FRET measurements of cell-traction forces and nano-scale clustering of adhesion ligands varied by substrate stiffness

Abstract

The mechanical properties of cell adhesion substrates regulate cell phenotype, but the mechanism of this relation is currently unclear. It may involve the magnitude of traction force applied by the cell, and/or the ability of the cells to rearrange the cell adhesion molecules presented from the material. In this study, we describe a FRET technique that can be used to evaluate the mechanics of cell-material interactions at the molecular level and simultaneously quantify the cell-based nanoscale rearrangement of the material itself. We found that these events depended on the mechanical rigidity of the adhesion substrate. Furthermore, both the proliferation and differentiation of preosteoblasts (MC3T3-E1) correlated to the magnitude of force that cells generate to cluster the cell adhesion ligands, but not the extent of ligand clustering. Together, these data demonstrate the utility of FRET in analyzing cell-material interactions, and suggest that regulation of phenotype with substrate stiffness is related to alterations in cellular traction forces.

Fig. 1.

Clustering of peptides resulting from cell adhesion was visualized by imaging FRET between fluorescently labeled peptides. (a) To use this technique, the adhesion peptide (G₄RGDASSK) was separately labeled with either Alexa Fluor 488 or Alexa Fluor 546, after conjugation of the peptides to the polymer. (b and c) Excitation of a control gel (unlabeled G₄RGDASSK-polymer and Alexa Fluor 488-G₄RGDASSK-polymer) at a wavelength of 488 nm resulted in a green emission limited to regions of gel containing attached cells. (d and e) In contrast, the excitation of a second control gel (unlabeled G₄RGDASSK-polymer and Alexa Fluor 546-G₄RGDASSK-polymer) at 488 nm resulted in minimal red emission. Finally, excitation of gels containing both Alexa Fluor 488-G₄RGDASSK-polymer and Alexa Fluor 546-G₄RGDASSK-polymer at 488 nm led to a reduction in the yield of green fluorescence (Φ_green)(f and g), but increased the yield of red fluorescence (Φ_red)(h). (i–k) Increasing the peptide density 10-fold increased the baseline energy transfer throughout the gels, and enhanced energy transfer in certain regions (arrows) and reduced fluorescence in other regions (asterisks) under the cells. b, d, f, and i are bright-field images of the cells. c, g, and j and e, h, and k are fluorescent images of the same field collected through the green and red channels, respectively. The white lines in all photos represent the cell boundaries. The elastic modulus (E) of the gel was kept constant at 60 kPa in all experiments.

Fig. 2.

Cells were untreated (a–c and g) or treated with Nocodazole (d–f and h) to depolymerize microtubules. Treatment of cells with Nocodazole significantly reduced the Φ_green (compare b with e), but increased Φ_red/Φ_green (compare c with f). a and d correspond to the bright field images of the adherent cells; b and e correspond to the fluorescent images collected through the green channel; c and f were prepared by overlaying the images collected through the green channel and those collected through the red channel. The effect of Nocodazole on the traction force that cells exert on the peptides was confirmed by immunostaining of cells to visualize vinculin associated with focal adhesions (g and h). Minimal vinculin aggregates indicating focal contacts were found in the untreated cells (g), whereas larger focal contacts at the cell edges were noted following Nocodazole treatment (h). E of the gels in these experiments was kept constant at 20 kPa.

Fig. 3.

E of the gels altered the capability of cells to cluster adhesion peptides, as visualized with FRET between fluorescently labeled adhesion peptides, and the traction force exerted by the cells. Increasing E from 20 to 60 kPa reduced Φ_green (compare b with Fig. 2b), but raised Φ_red/Φ_green (compare c with Fig. 2c). Increasing E further to 110 kPa increased Φ_green (e), but reduced Φ_red/Φ_green (f). a and d correspond to the bright-field images of the cells; b and e correspond to the fluorescent images collected through the green channel; c and f were prepared by overlaying fluorescent images collected through the green channel and those collected through the red channel. (g) Quantification of the fluorescent yield with image analysis software demonstrated that the cellular average degree of energy transfer calculated from Φ_green was maximized at E of 60 kPa. (h) The ratio Φ_red/Φ_green was also maximized at E of 60 kPa. (i) The normalized distance between peptides (D) was minimized at E of 60 kPa. (j) The normalized force that cells exerted to displace peptides (F) increased in proportion to E. Values of D and F were normalized by the distance between peptides and cell traction force on the softest gel (E ≈ 20 kPa). Differences in the values in g–j for cells on intermediate stiffness gels versus the other two conditions were statistically significant (P < 0.05).

Fig. 4.

The overall morphology and extent of spreading of cells adherent to gels were not altered with increases in the E of the gels from 20 (a) to 60 (b) and 110 (c) kPa. Membranes were stained with octadecyl rhodamine B chloride to visualize cells. In contrast, formation of focal adhesions, as visualized with immunofluorescence localization of vinculin, was enhanced when E was increased from 20 (d) to 110 (e) kPa.

Fig. 5.

A number of cellular activities, including proliferation, apoptosis, and differentiation, were regulated by the E of the gels. Raising E led to more rapid cell growth, as measured with the increase in cell number over time (a), and the level of [³H]thymidine incorporation (b). In a, filled circles, open circles, and filled squares represent E of 20, 60, and 110 kPa, respectively. (c) A larger fraction of cells cultured on the soft gel (E ≈ 20 kPa) were apoptotic, as indicated by the positive staining of annexin on the membrane of unpermeabilized cells. (d) Increasing E to 110 kPa led to a reduction in the fraction of apoptotic cells. The apoptosis assay was performed after 5 days in culture, and the green immunofluorescent staining of cell membranes results from the presence of annexin on the exterior surface of cell membranes during the early stages of apoptosis. In contrast, cell differentiation was enhanced with decreases in E, as measured with the level of osteocalcin secretion from the cells (e) and mineralization of these cultures (f). In a, b (days 5 and 7), e, and f, the values for cells on the gels of 60 and 110 kPa were statistically different (P < 0.05), as compared to values for cells on the softest gels; in e and f, the values for cells on the stiffest gels were also statistically different (P < 0.05), as compared to cells on the intermediate stiffness gels.