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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01087

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

¹ Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA
² Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA

View larger version (48K): [in a new window]   Fig. 1. Dual calcium and traction force imaging in a keratocyte moving on a gelatin substratum. (A) Fluorescence image of a fish keratocyte (white outline) loaded with the calcium indicator, Calcium Green-1-dextran moving in the direction indicated (open arrow). (B) Image of fluorescent beads (0.19-0.20 µm diameter) in their displaced positions within a 3.0% gelatin substratum, upon which the keratocyte in A is moving. (C) Image of fluorescent beads in their undisplaced positions (red) superimposed on panel B (blue beads) and the cell outline in panel A. Comparison of red and blue bead images represents the size and direction of bead displacements caused by the cell in its current position. Beads that do not undergo displacement appear white. The largest bead displacements occur perpendicular to the lateral cell edges (large open arrows) while smaller displacements can be seen within the leading lamella in the direction indicated (small open arrows).

View larger version (28K): [in a new window]   Fig. 2. Generation of traction vector maps using the gelatin traction force assay. (A) A vector map of the experimental bead displacements generated by the optical flow algorithm (Marganski and Dembo, 2003) with an outline of the cell edge and cell body superimposed upon it. The direction and size of the vectors indicate the direction and magnitude of bead displacements occurring in the plane of the substratum, generated by a moving keratocyte. (B) A vector map of traction stress computed from the bead displacements in panel A. Arrows indicate the size and direction of traction stresses. (C) A contour map showing areas of similar traction magnitude. Regions of low and high traction stress are represented by cool (blue-light green) and warm (yellow-purple) colors, respectively.

View larger version (44K): [in a new window]   Fig. 3. The temporal relationship between calcium transients and increases in traction stress. Panels A-D are color-coded vector maps obtained at time points before (A), during (B) and after a calcium transient (C,D). (E) Plots of Calcium Green-1 fluorescence (CG-1) and values for the 90th percentile traction stress (TF 90) that were obtained from a running average of five data points. Following a calcium transient, an increase in traction stress occurs that is maintained following a decrease in [Ca2+]i to baseline. Retraction (R) of the lateral rear edge results in a large decrease traction stress (D) yet this is elevated compared to pre-transient levels. (F) Cell outlines corresponding to the vector maps in panels A-D showing cell shape and movement with respect to the substratum.

View larger version (31K): [in a new window]   Fig. 4. The kinetic relationship between calcium transients and increases in traction stress in cells displaying calcium transients versus those that do not. Panels A-D are relative plots of Calcium Green-1 fluorescence and 90th percentile traction stress, where the first data point is set to 100% and retractions are indicated by R. Panel A is a relative plot of CG-1 fluorescence and traction stress from the transient shown in Fig. 3. Measurements of the increase in CG-1 fluorescence, m(Ca), and magnitude of traction stress, m(ts) are indicated by vertical lines. The duration of the calcium transient d(ts) and duration of increased traction stress, d(Ca) are indicated by horizontal lines. The initiation of a calcium transient and onset of increasing traction stress are indicated by closed and open arrowheads, respectively. The peak in CG-1 fluorescence and maximum traction stress are also indicated, respectively, by the closed and open arrows. Panel B is a relative plot of CG-1 fluorescence and traction stress from the transient shown in Fig. 5. Panel C shows an example of three calcium transients, each of which lead to a discrete increase in traction stress, such that a `stepping-up' in traction stress occurs. Panel D shows a representative example of six cells in which no transients occur and subsequently no changes in traction stress are seen. Panel E shows a histogram of the average temporal fluctuation in traction stress in cells displaying transients (n=17) and in control cells that do not display transients (n=6). Temporal fluctuation of either CG-1 fluorescence or traction stress was obtained by normalizing the standard deviation of a measurement with respect to the mean of all measurements, for the entire period of observation, for each cell. Panel F shows the average percent increase in traction stress occurring from the pre-transient value (set to 0%) to the maximum value (peak) for transients where no retraction occurred (gray bar) and for transients followed by a retraction (shaded bar). The average percent decrease in traction stress occurring between pre-transient values to post-transient values, where no retraction occurred (black bar) and for transients followed by a retraction (open bar). Asterisks indicate statistically significant differences (P<0.05).

View larger version (58K): [in a new window]   Fig. 5. Calcium transients induce distinct changes in the spatial organization of the highest traction stresses. Panels A-F are vector maps showing changes in distribution of the highest traction stresses (yellow-purple regions) during the calcium-induced rise in 90th percentile stress shown in panel G. Panel H shows the relative change in area of highest traction stresses within the square regions at the lateral rear edge (purple), the front lateral edge (yellow) and the leading edge (black) of the cell (panel F) moving in the direction indicated (bold arrow).

View larger version (25K): [in a new window]   Fig. 6. Diagram illustrating the changes in size and spatial distribution of regions producing the highest traction stress. Colored zones within the keratocyte represent the temporal sequence (blue, yellow, red) in which regions of highest traction stress enlarge, following a calcium transient. The graph shows the duration of each region, with respect to the increase in 90th percentile traction stress (red line), represented by the length of matching colored arrows. The numbered boxes represent different stages of the mechano-chemical feedback cycle as explained in the text.

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