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.

Matrix elasticity, cytoskeletal forces and physics of the nucleus: how deeply do cells ‘feel’ outside and in?

“…The collective results from the FEM modeling are similar to results from limited studies in which the spread area of MSCs was measured for a set of nominally ~1-m thin gels versus thick gels (Engler et al, 2008). Importantly, the stiffness ranges for mechanosensitivity match matrix compliance at which various cell types -smooth-muscle cells (Engler et al, 2004c), fibroblasts (Rajagopalan et al, 2004) and malignant phenotypes (Paszek et al, 2005) -show many distinctive responses in cytoskeletal organization, signaling, etc.…”

Force field evolution during human blood platelet activation

“…Compared to fibroblasts (Dembo and Wang, 1999;Munevar et al, 2001;Rajagopalan et al, 2004), fish keratocytes (Oliver et al, 1999) and airway smooth muscle cells (Tolić-Nørrelykke and Wang, 2005) generating traction forces of 0.8-3.03610 3 Pa, 10 2 Pa and again 10 2 Pa, respectively, platelets are among the strongest cells measured by TFM. Although actomyosin is recognized as the main generator for contractile forces in all of the above mentioned cell types (Li et al, 2005;Gunst and Tang, 2000;Pollard et al, 1977), different mechanisms of actomyosin rearrangement may account for the differences in traction forces and, not surprisingly, the arrangement of the force fields.…”

“…Polyacrylamide (PAA) substrates were prepared according to references (Rajagopalan et al, 2004;Brandley and Schnaar, 1988). Briefly, a polymerization solution was mixed containing 8% monomeric acrylamide (BioRad, Hercules, CA, USA); 0.04% crosslinker bis-acrylamide (Bio-Rad); 50 mM HEPES; 13.4 mM catalyzer TEMED (Bio-Rad), 10 mM acrylic acid NHS-esther (Sigma-Aldrich, St Louis, MO, USA), 2 ml/ml red fluorescent marker beads (0.1 mm diameter, 2% solids, excitation 580 nm, emission 605 nm; Invitrogen, Carlsbad, CA, USA) and 0.05% ammonium persulfate to initiate polymerization.…”

“…The experimental technique has been amply used to study contractile forces generated by both migrating cells, such as fibroblasts (Dembo and Wang, 1999;Pelham and Wang, 1999;Munevar et al, 2001;Beningo et al, 2001;Rajagopalan et al, 2004) or fish keratocytes (Oliver et al, 1999;Lee et al, 1994), and stationary contractile cells, such as airway smooth muscle cells (Butler et al, 2002;Tolić-Nørrelykke and Wang, 2005). Average traction forces for the three cell types are typically on the order of 0.8-3.03610 3 Pa, 10 2 Pa and again 10 2 Pa, respectively.…”

Contractile cell forces exerted on rigid substrates

“…In light of a recent study (Rajagopalan et al, 2004), however, this result is more than reasonable, as the cell contractility can vary from experiment to experiment by a factor of 25 for individual cells. Because we integrated over about 100 fi broblasts, one could expect variations by a factor of 2.5.…”