. Consequently, we assume that the increase in apical tension can be attributed to a higher acto-myosin contraction and the formation of actin stress fibers (Table 1).

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Figure 3. Mechanical properties of NMuMG cells under various conditions.

(A) Comparison of tension values T_x (kernel density functions), in which T_x can be T₀ or T_t, obtained from either indentation (grey, n = 108) or tether pulling (black, n = 109) experiments of untreated NMuMG cells. (B) Results from tether pulling experiments with untreated NMuMG cells (black, n = 109), NMuMG cells treated 48 h with TGF-β1 (blue, n = 274), single untreated NMuMG cells (green, n = 63) and fibroblasts (turquoise, n = 143). n depicts the number of tethers used for calculation. (C) Analysis of force indentation curves using a tension model [28], [30], [43]. Membrane tension (kernel density function) obtained from indentation experiments of untreated NMuMG cells (grey, labeled with number 1, n = 108), NMuMG cells treated 24 h with TGF-β1 (red, labeled with number 2, n = 52), NMuMG cells treated 48 h with TGF-β1 (blue, labeled with number 3, n = 95), single untreated NMuMG cells (green, labeled with number 4, n = 34) and NMuMG cells treated 48 h with TGF-β1 and 10 µg/ml cytochalasin D for 10 min (brown, labeled with number 5, n = 61) n depicts the number of curves used for calculation. In each experiment the velocity for cantilever approach and retraction during either indentation or tether pulling was 1 µm/s. For each category at least 4 cells were analyzed. (D) Fluorescence images of untreated NMuMG cells (left image) and NMuMG cells treated for 48 h with TGF-β1 (right image). Actin staining is shown in red, whereas the ERM protein moesin is stained in green. Scale bars: 20 µm.

https://doi.org/10.1371/journal.pone.0080068.g003

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Table 1. Tension values of NMuMG cells under various conditions obtained from AFM indentation measurements.

https://doi.org/10.1371/journal.pone.0080068.t001

The assumption is supported by the fact that after incubation of mesenchymal-like NMuMG cells with the actin-depolymerizing agent cytochalasin D, a shift to lower tension values is found (Fig. 3C). This important result shows the strong influence of the cytoskeleton on apical tension during the transition. Interestingly, membrane tension remains unaffected after application of cytochalasin D. Besides stress fiber formation, upregulation and clustering of moesin at the ventral membrane side of NMuMG cells, which has already been described by Haynes et al. [22] and which can also be observed in our fluorescence micrographs (Fig. 3D) might have a strong impact on cellular mechanics by increasing the coupling strength between the membrane and the underlying actin composite. Similarly, Larson and coworkers [44] described the strong influence of ERM molecules on cortical mechanics in mouse oocytes during completion of meiosis and generation of cell polarity. Performing tether pulling experiments with concanavalin A coated cantilevers (Fig. 2B) [45] leads to a clear confinement of membrane tension from cortical tension as during these measurements only the cellular membrane is addressed (equation 6). Moreover, we corrected membrane tension T_t for viscous contributions by using different cantilever retraction velocities (Fig. S9) according to Hochmuth et al. (1982) [46] and Krieg et al. (2008) [35] (eq. 5). By doing so, we calculated membrane tensions of 0.005 mN·m⁻¹ and 0.052 mN·m⁻¹ for NMuMG cells within either the epithelial or the mesenchymal-like state, respectively, as well as a viscosity coefficient η of 7.0·10⁻⁷ N·s·m⁻¹ (Fig. S9). Comparable values for η have been found by Marcus and Hochmuth (2002) [47] and Sun et al. (2007) [48] for neutrophils and bovine aortic endothelial cells, respectively. Although both parameters T₀ and T_t raise dramatically during EMT, the actin cortex highly dominates apical tension in either state. Whereas in case of NMuMG cells within the epithelial state, the contribution of membrane tension and cortical tension to the overall apical tension are 6% and 94%, respectively, their impact only slightly shifts to 8% and 92% after completion of EMT. Moreover, the calculated tension values from tether pulling experiments (Fig. 3B) show a bimodal distribution after transition (48 h incubation time) with a second peak at about 0.25 mN/m being in the same regime than tension values found for single NMuMG cells as well as for fibroblasts. Therefore, we assume that besides apical tension also membrane tension of cells that undergo EMT approaches that of single NMuMG cells.

Viscoelastic alterations of NMuMG cells during EMT

The complex structure of cellular systems including the highly dynamic architecture of the cytoskeletal filaments underlines the necessity of suitable models for a more profound understanding of the cells' mechanical properties. Especially treating cells as purely elastic bodies is inadequate in most of the cases as they are known to exhibit viscoelastic behavior. Therefore, we recorded frequency-dependent rheological data according to a method introduced by Shroff and coworkers [49]. Upon indenting the cells, the AFM cantilever is excited sinusoidally at a given frequency (5 Hz to 100 Hz, Fig. 4A) and the obtained force curves were analyzed using the soft glassy rheology model. The model provides us with information about the elastic (storage modulus, G′) and viscous (loss modulus, G″) properties of the cells (Fig. 4B,C) [37]. Mainly, the frequency dependence of the elastic modules allows to infer, whether cells are solid-like or behave as a liquid thus displaying a more invasive potential. In case of a solid-like behavior the storage modulus G′ is generally higher than the loss modulus G″ within the recorded frequency range. A more liquid-like behavior provides the contrary result. The soft glassy rheology model treats the cytoskeleton as a network of individual structural elements, i.e. actin fibers, which are trapped in harmonic energy wells. Active motion, driven by the motor proteins, promotes hopping of the elements from one energy well to another. Most of the energy is restored by trapping the elements in a neighboring harmonic potential, while some of the energy is dissipated in the framework of heat. Upon administration of this model the calculated loss tangent η (η = G″/G′) represents mechanical friction coupled to the rate of crosslink-cycling, G′ relates to the number of acto-myosin bridges and the cortical tension, which promote the storage of energy, and α reflects the so called ‘noise temperature’ of the material. Applying this model to the data, it becomes apparent that the most prominent change in viscoelastic spectra is the substantial but only temporal increase in α, which is simply the slope of lg(G′) vs. lg(ω) shown in Fig. 4B [50], [51]. Soft glassy rheology (SGR) interprets α, the power law exponent, as the effective lattice temperature that similar to k_BT supplies energy for remodeling of the cytoskeleton thus facilitating hopping from energy well to energy well. In other words the power-law exponent α indicates how far the elements (actin cortex) are away from their equilibrium state. Although the energy wells are somewhat abstract with respect to cell biology and do not necessarily refer to spatially defined molecular entities they basically represents constraint to molecular motion. In case of untreated NMuMG cells both modules G′ and G″ rise with increasing frequency according to a weak power law (Fig. 4B,C). Interestingly, upon induction of the EMT, the storage modulus becomes largely independent of frequency, whereas the loss modulus doesn't change significantly (Fig. 4C). The loss modulus representing the amount of dissipated energy within the sample and therefore hinting at the viscous signature of the cells only increases after completion of the EMT (Fig. 4F blue and black curve, respectively). Moreover after finalization of the transition NMuMG cells become much stiffer (Fig. 4B). These results underscore that mainly elastic properties of NMuMG cells are altered during transition from the epithelial to the mesenchymal phenotype, most likely due to an increase in tension mediated by an altered cytoskeletal structure and the concomitant decrease in membrane area. These findings are also reflected in the parameters obtained by applying the power law structure damping model [51] (Table S1). During these measurements only force curves recorded on the center of the cellular bodies are considered as the cell borders are generally stiffer leading to a decrease in significance of the obtained results (Fig. 4D–F).

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Figure 4. Viscoelastic properties of NMuMG cells during epithelial-to-mesenchymal transition.

(A) Scheme of microrheological setup. Upon indentation of the sample, the AFM cantilever is excited sinusoidally and force curves are recorded within a predefined area. (B) Calculated storage modules of untreated NMuMG cells (black, n = 106), NMuMG cells during EMT (green, n = 90) and NMuMG cells within the final mesenchymal-like state (blue, n = 73). (C) Calculated loss modules. Color coding as in B. The dashed lines show the corresponding fits according to the power-law structural damping model [51]. n depicts the number of curves used for calculation. (D–F) AFM deflection images of living NMuMG cells during the transition. Untreated NMuMG cells (D), NMuMG cells treated 24 h with TGF-β1 (E) and NMuMG 48 h after administration of TGF-β1 (F). White points mark the positions of the recorded force curves on the cellular bodies used for analysis omitting the stiffer cell-cell borders.

https://doi.org/10.1371/journal.pone.0080068.g004

Time-dependent delocalization and down-regulation of E-cadherin

During EMT intercellular junctions are strongly impaired as observed for NMuMG cells (Fig. 5A–C). The significant decline in apical tension (p-value<0.01) after administration of EDTA emphasizes the importance of cell-cell contacts for cellular mechanics (Fig. S10). Calcium depletion also interferes with cytoskeleton integrity, which also reduced the measured cortical tension. Therefore, a more profound understanding of the temporal alterations of cell-cell adhesion during EMT including a quantification of the exerted forces between the cells even down to the single-molecule level is of fundamental importance. Especially, the concerted and time-dependent down-regulation of the adherens junction protein E-cadherin is essential for epithelial-to-mesenchymal transition [52]. Whereas the amount of E-cadherin remains unchanged within the first 2–3 days after cytokine addition, the localization of the protein is completely altered. The strong membrane displacement of E-cadherin implicates that binding to the actin cytoskeleton is abrogated (Fig. 5B). Interestingly, a complete down-regulation is observed only 8 d after cytokine addition, as described previously [53] (Fig. 5C). These findings were confirmed with the help of a fluorescence intensity analysis. Upon summing up the intensity values of the E-cadherin fluorescence images (Fig. 5A–C), we calculated a value of 3759 a.u. for NMuMG cells in the epithelial state, 4610 a.u. for NMuMG cells 48 h after EMT induction and a value of 976 a.u. for NMuMG cells within the mesenchymal state. The fluorescence images were recorded using the same time (300 ms) and intensity for illumination. As cadherins are known for mediating a physical coupling between neighboring cells [13], a strong influence on cellular mechanics is expected as already reviewed by Cavey and Lecuit in 2009 [54]. Therefore, we investigated the overall interaction force of two opposing cells, either two untreated NMuMG cells (epithelial state) or two NMuMG cells incubated with TGF-β1 for at least 8 d (mesenchymal-like state), as well as their interaction on the single molecule level via single cell force spectroscopy (SCFS), and single molecule force spectroscopy. Figure 5D shows a typical force retraction curve of two epithelial cells being brought into contact. The maximum adhesion force decreases (Fig. 5F) significantly from about 171 pN to 120 pN after cytokine stimulation (p<0.001). More specific than the overall adhesion force is the occurrence of single rupture events documenting individual unbinding events of E-cadherin dimers (Fig. 5D in-lay). To evaluate whether the single rupture events prior to EMT can indeed be attributed to homomeric E-cadherin interactions, single E-cadherin molecules covalently bound to an AFM cantilever via a thiol linker are used for adhesion mapping of the cellular surface. The obtained histograms taken from force volume data of several cells show a characteristic maximum for untreated NMuMG cells at about 35 pN being in good accordance with SCFS data (vide infra). This force maximum vanishes upon addition of an E-cadherin antibody (red curve, Fig. 5H).

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Figure 5. Alterations in E-cadherin expression and localization during EMT.

(A) NMuMG cells in the epithelial state showing localization of E-cadherin at the cell-cell borders. (B) NMuMG cells incubated 48 h with the cytokine TGF-β1. Here a delocalization and increased intracellular uptake of E-cadherin is observable. (C) NMuMG cells incubated 10 d with the cytokine TGF-β1. After this long incubation time, expression of E-cadherin is completely down-regulated. Staining of the nucleus was carried out with DAPI (blue). A monoclonal Alexa Fluor488-conjugated IgG2a antibody was used to stain E-cadherin (green). Scale bar: 20 µm. (D) Typical retraction curve from single cell force spectroscopy measurements showing the interaction of two untreated NMuMG cells. The arrow marks a characteristic rupture event attributed to a specific homomeric cadherin interaction. Inlay displays the rupture event in higher magnification. A contact time of 1 sec between the cells was chosen. (E/G) Histograms of single rupture forces obtained from single cell force spectroscopy measurements using a contact time of 1 sec in either case (D). Force curves were recorded with an effective loading rate (force per time) of v_r = 2.3 nN/s for epithelial cells and of v_r = 0.7 nN/s for cells within the mesenchymal-like state, respectively. Strength of homomeric cadherin binding of either two untreated NMuMG cells (E, n = 149) or two NMuMG cells treated 8–10 d with TGF-β1 (G, n = 230) were investigated. (F) Maximum adhesion force of either two epithelial cells (NMuMG −, n = 175) or two mesenchymal-like cells (NMuMG +, n = 285) obtained from the minima in the retraction curves (Fig. 5D).*** P<7.7·10⁻¹⁶ (Wilcoxon rank sum test). Both categories are significantly different from each other. (H) Kernel density function of single rupture forces. A single E-cadherin molecule was brought into contact with an epithelial cell (black, n = 327) or with an epithelial cell pre-incubated for 1 h under physiological conditions with an E-cadherin antibody (red, n = 373) to abolish specific interactions. Contact time between the single molecules and the cells was 1 sec under both conditions. The effective loading rate v_r was 0.2 nN/s.

https://doi.org/10.1371/journal.pone.0080068.g005

Analyzing the recorded force distance curves obtained from cell-cell adhesion measurements, we detect a bimodal distribution of the single rupture forces hinting at the detachment of more than one E-cadherin molecule during cantilever retraction. However as inferred from single molecule experiments using single E-cadherin constructs attached to the cantilever and the underlying substrate, we also found a bimodal distribution (Fig. S10). It is therefore conceivable that instead of a detachment of multiple E-cadherin proteins a multistate unbinding of single molecules takes place as already inferred from Leckband and coworkers [55]. Therefore, we compared only the first peaks at low forces within the histograms of untreated and treated NMuMG cells. By doing so, we clearly see a decline of the unbinding force from 40 pN to 28 pN during EMT (Fig. 5E,G). Differences in the detected rupture forces by either single-cell or single-molecule force measurements can be attributed to the absence of an intracellular domain in the latter case contributing to the strength of cadherin-cadherin bonds [56] as well as dissimilar loading rates used during the experiments. However, similar to the findings of Evans and Calderwood [57], we revealed only a small influence of the effective loading rate on rupture force of the homomeric E-cadherin bond (Fig. S11B), which is too weak to explain the shift of the interaction forces of single unbinding events from 40 pN down to 28 pN during EMT. The weak dependence of the rupture force on loading rate is a good example for a persistent cellular adhesion binding in contrast to transient connectors as VCAM-1 [49]. The number of successful adhesion events during each set of experiments only slightly varies, especially in case of the cell-cell interaction experiments (Table S2).

In summary, these results support the hypothesis and expectation that mainly down-regulation of E-cadherin is responsible for the weakening of intercellular interactions during epithelial-to-mesenchymal transition.