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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02760

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Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels

Benjamin D. Matthews^1,2, Darryl R. Overby^1,*, Robert Mannix¹ and Donald E. Ingber^1,

¹ Vascular Biology Program, Departments of Pathology and Surgery, Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
² Department of Pediatrics, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA 02115, USA

View larger version (34K): [in a new window]   Fig. 1. Visualization and quantification of stress-induced displacements of integrin-bound magnetic microbeads on the surface membrane of cultured cells. (A) Differential interference contrast view of an adherent endothelial cell with an arrow indicating a single RGD-coated magnetic bead (4.5 µm diameter) bound to integrins on its apical surface. Bar, 20 µm. (B) A series of bright-field images recorded over 9 seconds showing bead position before (top), during (middle) and after (bottom) application of a 3-second force (130 pN) pulse (arrow indicates direction of force). (C) Bead displacement (µm) as a function of time before, during and after the 3-second force pulse (thick black line), as determined using computerized image analysis.

View larger version (13K): [in a new window]   Fig. 2. Effects of modulators of cytoskeletal prestress and focal adhesion assembly on the immediate viscoelastic response of individual magnetic beads bound to surface integrin receptors. A 3-second force (130 or 350 pN) pulse induced less displacement when applied to RGD-beads bound to integrins on control cells compared with cells treated immediately prior to force application with BDM, Y27, C3 transferase, PP2 or gadolinium. Data represent the mean ± s.e.m.; *P<0.05 compared with levels in the control.

View larger version (24K): [in a new window]   Fig. 3. Application of high (nN) levels of stress to cell-surface integrins increases intracellular calcium. (A) Phase-contrast view of an adherent endothelial cell with attached RGD-bead (white arrow) bound to integrins on the apical cell surface. Black arrowhead indicates position of the tip of the electromagnet. (B) A time series of pseudocolored fluorescence images of the cell shown in A after mechanical stress (5 nN) was applied with the magnet. These pseudocolored images demonstrate a transient stress-induced increase in [Ca 2+]i as a brief shift in color from blue to yellow, as detected using Fura-2AM ratio-imaging (color bar indicates [Ca 2+]i in nM). The times after start of the time-lapse series are indicated in seconds; the force pulse was applied at 9 seconds in this series. (C) Plot of average [Ca 2+]i for control () and gadolinium chloride-treated () cells as a function of time; the inhibition of stress-induced calcium influx by gadolinium was statistically significant (P<0.002; error bars indicate s.e.m.). Black arrow indicates when the 3-second force pulse was applied.

View larger version (22K): [in a new window]   Fig. 4. Early cellular strengthening response to oscillatory force. (A) Schematic of experimental design. Six consecutive 3-second force pulses (130 pN) were applied to bound RGD-beads, separated by brief (4-second) intervals. (B) Representative example of bead displacements measured in response to the pulsatile force regimen shown in A. Note strengthening of adhesions following pulses 2 and 3, as indicated by decreased displacements. (C) Average relative RGD-bead displacements induced by the pulsatile force regimen. Black bars, control; white bars, BDM; gray bars, Y27632; hatched bars, 4°C; *P<0.05 where indicated compared with the level in the first pulse respective experimental condition; note that only controls exhibited significant attenuation.

View larger version (20K): [in a new window]   Fig. 5. Adaptive strengthening of bead-integrin-cytoskeleton linkages induced by prolonged static force. (A) Schematic of experimental design. Brief (3 second) force pulses (black bars) were applied to surface-bound RGD-beads before and after static forces that were sustained for different times (grey bar) in order to determine if prolonged forces induce stable cell strengthening. (B) Prolonged force (250 pN) of 15 seconds duration induced strengthening of RGD-bead adhesions. Relative bead displacements represent displacements produced by the second 3-second force pulse divided by that induced by the first force pulse (white bar) (*P<0.05; grey bars indicate no significant difference compared with pre-force levels). (C) Strengthening of RGD-bead adhesions in response to application of force (130 or 350 pN) prolonged for 2 minutes in the absence (black bars) or presence (white bars) of BDM, Y27632, gadolinium, C3 transferase, PP2 or exposure to 4°C (*P<0.05 compared with pre-force levels).

View larger version (21K): [in a new window]   Fig. 6. Large-scale translocation of bead adhesions in response to prolonged force. The position of individual surface-bound magnetic beads coated with RGD peptide or K20 antibody was recorded in the absence or presence of sustained (2 minutes) application of force (130 pN) in a centripetal direction, opposite to that in which the beads normally move on the surface of the cell. (A) Measured displacements of RGD-beads in the absence of force (; –F) and in the presence of force (+ F) without () or with Y27 () or BDM (). Displacements of K20-beads in the absence of force (; –F) or presence of force (+F) without () or with soluble RGD peptide (sRGD, ). (B) Representative X-Y plot of positions of a representative RGD-bead before (), during (black line), and after () 2 minute application of force (130 pN). The bead was initially displaced toward the magnet, but was then actively retracted against the magnetic gradient and towards the nucleus during the course of force application (arrow indicates direction of applied force).

View larger version (23K): [in a new window]   Fig. 7. Contributions of Rho, mechanosensitive ion channels and Src tyrosine kinases to the adaptive repositioning response to prolonged stress. RGD-beads bound for 10 minutes to cells were exposed to a prolonged (60 seconds in A,B; 30 seconds in C) stress (350 pN) applied in a centripetal direction opposite to that which beads normally move on the cell surface. (A) Bead displacements relative to the nucleus were measured in cells exposed to the BioPORTER proteofection reagent in the absence () or presence () of C3 transferase. (B) Bead displacements measured in the absence () or presence () of gadolinium chloride. (C) Bead displacements measured in the absence () or presence () of PP2. Error bars indicate s.e.m.; P<0.05 at times greater than 30 seconds compared with controls in all graphs.

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