[10] Measurements of cell-generated deformations on flexible substrata using correlation-based optical flow

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Primary bovine osteoblasts and human osteosarcoma cells exposed to direct-current electric fields undergo processes of retraction and elongation ultimately resulting in the realignment of the long cellular axis perpendicular to the electric field. The time taken for this reorientation was inversely correlated to field strength within a certain range. Cellular force output during reorientation was analyzed using a simple modification of traction force microscopy. The first detectable reaction was an increase in average traction force magnitude occurring between 10 and 30 seconds of electric field exposure. In the following 2 to 15 minutes traction forces at margins tangential to the electric field decreased below their initial values. Phase-contrast microscopy revealed elongating protrusions at these margins several minutes later. We could not correlate the initial traction changes with any change in intracellular free calcium levels measured using the fluorescent dye Fura-2 AM.

Section: Methodsmentioning

confidence: 99%

Dynamic changes in traction forces with DC electric field in osteoblast-like cells

Curtze

Dembo

Miron

et al. 2004

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“…Substratum deformation was calculated by comparing the positions of marker beads between the displaced and reference images, using a correlation-based optical flow algorithm (Dembo and Wang, 1999;Marganski et al, 2003). Custom software (LIBTRC) was then used for calculating and generating traction vector maps as previously described (Dembo and Wang, 1999).…”

Section: Measurement Of Substratum Deformation and Generation Of Tracmentioning

confidence: 99%

“…Recently, both wild-type and myosin II null Dictyostelium cells were found to deform weakly crosslinked silicone substrata (Uchida et al, 2003). However, the spatial resolution of silicone traction force assays is poor because of their low 'stress contrast' (Dembo and Wang, 1999;Marganski et al, 2003). This means that large traction stresses will 'overshadow' smaller ones, especially if they are close together, and it is likely to pose a problem for imaging traction stresses in small cell types like Dictyostelium with an average diameter of 10 m. Gelatin substrata have a spatial resolution that is an order of magnitude greater than crosslinked silicone films, and are thus well suited for traction force microscopy (TFM) in Dictyostelium.…”

Section: Introductionmentioning

confidence: 99%

“…This means that large traction stresses will 'overshadow' smaller ones, especially if they are close together, and it is likely to pose a problem for imaging traction stresses in small cell types like Dictyostelium with an average diameter of 10 m. Gelatin substrata have a spatial resolution that is an order of magnitude greater than crosslinked silicone films, and are thus well suited for traction force microscopy (TFM) in Dictyostelium. Here we have used a gelatin traction force assay to detect the traction forces produced by wild-type, mlcE -and mhcA -Dictyostelium and generated vector maps of the traction stresses produced by these cell types using custom traction mapping software, LIBTRC (Dembo and Wang, 1999;Marganski et al, 2003). We have correlated changes in the magnitude and distribution of traction stresses with measures of cell speed, area and shape, during cycles of protrusion and retraction.…”

Section: Introductionmentioning

confidence: 99%

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Traction force microscopy in Dictyostelium reveals distinct roles for myosin II motor and actin-crosslinking activity in polarized cell movement

Lombardi

Knecht

Dembo

et al. 2007

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Continuous cell movement requires the coordination of protrusive forces at the leading edge with contractile forces at the rear of the cell. Myosin II is required to generate the necessary contractile force to facilitate retraction; however, Dictyostelium cells that lack myosin II (mhcA–) are still motile. To directly investigate the role of myosin II in contractility we used a gelatin traction force assay to measure the magnitude and dynamic redistribution of traction stresses generated by randomly moving wild-type, myosin II essential light chain null (mlcE–) and mhcA– cells. Our data show that for each cell type, periods of rapid, directed cell movement occur when an asymmetrical distribution of traction stress is present, in which traction stresses at the rear are significantly higher than those at the front. We found that the major determinants of cell speed are the rate and frequency at which traction stress asymmetry develops, not the absolute magnitude of traction stress. We conclude that traction stress asymmetry is important for rapid, polarized cell movement because high traction stresses at the rear promote retraction, whereas low traction at the front allows protrusion. We propose that myosin II motor activity increases the rate and frequency at which traction stress asymmetry develops, whereas actin crosslinking activity is important for stabilizing it.

“…1C) shows the size and direction of bead displacements. Displacement of the substratum is calculated by comparing the positions of marker beads between the disturbed and reference image, using a correlation-based optical flow algorithm (Marganski et al, 2003). This program determines substratum deformation by finding the best match in the pattern of fluorescent intensity between a small square sub-region of the reference image, and a similar region in the disturbed image.…”

Section: Measurement Of Substrate Displacementmentioning

confidence: 99%

Calcium transients induce spatially coordinated increases in traction force during the movement of fish keratocytes

Doyle

Marganski

Lee

2004

The coordination of protrusion with retraction is essential for continuous cell movement. In fish keratocytes the activation of stretch-activated calcium channels, and the resulting increase in intracellular calcium, trigger release of the rear cell margin when forward movement is impeded. Although it is likely that retraction involves a calcium-dependent increase in cytoskeletal contractility, it is not known how the timing, magnitude and localization of contractile forces are organized during retraction. We have addressed this question using a new gelatin traction force assay in combination with calcium imaging to determine what changes in cytoskeletal force production accompany calcium-induced retraction. We find that individual calcium transients are followed within seconds by a rapid increase in traction stress that is maintained, or increases in a stepwise manner, until retraction occurs. Increases in traction stress are accompanied by a distinct sequence of changes in the spatial distribution of large traction stresses. Regions of increased traction stress enlarge at the lateral cell margins and expand forward along the cell margin. In particular, rearward facing propulsive' tractions at the leading edge of the cell, which are normally very low, increase several fold. Following retraction, a precipitous drop in traction stress is observed. Such distinct variations in traction stress are not observed in cells when calcium transients are absent. These results suggest a mechanism by which global increases in intracellular calcium can locally regulate contractile force production, in order to maintain a rapid highly directed mode of movement.