Direct comparison of the spread area, contractility, and migration of balb/c 3T3 fibroblasts adhered to fibronectin- and RGD-modified substrata

Abstract

Native proteins are often substituted by short peptide sequences. These peptides can recapitulate key, but not all biofunctional properties of the native proteins. Here, we quantify the similarities and differences in spread area, contractile activity, and migration speed for balb/c 3T3 fibroblasts adhered to fibronectin- (FN) and Arg-Gly-Asp (RGD)-modified substrata of varying surface density. In both cases spread area has a biphasic dependence on surface ligand density (sigma) with a maximum at sigma approximately 200 molecules/microm2, whereas the total traction force increases and reaches a plateau as a function of sigma. In addition to these qualitative similarities, there are significant quantitative differences between fibroblasts adhered to FN and RGD. For example, fibroblasts on FN have a spread area that is on average greater by approximately 200 microm2 over a approximately 40-fold change in sigma. In addition, fibroblasts on FN exert approximately 3-5 times more total force, which reaches a maximum at a value of sigma approximately 5 times less than for cells adhered to RGD. The data also indicate that the differences in traction are not simply a function of the degree of spreading. In fact, fibroblasts on FN (sigma approximately 2000 microm(-2)) and RGD (sigma approximately 200 microm(-2)) have both similar spread area (approximately 600 microm2) and migration speed (approximately 11 microm/h), yet the total force production is five times higher on FN than RGD (approximately 0.05 dyn compared to approximately 0.01 dyn). Thus, the specific interactions between fibroblasts and FN molecules must inherently allow for higher traction force generation in comparison to the interactions between fibroblasts and RGD.

FIGURE 1

Quantitation of the coupling efficiency of FN (A) and RGD (B) to polyacrylamide substrata. The relationship between the amount of input ligand and the amount immobilized on the substratum determined by radiolabeling is shown. Linear curve fitting indicates that the coupling efficiency is ∼6.8% (R ∼ 0.98) for FN (A) and ∼3.5% (R ∼ 0.99) for RGD (B). These curves were then used to convert the input concentration of FN or RGD (in μg/ml) into an immobilized surface density (in molecules/μm²).

FIGURE 2

Traction force microscopy of a typical balb/c 3T3 fibroblast adhered to a substratum with σ_FN ∼ 200 μm⁻² (A and B) and σ_RGD ∼ 200 μm⁻² (C and D). The bar equals 9.4 μm and indicates the length scale. The arrow equals 9.4 μm for the experimental substratum displacement fields (A and C) and 96 kdyn/cm² for the most-likely cellular traction fields (B and D). The cell adhered to the FN-modified substratum is characterized by A ∼ 900 μm², |T| ∼ 8.7 kdyn/cm², and |F| ∼ 0.08 dyn, whereas the cell adhered to the RGD-modified substratum is characterized by A ∼ 430 μm², |T| ∼ 3.8 kdyn/cm², and |F| ∼ 0.02 dyn.

FIGURE 3

Comparison of FN (□) and RGD (¡) surface densities on the population average spread area 〈A〉. Values are reported as an average ± SE. See Table 1 for the sample size n for each condition. Asterisk indicates that statistically significant differences (p < 0.05) are present between the FN and RGD data at the particular surface density of ligand.

FIGURE 4

Comparison of FN (□) and RGD (¡) surface densities on the population average total absolute traction force 〈|F|〉. Values are reported as an average ± SE. See Table 1 for the sample size n for each condition. Asterisk indicates that statistically significant differences (p < 0.05) are present between the FN and RGD data at the particular surface density of ligand.

FIGURE 5

Comparison of FN (□) and RGD (¡) surface densities on the population average traction magnitude 〈|T|〉. Values are reported as an average ± SE. See Table 1 for the sample size n for each condition. Asterisk indicates that statistically significant differences (p < 0.05) are present between the FN and RGD data at the particular surface density of ligand.

FIGURE 6

Comparison of FN (□) and RGD (¡) surface densities on the population average migration speed 〈S〉. Values are reported as an average ± SE. See Table 1 for the sample size n for each condition.

FIGURE 7

Contractility differences with equal spread area and migration speed. Phase contrast images and corresponding traction fields of a balb/c 3T3 fibroblast adhered to an FN- (A and C) and RGD- modified (B and D) substratum. The bar is 8 μm for the phase contrast images (A and B) and 120 kdyn/cm² for the most-likely cellular traction fields (C and D). The surface density is ∼2000 μm⁻² for FN and ∼200 μm⁻² for RGD. These surface densities were chosen because the population average spread area (〈A〉 ∼ 600 μm²) and speed (〈S〉 ∼ 11.1 μm/h) are similar for cells on FN- and RGD-modified substrata, but the population average contractile output is different. Furthermore, the two cells shown were chosen because they represent the closest to the population mean in terms of 〈A〉, 〈|F|〉, and 〈|T|〉. The contractile output of the cell on FN is characterized by |T| ∼ 8.6 kdyn/cm² and |F| ∼ 0.04 dyn, whereas the cell on RGD is characterized by |T| ∼ 3.8 kdyn/cm² and |F| ∼ 0.02 dyn.

FIGURE 8

Actin and paxillin distribution for balb/c 3T3 fibroblasts of similar area (|A| ∼ 600 μm²) adhered to FN-PAAM (A and B; σ_FN ∼ 2000 μm⁻²), and to GRGDSP-PAAM (C and D; σ_RGD ∼ 200 μm⁻²). Scale bar = 10 μm.