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. 1999 Apr;10(4):935-45.
doi: 10.1091/mbc.10.4.935.

High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate

R J Pelham Jr  1 , Y l Wang
Affiliations
Free PMC article

High resolution detection of mechanical forces exerted by locomoting fibroblasts on the substrate

R J Pelham Jr et al. Mol Biol Cell. 1999 Apr.
Free PMC article

Abstract

We have developed a new approach to detect mechanical forces exerted by locomoting fibroblasts on the substrate. Cells were cultured on elastic, collagen-coated polyacrylamide sheets embedded with 0. 2-micrometer fluorescent beads. Forces exerted by the cell cause deformation of the substrate and displacement of the beads. By recording the position of beads during cell locomotion and after cell removal, we discovered that most forces were radially distributed, switching direction in the anterior region. Deformations near the leading edge were strong, transient, and variable in magnitude, consistent with active local contractions, whereas those in the posterior region were weaker, more stable, and more uniform, consistent with passive resistance. Treatment of cells with cytochalasin D or myosin II inhibitors caused relaxation of the forces, suggesting that they are generated primarily via actin-myosin II interactions; treatment with nocodazole caused no immediate effect on forces. Immunofluorescence indicated that the frontal region of strong deformation contained many vinculin plaques but no apparent concentration of actin or myosin II filaments. Strong mechanical forces in the anterior region, generated by locally activated myosin II and transmitted through vinculin-rich structures, likely play a major role in cell locomotion and in mechanical signaling with the surrounding environment.

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Figures

Figure 1
Figure 1
Distribution of substrate deformation caused by a motile 3T3 cell. (A) The dotted line represents the cell boundary at t = 0 min, and the solid line represents the cell boundary at t = 30 min. Arrows show changes in the position of substrate-embedded beads as the cell migrated forward during this period of time. (B) At t = 30 min, the cell was detached from the substrate with trypsin. Arrows were constructed from the bead positions after cell detachment to those before detachment and reflect the net forces exerted by the cell. To facilitate plotting, the scale of the arrows was amplified twice relative to the scale of the cell (this also applies to all subsequent figures). The arrows show a radial pattern converging in a region just in front of the nucleus. Bar, 20 μm.
Figure 2
Figure 2
Changes in substrate deformation in the anterior region of motile cells. (A) The movement of substrate-embedded beads under the anterior portion of a motile cell was recorded at the time (in minutes) indicated in the top left corner. Small arrows indicate the positions of the several beads relative to their neutral positions at t = 0. The direction of cell movement is indicated by a large arrow at t = 0. The boundary of the cell is indicated by a dotted line at t = 20 and 30 min. The position of the nucleus is indicated by a dashed circle at t = 45 min. Strong deformation developed in a region ∼5–10 μm from the active leading edge and dissipated as the nucleus moved over the beads. Bar, 10 μm. (B) The magnitude of substrate deformation is plotted as a function of time for three beads from three different locomoting cells. The deformation reached its maximum in ∼25 min and then dissipated over the next ∼30 min without showing a measurable plateau. (C) The track of a bead moving under the anterior region of a cell follows a smooth course, with no detectable fluctuation in direction. Bar, 1 μm.
Figure 3
Figure 3
Changes in substrate deformation in the posterior region of motile cells. (A) The movement of substrate-embedded beads under the posterior portion of a motile cell was recorded at the time (in minutes) indicated in the top left corner. Small arrows indicate the positions of the several beads relative to their positions at t = 0. The direction of cell movement is indicated by a large arrow at t = 0. Weak deformation developed in the tail region and was maintained until the cell migrated away from the beads. Bar, 10 μm. (B) The magnitude of substrate deformation is plotted as a function of time for three beads from three different locomoting cells. The deformation reached its plateau in ∼20 min and was maintained at a low, constant level for 50–60 min until the cell moved beyond the beads.
Figure 4
Figure 4
Effects of drugs on mechanical forces generated by the cell. (A) A cell and beads in the underlying substrate were observed in the presence of 2 μM cytochalasin D for 30 min. The cell stopped forward movement and retracted its boundary (from dotted to solid line), while all the beads moved away from the center of the cell, suggesting the relaxation of forces throughout the cell. (B) The cell was then detached by trypsin to detect any residual deformation at steady state. No bead movement was detected. The dots represent the stationary positions of the beads. (C–F) Similar experiments were performed with 20 μM KT5926 (C and D) and 1 μM nocodazole (E and F). A fraction of thebeads moved away from the center of the cell upon treatment with KT5926, as shown by arrows (C), while others maintained their positions, as shown by dots. No newly developed inward force was detected. The rate of forward movement of the nucleus was reduced by 75% (from dotted to solid line), while the protrusion of the leading edge appeared similar to that in control cells. The forces maintained a radial pattern as in control cells but with an average magnitude reduced by 80–90% (D). Nocodazole had no apparent effect on the overall pattern of forces as compared with that in control cells (E and F). Bar, 20 μm.
Figure 5
Figure 5
Distribution of actin filaments and myosin II in relation to strong substrate deformations in the anterior region. (A and C) Cells and beads in the underlying substrate were observed for 20 min. Movements of the beads are indicated by arrows. Dotted and solid lines indicate the starting and ending positions, respectively, of the cell boundary and nucleus. (B and D) The cells were then fixed and stained with rhodamine–phalloidin (B) or antibodies against myosin II (D) to determine the relationship between actin–myosin II organization and mechanical forces. Actin filaments are distributed similarly in areas of large and small substrate deformations (A and B, rectangles). Fine actin bundles are present throughout most regions of the cell. In addition, the concentration of myosin II was similar in regions of strong and weak forces (C and D, rectangles). Bars, 20 μm.
Figure 6
Figure 6
Distribution of vinculin in relation to large substrate deformations in the anterior region. (A) A cell and beads in the underlying substrate were observed for 12 min. Movements of the beads are indicated by arrows. Solid and dotted lines indicate the starting and ending positions, respectively, of the cell boundary and nucleus. (B) The cell was then fixed and immunofluorescence stained for vinculin to determine the relationship between vinculin organization and mechanical forces. Vinculin is concentrated at elongated plaque structures in the region of strong mechanical forces (B; arrow). Bar, 20 μm.
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