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.
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 KA 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.
Figure 3.
Mechanical properties of NMuMG cells under various conditions.
(A) Comparison of tension values Tx (kernel density functions), in which Tx can be T0 or Tt, 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.
Table 1.
Tension values of NMuMG cells under various conditions obtained from AFM indentation measurements.
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.
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 vr = 2.3 nN/s for epithelial cells and of vr = 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 vr was 0.2 nN/s.
Figure 6.
Scheme illustrating the structural alterations during epithelial-to-mesenchymal transition.
Within the epithelial state, membrane tension is mainly dominated by cell-cell interactions (tight and adherens junctions), the adhesion of the cytoskeleton to the membrane via ERM proteins and the buffering membrane reservoir (e.g. folds and microvilli). Upon transformation, actin stress fibers are formed and cell-substrate adhesion increases accompanied by a loss of membrane area. Structural changes (e.g. loss of cortical actin and intercellular junctions) include a reduction in surface area leading to homeostasis of membrane tension (cell in the middle). Destabilization of the cadherin complex [76], followed by E-cadherin delocalization and internalization take place during the transition. Within these early time regimes, the cells can be treated as liquid droplets surrounded by a membrane with static tension. However, within the final mesenchymal-like state membrane protrusions are stretched out allowing the cell to increase its spreading area on the surface. Due to this enforced cell-substrate adhesion in conjunction with an increased stress fiber formation, the mechanical response of mesenchymal-like cells has to be described as a viscoelastic solid.