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. 2002 Jul 8;158(1):153-64.
doi: 10.1083/jcb.200201105. Epub 2002 Jul 8.

Effects of cell tension on the small GTPase Rac

Akira Katsumi  1 , Julie Milanini, William B Kiosses, Miguel A del Pozo, Roland Kaunas, Shu Chien, Klaus M Hahn, Martin Alexander Schwartz
Affiliations
Free PMC article

Effects of cell tension on the small GTPase Rac

Akira Katsumi et al. J Cell Biol. .
Free PMC article

Abstract

Cells in the body are subjected to mechanical stresses such as tension, compression, and shear stress. These mechanical stresses play important roles in both physiological and pathological processes; however, mechanisms transducing mechanical stresses into biochemical signals remain elusive. Here, we demonstrated that equibiaxial stretch inhibited lamellipodia formation through deactivation of Rac. Nearly maximal effects on Rac activity were obtained with 10% strain. GAP-resistant, constitutively active V12Rac reversed this inhibition, supporting a critical role for Rac inhibition in the response to stretch. In contrast, activation of endogenous Rac with a constitutively active nucleotide exchange factor did not, suggesting that regulation of GAP activity most likely mediates the inhibition. Uniaxial stretch suppressed lamellipodia along the sides lengthened by stretch and increased it at the adjacent ends. A fluorescence assay for localized Rac showed comparable changes in activity along the sides versus the ends after uniaxial stretch. Blocking polarization of Rac activity by expressing V12Rac prevented subsequent alignment of actin stress fibers. Treatment with Y-27632 or ML-7 that inhibits myosin phosphorylation and contractility increased lamellipodia through Rac activation and decreased cell polarization. We hypothesize that regulation of Rac activity by tension may be important for motility, polarization, and directionality of cell movement.

Figures

Figure 1.
Figure 1.
Equibiaxial stretch inhibits lamellipodia formation. Rat VSM cells were plated for 3h on collagen-coated silicone membranes in the equibiaxial stretch device. Cells were then stretched to increase area by 15%. (A) At indicated time points cells were fixed and stained with rhodamine-phalloidin. Cells with representative morphologies are shown. Bar, 20 μm. (B) The portion of the cell perimeter occupied by lamellipodia is expressed as percent of the total perimeter. Values are means ± SEM, for 20 cells per data point from three independent experiments. (C) Time-lapse images of VSM cells were recorded and total cell areas before and at various times after stretch were quantified. Values are means ± SEM for 27 cells.
Figure 2.
Figure 2.
Effects of equibiaxial stretch on Rac and Rho. (A) VSM cells with or without 15% stretch for the indicated time were lysed and pull-down assays performed. Rac was detected by Western blotting. (Top) Rac bound to PBD. (Bottom) Total cell lysates probed for Rac. For densitometric quantification, relative Rac activity was calculated from the amount of PBD-bound Rac normalized to the amount of total Rac. Results are means ± SEM from six experiments. * denotes a P value of <0.02 compared with unstretched cells. (B) VSM cells were stretched equibiaxially to increase area by the indicated amounts. After 5 min, GTP-Rac was assayed as in A. (Top) Rac bound to PBD. (Bottom) Total cell lysate probed for Rac. Densitometric quantification shows relative Rac activity calculated from the amount of PBD-bound Rac, normalized to the amount of total Rac. Results are means ± SEM from four experiments. * and ** denote P values of <0.02 and <0.01, respectively. (C) VSM cells with or without stretch were lysed and pull-down assays performed. Rho was detected by Western blotting. (Top) Rho bound to RBD. (Bottom) Total cell lysates probed for Rho. For densitometric quantification, relative Rho activity was calculated from the amount of RBD-bound Rho normalized to the amount of total Rho. Results are means ± SEM from four experiments.
Figure 2.
Figure 2.
Effects of equibiaxial stretch on Rac and Rho. (A) VSM cells with or without 15% stretch for the indicated time were lysed and pull-down assays performed. Rac was detected by Western blotting. (Top) Rac bound to PBD. (Bottom) Total cell lysates probed for Rac. For densitometric quantification, relative Rac activity was calculated from the amount of PBD-bound Rac normalized to the amount of total Rac. Results are means ± SEM from six experiments. * denotes a P value of <0.02 compared with unstretched cells. (B) VSM cells were stretched equibiaxially to increase area by the indicated amounts. After 5 min, GTP-Rac was assayed as in A. (Top) Rac bound to PBD. (Bottom) Total cell lysate probed for Rac. Densitometric quantification shows relative Rac activity calculated from the amount of PBD-bound Rac, normalized to the amount of total Rac. Results are means ± SEM from four experiments. * and ** denote P values of <0.02 and <0.01, respectively. (C) VSM cells with or without stretch were lysed and pull-down assays performed. Rho was detected by Western blotting. (Top) Rho bound to RBD. (Bottom) Total cell lysates probed for Rho. For densitometric quantification, relative Rho activity was calculated from the amount of RBD-bound Rho normalized to the amount of total Rho. Results are means ± SEM from four experiments.
Figure 3.
Figure 3.
Effects of stretch on V12Rac- and Tiam1-transfected VSM cells. (A–D) VSM cells were transfected with 2 μg of pEGFP-C1-V12Rac. (E–H) VSM cells were cotransfected with 1.6 μg of pcDNAINeo-Tiam1 and 0.4 μg of pEGFP. At 24 h after transfection, cells were plated on collagen I–coated silicone membrane for 3 h and stretched equibiaxially by 15% for 5min. Fluorescence images of actin filaments (A, C, E, and G) and GFP (B, D, F, and H) in the same cells were shown. Results are representative of three experiments. Bar, 20 μm. (I) The portion of the cell perimeter occupied by lamellipodia is expressed as percent of the total perimeter. Values are means ± SEM, for 20 cells per data point. * indicates P < 0.01.
Figure 4.
Figure 4.
Effects of uniaxial stretch on membrane protrusive activity. (A) VSM cells were plated on collagen I–coated silicone membrane for 75 min. Cells were then stretched uniaxially by 8%, and phase-contrast images recorded. (B) Schematic representation of the cell in (A). The area that increased between 1 and 5 min after stretch is shown in red, and the area retracted during the same period is shown in blue. (C) Average increases or decreases in area during the specified interval after stretch were calculated for ends and the sides. Values are mean ± SEM for 80 cells from four independent experiments.
Figure 5.
Figure 5.
Stress fiber alignment by uniaxial stretch. (A) VSM cells were plated on collagen I–coated silicone membrane in uniaxial stretch devices for 75 min. Cells were then stretched by 8% for the time indicated, fixed, and stained with rhodamine-phalloidin. The arrow indicates the direction of stretch. Bar, 20 μm. (B) Cells were scored for whether the main direction of stress fibers was parallel or perpendicular (± 45°) to the direction of stretch. For each time point, 100 cells were scored. Results are means ± SEM from three independent experiments. * and ** denote P < 0.05 or 0.02, respectively, compared with control. (C) VSM cells were transfected with pEGFP-C1-V12Rac. Cells were plated on collagen I–coated silicone membrane in uniaxial stretch devices for 75 min. Cells were then stretched by 8% for the time indicated. Fluorescence images of actin filaments and GFP in the same cells were shown. The arrow indicates the direction of stretch. Bar, 20 μm. (D) The alignment of stress fibers was assessed as before for 100 cells per time point. Results are means ± range from two independent experiments.
Figure 5.
Figure 5.
Stress fiber alignment by uniaxial stretch. (A) VSM cells were plated on collagen I–coated silicone membrane in uniaxial stretch devices for 75 min. Cells were then stretched by 8% for the time indicated, fixed, and stained with rhodamine-phalloidin. The arrow indicates the direction of stretch. Bar, 20 μm. (B) Cells were scored for whether the main direction of stress fibers was parallel or perpendicular (± 45°) to the direction of stretch. For each time point, 100 cells were scored. Results are means ± SEM from three independent experiments. * and ** denote P < 0.05 or 0.02, respectively, compared with control. (C) VSM cells were transfected with pEGFP-C1-V12Rac. Cells were plated on collagen I–coated silicone membrane in uniaxial stretch devices for 75 min. Cells were then stretched by 8% for the time indicated. Fluorescence images of actin filaments and GFP in the same cells were shown. The arrow indicates the direction of stretch. Bar, 20 μm. (D) The alignment of stress fibers was assessed as before for 100 cells per time point. Results are means ± range from two independent experiments.
Figure 6.
Figure 6.
FRET assay for GTP-Rac. VSM cells containing wild-type Rac-GFP and Alexa-PBD protein were analyzed. (A) Corrected FRET images are representative of five independent experiments. A color scale corresponding to FRET intensity on a scale of 0–92 is displayed. Red represents high and blue low FRET signal. (B) Scoring polarization. Cells with positive FRET signals near edges were divided into quadrants as for Fig 4. They were scored to assess whether FRET was predominantly localized to ends, as opposed to remaining unpolarized or localizing to the sides. Values represent the percent of cells where FRET is predominantly at the ends. Approximately 50 cells were scored for each time point. (C) Positive zones at cell edges were outlined and the sum of all pixel intensities within these areas was calculated. The fraction of the total FRET signal within the ends and side quadrants were calculated for each cell and expressed as percent of the total. Values are means ± SEM, for ∼50 cells at each point.
Figure 7.
Figure 7.
Effects of Y-27632 and ML-7 on Rac activity. VSM cells were spread on collagen I–coated coverslip for 16 h, and were incubated with indicated concentrations of Y-27632 (A) or ML-7 (B) for 1 h. Cells were then lysed and GTP-bound Rac and total Rac was detected using the pull-down assay. For densitometric quantification, values are means ± SEM from three experiments. * and ** denote P < 0.02 or <0.05 relative to control, respectively. (C) VSM cells treated with indicated amounts of Y-27632 or ML-7 for 1 h were fixed and stained with rhodamine-phalloidin. Bar, 20 μm.
Figure 8.
Figure 8.
Y-27632 stimulates lamellipodia and disrupts polarity. VSM cells were plated on coverslips coated with collagen I. (A) Time-lapse images of cells at the indicated time points before and after the addition of 10 μM Y-27632 are shown. The total (B) and tail (C) lamellipodia was quantified as described in the Materials and methods. Values are means ± SEM for 10 cells per each condition from four independent experiments. ** denotes P < 0.02.
Fig. 9.
Fig. 9.
ML-7 stimulates lamellipodia and disrupts polarity. VSM cells were plated on coverslips coated with collagen I. Time-lapse images of cells at the indicated time point after the addition of 1 μM ML-7 are shown (A). The total (B) and tail (C) lamellipodia was quantified as described in the Materials and methods. Values are means ± SEM for 10 cells per each condition from four independent experiments. ** denotes a P < 0.02.
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