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. 1999 Dec 13;147(6):1313-24.
doi: 10.1083/jcb.147.6.1313.

Keratocytes pull with similar forces on their dorsal and ventral surfaces

C G Galbraith  1 , M P Sheetz
Affiliations

Keratocytes pull with similar forces on their dorsal and ventral surfaces

C G Galbraith et al. J Cell Biol. .

Abstract

As cells move forward, they pull rearward against extracellular matrices (ECMs), exerting traction forces. However, no rearward forces have been seen in the fish keratocyte. To address this discrepancy, we have measured the propulsive forces generated by the keratocyte lamella on both the ventral and the dorsal surfaces. On the ventral surface, a micromachined device revealed that traction forces were small and rearward directed under the lamella, changed direction in front of the nucleus, and became larger under the cell body. On the dorsal surface of the lamella, an optical gradient trap measured rearward forces generated against fibronectin-coated beads. The retrograde force exerted by the cell on the bead increased in the thickened region of the lamella where myosin condensation has been observed (Svitkina, T.M., A.B. Verkhovsky, K.M. McQuade, and G. G. Borisy. 1997. J. Cell Biol. 139:397-415). Similar forces were generated on both the ventral (0.2 nN/microm(2)) and the dorsal (0.4 nN/microm(2)) surfaces of the lamella, suggesting that dorsal matrix contacts are as effectively linked to the force-generating cytoskeleton as ventral contacts. The correlation between the level of traction force and the density of myosin suggests a model for keratocyte movement in which myosin condensation in the perinuclear region generates rearward forces in the lamella and forward forces in the cell rear.

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Figures

Figure 1
Figure 1
Keratocytes generate large traction forces orthogonal to the direction of migration. Keratocytes generate forces perpendicular to the direction of migration as indicated by wrinkles that are parallel to the direction of motion. Deformable substratum were made by cross-linking Dow Corning 710 fluid. The stiffness of the substrata was decreased by exposure to 254 nm UV light (Burton and Taylor 1997). Keratocytes generate wrinkles that are parallel to the direction of motion, indicating that the largest traction forces are perpendicular to the direction of motion. Bar, 10 μm.
Figure 2
Figure 2
Traction forces on either side of the keratocyte nucleus measured with the micromachined substratum. Traction forces measured on either side of the nucleus are large and orthogonal to the direction of motion, ∼12 nN. Bar, 5 μm.
Figure 3
Figure 3
Lamella of keratocytes generate a small rearward traction force against the micromachined substratum. Bar, 5 μm. Once the lamella of a keratocyte is over the measurement pad (t = 30 s), it generates a traction force of ∼5 nN (t = 45 s) in the direction opposite that of migration, indicated as a negative force. The direction of the force changes and the magnitude increases as the thickened region of the lamella crosses the pad (t = 50 s).
Figure 4
Figure 4
Keratocytes bind to FN-coated surfaces. Individual keratocytes were plated on coverslips coated with either FN 120 kD (5 μg/ml) or BSA (2 mg/ml). The percentage of cells that bound and spread on FN-coated substrata were significantly greater than the fraction that bound to BSA-coated substrata. The binding to FN was significantly inhibited by the addition of 1 mM GRGdSP; moreover, the addition of 1 mM GRGDNP decreased the percentage of cells bound on FN to the same level as control.
Figure 5
Figure 5
Keratocytes bind and transport FN-coated beads rearward. The majority of the FN-coated beads were transported rearward by the cell once they were released from the trap. In contrast, the majority of control BSA-coated beads did not bind to the surface of the cell. FN-coated beads, n = 133; BSA-coated beads, n = 87.
Figure 6
Figure 6
Keratocytes bind FN-coated beads preferentially at the leading edge. Ligand-coated beads were presented to different regions of the keratocyte lamella. a, Approximately 70% of FN-coated beads bound and moved rearward when presented at the leading edge of the cell. The percentage of bound beads decreased to ∼30% when the beads were presented 0.5–1.0 μm behind the leading edge. FN-coated beads placed on the leading edge, n = 43; FN-coated beads placed behind the leading edge, n = 43; BSA-coated beads, n = 40. b, Immunofluorescent labeling of a fish keratocyte stained with an antibody against the cytoplasmic region of the β1 integrin. Note the preferential localization of integrin along the leading edge of the lamella. Bar, 10 μm.
Figure 7
Figure 7
Displacement of FN-coated beads within the laser trap determines the force exerted by the dorsal surface of keratocytes. A 50-mW laser trap was used to place and hold a 1 μm bead coated with FNIII 7–10 on the lamella of a keratocyte. The cell displaces the bead with a force of 158 pN (t = 7.7 s) before it exerts enough force to escape the trap. Once the bead escapes the trap, it travels rearward at approximately the same velocity as the cell travels forward.
Figure 8
Figure 8
Beads that escape the force of a laser trap are more likely to be recaptured before they reach the thickened region of the lamella. A 50-mW laser trap was used to place and hold a 1-μm bead coated with FNIII 7–10 on the lamella. The bead was more likely to escape the trap if it was initially placed on the cell edge (t = 2.4 s). The same power trap could easily recapture the bead in the thin region of the lamella, as indicated by the sharp change in bead position (t = 10.3 s). However, the bead could not be recaptured if it reached the thickened region of the lamella (t = 29.9 s) or the nucleus.
Figure 9
Figure 9
Density of F-actin in the keratocyte lamella. a, A line intensity histogram of a phalloidin-labeled keratocyte shows that the density of the F-actin network decreases away from the leading edge. The density increases again to ∼75% of its maximal value in the perinuclear region. b, Oregon green phalloidin-labeled keratocyte. Bar, 10 μm.
Figure 10
Figure 10
Model of keratocyte migration. Actin–myosin condensation in the perinuclear region generated rearward traction forces in the lamella by pulling on the orthogonal actin in this region. The condensation also generates forward forces that push the cell body forward. Finally, the perinuclear bundles generate the pincer forces that are orthogonal to the direction of migration. a, Orientation of traction forces measured by micromachined substrata and optical gradient trap. b, Orientation of actin and myosin network in keratocyte cytoskeleton (based on data from Svitkina et al. 1997).
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