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First published online 4 January 2005
doi: 10.1242/jcs.01590

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Cyclic changes in keratocyte speed and traction stress arise from Ca²⁺-dependent regulation of cell adhesiveness

Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, USA

View larger version (24K): [in a new window]   Fig. 2. The interrelationship between traction stress, cell shape and speed during one cycle of feedback. (A) An example of a single cycle of mechano-chemical feedback showing relative changes in traction stress (red), cell speed (green), shape factor (orange) and calcium indicator fluorescence (blue). Retraction of the rear cell margin is indicated (R). (B) The average relative changes in traction stress (black bar), cell speed (open bar) and shape (gray bar), associated with eight individual transients, for each phase of the feedback cycle. These are designated the pre-transient (1), transduction (2) and post-transient phases (3) and are also indicated in A.* indicates a significant difference (P<0.05) from pre-transient and transduction measures. indicates a significant difference (P<0.05) from measures of traction stress in all other phases.

View larger version (28K): [in a new window]   Fig. 7. Summary histogram of relative magnitudes and rates of change in traction stress and cell speed under all experimental conditions and for different phases of the feedback cycle. (A) Percent change in traction stress (black) and cell speed (gray), before increases in [Ca2+]i and after calcium-induced retractions, and treatments that inhibit calcium transients. (B) Rate of change (percent/second) of the parameters shown in A. In all cases an inverse relationship exists between traction stress and cell speed, except for EDTA treatment.

View larger version (28K): [in a new window]   Fig. 1. Cyclic changes in traction stress, cell speed and shape that accompany a single calcium transient. (A) Plots of the absolute change in 90th percentile traction stress (red), cell speed (green) and shape factor (orange) and [Ca2+]i as shown by calcium green-1 dextran fluorescence (CG-1: blue). (B-E) Contour maps of traction stress magnitude at corresponding time points marked in A. Shape factor decreases slightly, indicating cell elongation, 38 seconds before the calcium transient. Traction stress begins to increase 15 seconds before the transient (B) and continues to increase, reaching a maximum 70 seconds after the onset of the transient (D). Note the region of increased traction stress that enlarges along the cell margin in B-D. During this time period cell movement is completely inhibited. Retraction starts 60 seconds (arrow) after the calcium transient starts, as shown by the increase in cell speed and the precipitous drop in traction stress (D-E), together with an increase in shape factor, or cell roundness, that occurs 10 seconds later. When retraction is complete (arrowhead) cell speed returns close to the pre-transient value and cell shape begins to elongate, marking the end of one cycle of mechano-chemical feedback.

View larger version (60K): [in a new window]   Fig. 3. Inhibition of calcium transients prevents retraction and cell movement but not the generation of traction stress. (A,B) Plots of the relative changes in traction stress (red), cell speed (green), cell shape (orange) and calcium indicator fluorescence (blue). Each graph was offset for clarity and values for shape factor were multiplied by 3, for illustrative purposes. The addition of EGTA (A) or Gd3+ (B) is indicated (arrow), as is the retraction (R). (C) IRM images of an entire cell moving in the direction indicated (bold white arrow), and insets showing a retracting cell margin on the right side, at various time points before and after the addition (bold black arrow) of EGTA. Regions of very close contact beneath the lamellipodium (white arrowheads) and retraction fibers at the retracting rear margin (black arrowhead) can be seen before EGTA addition. retraction fibers disappear, indicating a reduction in the rate of retraction. A region of very close contact at the rear cell margin begins to enlarge 30 seconds later (arrow) and becomes more distinct after 2-3 minutes, by which time retraction is inhibited. Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 4. Immunofluorescence staining of the actin cytoskeleton and vinculin in untreated keratocytes and cells treated with EGTA or gadolinium. Phalloidin staining of the actin cytoskeleton in untreated cells (A) and those treated with EGTA (C) or Gd3+ (E). Note in A the prominent centrally located stress fibers (white arrow). (B) Anti-vinculin antibody staining of untreated keratocytes showed a uniform distribution of vinculin puncta throughout the cell that increased in density a short distance behind the leading edge. Anti-vinculin antibody staining of keratocytes treated with EGTA (D) or Gd3+ (E) displayed large aggregations of vinculin within focal adhesion-like structures at the lateral rear cell margins (white arrowheads). The cell body appears brighter due to its increased thickness compared with the lamella. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 5. Effects of inducing cell detachment by chelating external Ca2+ and Mg2+ with EDTA. (A) The relative changes in traction stress (red), cell speed (green), cell shape (orange) and calcium indicator fluorescence (blue). Each graph was offset for clarity, and values for shape factor were multiplied by 3, for illustrative purposes. The addition of EDTA (A) is indicated (arrow), as well as retraction of the rear (R). (B) IRM observations of keratocytes before and after treatment with EDTA. Bar, 10 µm. Addition of EDTA (A) leads to an immediate drop in traction stress, together with an increase in cell speed and shape factor, indicative of rapid cell detachment. IRM observations of a moving keratocyte in the direction indicated (bold white arrow) show cell detachment beginning at the leading edge 30 seconds after treatment, followed by retraction of the entire cell margin at 2 minutes.

View larger version (66K): [in a new window]   Fig. 6. Effects of inducing retraction by increasing [Ca2+]i with calcimycin. (A) The relative changes in traction stress (red), cell speed (green), cell shape (orange) and calcium indicator fluorescence (blue). Each graph was offset for clarity, and values for shape factor were multiplied by 3, for illustrative purposes. The addition of calcimycin (A) is indicated (arrow), as well as retraction of the rear (R). The addition of calcimycin leads to a rapid increase in calcium indicator fluorescence, to a higher level and of longer duration than the SAC-mediated calcium transients. Traction stress increases slightly at 60 seconds while cell speed decreases. Retraction begins at 80 seconds, as shown by the rapid increase in cell speed and slower cell rounding. (B) IRM observations of a treated keratocyte moving in the direction indicated (arrow) confirm that a brief period of adhesion strengthening occurs at the lateral rear and front edges (black arrowheads) between 0 and 20 seconds after calcimycin addition. The intervening time points are shown as insets. Note the increasing thickness of the line of very close contact, along the cell margin. In this example, retraction starts at the lateral cell edges and continues along the entire cell margin, until completed at 2 minutes.

View larger version (19K): [in a new window]   Fig. 8. Summary histogram of the effects of calcium transient frequency on cell speed. In cells that are less adhesive transient frequency has no significant effect on the average 90th percentile traction stress (dark bar) or cell speed (stippled bar). By contrast, transient frequency is associated with a significant increase in average 90th percentile traction stress and cell speed in cells that are more adhesive. * indicates a significant difference (P<0.05) from all other measures of cell speed. # indicates a significant difference (P<0.05) between these two measures only.

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