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First published online 19 November 2003
doi: 10.1242/jcs.00795

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Regulation of mechanical interactions between fibroblasts and the substratum by stretch-activated Ca²⁺ entry

¹ Department of Physiology, University of Massachusetts Medical School, 377 Plantation Street, Suite 327, Worcester, MA 01605, USA
² Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215, USA

View larger version (39K): [in a new window]   Fig. 1. Increases in traction forces and intracellular Ca2+ following the application of stretching forces. Mechanical forces were applied to a migrating NIH3T3 cell by stretching the flexible substratum near the leading edge with a micro needle. Arrows in B and C indicate the direction of stretching. (A-C) Phase contrast and (D-F) traction stress images were recorded prior to (A,D) and following the removal of the needle, at time points indicated (B,C,E,F). Asterisks mark a fiduciary reference point on the substratum (A-C). Traction stress images were rendered with different colors representing the magnitude, from 2.50x101 dynes/cm2 (violet) to >3.99x104 dynes/cm2 (red). To detect the Ca2+ concentration, cells loaded with Fluo-4-dextran were recorded before (G,H) or after (I,J) the application of stretching forces. While the cell stretched in control balanced salt solution showed a clear increase in fluorescence intensity 10 minutes after stretching (G,I), the cell stretched in 100 µM Gd3+ showed a slight decrease in fluorescence intensity (H,J). Scale bars: 20 µm.

View larger version (85K): [in a new window]   Fig. 2. Response of cell migration and traction forces to the application of Gd3+. Phase contrast images (A-C) of a migrating NIH3T3 fibroblast, recorded before and at specified time points after the application of 100 µM Gd3+. At steady state, the cell showed no apparent effect on the spread morphology or local protrusive activities (arrows, C). However, forward migration of the cell was inhibited. Asterisks mark a fiduciary reference point on the substratum. The corresponding traction stress images (D-F), with different colors representing the magnitude, from 1.04x102 dynes/cm2 (violet) to >1.00x105 dynes/cm2 (red), showed a striking decrease in traction stress following Gd3+ application. Scale bar: 20 µm.

View larger version (14K): [in a new window]   Fig. 3. Average migration speed in response to inhibition of stretch-activated Ca2+ entry and Ca2+ depletion. NIH3T3 cells incubated globally in gadolinium ion showed a marked decrease in average migration speed compared with control cells. Cell migration speed also decreased sharply upon application of Gd3+ to the leading edge, but not when applied to the trailing edge. Global application of EGTA similarly caused a sharp decrease in average migration speed. The rate measured in 5 mM EGTA included some component of nuclear displacement as a result of limited cell retraction. Error bars indicate standard error of the mean.

View larger version (70K): [in a new window]   Fig. 4. Response of cell migration and traction forces to the application of EGTA. Phase contrast images (A-C) of a migrating NIH3T3 fibroblast, recorded immediately before (A) and after (B,C) the application of 5 mM EGTA, showed inhibition of local protrusive activities, and partial retraction. Asterisks mark a fiduciary reference point on the substratum. The corresponding traction stress images showed a striking decrease in traction forces (D-F). Different colors represent the magnitude of traction stress, from 9.5x101 dynes/cm2 (violet) to >1.00x105 dynes/cm2 (red). Scale bar: 20 µm.

View larger version (78K): [in a new window]   Fig. 5. Response of cell migration and traction forces to local application of Gd3+ near the leading edge. Phase contrast images (A-C) of a migrating NIH3T3 fibroblast, recorded immediately before (A) and at specified time points after the application of 100 µM Gd3+ near the leading edge (arrows, B,C), showed no inhibition of local protrusive activities (arrows), although the cell body failed to migrate forward. Asterisks mark a fiduciary reference point on the substratum. The corresponding traction stress images (D-F), with different colors representing the magnitude, from 4.25x101 dynes/cm2 (violet) to >4.50x104 dynes/cm2 (red), showed a global decrease in traction stress following the local Gd3+ application. Scale bar: 20 µm.

View larger version (75K): [in a new window]   Fig. 6. Response of cell migration and traction forces to local application of Gd3+ near the trailing edge. Phase contrast images (A-C) of a migrating NIH3T3 fibroblast, recorded immediately before (A) or at specified time points after the application of 100 µM Gd3+ near the trailing edge, showed no apparent effect on cell morphology or migration. Asterisks mark a fiduciary reference point on the substratum. The corresponding traction stress images (D-F), with different colors representing the magnitude, from 7.63x101 dynes/cm2 (violet) to >4.75x104 dynes/cm2 (red), showed no apparent effect on traction stress following the local Gd3+ application. Scale bar: 20 µm.

View larger version (77K): [in a new window]   Fig. 7. Response of focal adhesions to Gd3+. IRM images of a NIH3T3 cell were recorded immediately before (A), or after the treatment with 100 µM Gd3+ at indicated time points (B,C). Note the transient increase in the darkness of the image (B, arrows), before the image returned to a pattern similar to that before the treatment (C). However, the adhesion plaques appeared less elongated compared with those before treatment, and more adhesion plaques were found near the center of the cell (C, arrows). Also there appeared to be some increase in the amorphous close contact compared to the image before treatment. Scale bar: 20 µm.

View larger version (43K): [in a new window]   Fig. 8. Effects of Gd3+ on vinculin and phosphotyrosine organization. NIH3T3 cells were treated with 100 µM Gd3+ (A-E) or control buffer (F-J) for 30 minutes before processing for immunofluorescent staining of vinculin (A,F), actin filaments (B,G), or phosphotyrosine (E,J; in different cells). C and H show combined actin-vinculin images. The control cell showed the characteristic localization of vinculin at focal adhesions (F), which were concentrated at the termini of stress fibers (H) and appeared as dark plaques in the IRM image (I). The Gd3+-treated cell showed a reduction in stress fiber number and intensity, and an increase in cortical actin intensity (B). Short stress fibers persist near the nucleus. Vinculin localization was much reduced at focal adhesions, and appeared primarily perinuclear (A). Staining for phosphotyrosine showed a similar decrease at focal adhesions in Gd3+ treated cells (E). IRM image of treated cell showed adhesion plaques throughout the cell (D), which appeared less elongated than those in control cells (I). Scale bar: 20 µm.