Traction forces generated by locomoting keratocytes.

Abstract

Abstract. Traction forces produced by moving fibroblasts have been observed as distortions in flexible substrata including wrinkling of thin, silicone rubber films. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards. However, traction forces could not be detected with faster moving cell types such as leukocytes and growth cones (Harris, A. K., D. Stopak, and P. Wild. 1981. Nature (Lond.). 290:249-251). We have developed a new assay in which traction forces produced by rapidly locomoting fish keratocytes can be detected by the two-dimensional displacements of small beads embedded in the plane of an elastic substratum. Traction forces were not detected at the rapidly extending front edge of the cell. Instead the largest traction forces were exerted perpendicular to the left and right cell margins. The maximum traction forces exerted by keratocytes were estimated to be ,x,2 x 10 -8 N. The pattern of traction forces can be related to the locomotion of a single keratocyte in terms of lamellar contractility and area of close cell-substratum contact.T o move cells must exert traction forces upon the substratum. This involves the temporal and spatial regulation of numerous force generating molecular motors. Yet an understanding of how and where moving cells generate traction forces, represents a major gap in our knowledge of cell locomotion. This paper presents the first measurements of the traction forces generated by rapidly moving cells.Traction forces produced by moving fibroblasts were first observed as distortions in flexible substrata that caused wrinkling of thin, silicone rubber films (Harris et al., 1980). These traction forces act inwards, relative to the extending lamella and retracting edge, leading to compression of the substratum such that wrinkles are formed perpendicular to the direction of lamellar extension. Wrinkles were thought to be formed by an actomyosin-based contraction of the cytoskeleton which is transmitted to the substratum via focal adhesions located just behind the extending edge and trailing cell edge. Traction forces generated by fibroblast lamellae were thought to represent the forces required to move the cell forwards along the substratum. It was therefore surprising to find that traction forces generated by faster moving cell types such as leukocytes and growth cones could not be detected (Harris, 1981), since it was assumed that larger traction forces would be required for faster locomotion. However, slow moving cells such as fibroblasts form strong focal adhesions to the substratum whereas faster moving cells tend to form weaker close contacts (Couchman and Reese, 1979).In addition large numbers of actin stress fibers are found in slower moving cells, implying greater cytoskeletal contractility. Therefore rapid cell locomotion appears to rely on both weaker cell substratum adhesions and cytoskeletal contractility.To learn more about the traction forces required for rapid locomotion, we have modified the traction ...

Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

“…It is conceivable that wrinkle-producing fibroblasts exert only the minimum force necessary to deform these weak surfaces; therefore, the use of elastomers with gradually increasing stiffness is crucial to determine their maximum contractile potential. Highly compliant elastomers have been wrinkled by a large proportion of ␣-SMAnegative fibroblasts consistent with the wrinkling capability of numerous cell types (Lee et al, 1994;Oliver et al, 1995). With the use of stiffer substrates, we determined a threshold of ϳ4 N that discriminates between these relatively weak traction forces and higher contractile forces (Roy et al, 1999); this threshold is only surpassed by ␣-SMA-positive fibroblasts.…”

“…Before seeding cells, these substrates were mechanically wrinkled with a deflecting flexible microneedle with a stiffness between 90 and 110 nN/ m (Lee et al, 1994;Fray et al, 1998). A second stiff needle (Ն50 N/ m) was used to fix the substrate at a distance of 200 m, simulating the substrate-pinching of bipolar cells.…”

“…To determine contractility on a single cell level, we used a modification of the silicone elastomer substrate assay originally developed by Harris et al (1980), combined with a modification of the force quantification method introduced by Lee et al (1994). The contractility of whole cell populations was quantified with the use of stress-released collagen lattices (Mochitate et al, 1991;Tomasek et al, 1992;Grinnell et al, 1999b).…”

“…Conversely, we have reduced ␣-SMA expression in LFs by blocking the activity of endogenous TGF␤1 with a specific neutralizing antibody, the soluble TGF␤-receptor type II (TGF␤-sR) or the recombinant ED-A fragment (rED-A) of cellular fibronectin (FN;Serini et al, 1998). Finally, in a direct approach to induce expression of ␣-SMA, we have transiently and stably transfected Swiss 3T3 fibroblasts with ␣-SMA cDNA.To determine contractility on a single cell level, we used a modification of the silicone elastomer substrate assay originally developed by Harris et al (1980), combined with a modification of the force quantification method introduced by Lee et al (1994). The contractility of whole cell populations was quantified with the use of stress-released collagen lattices (Mochitate et al, 1991;Tomasek et al, 1992;Grinnell et al, 1999b).…”

On a poroviscoelastic model for cell crawling

“…The rear of the cell exerts a force on the substrate in the direction of cell motion. A similar experiment in Lee et al (1994) found large, lateral forces directed inwards in the flanks and rear of the cell. However, they did not observe rearward directed force under lamellipodia, 2 they found a very small force in the direction of motion at the leading edge of the cell.…”