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First published online 5 August 2008
doi: 10.1242/jcs.030320

Journal of Cell Science 121, 2795-2804 (2008)
Published by The Company of Biologists 2008
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Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner

Hiroaki Hirata1,2, Hitoshi Tatsumi3 and Masahiro Sokabe1,2,3,*

1 Cell Mechanosensing Project, ICORP/SORST, Japan Science and Technology Agency, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan
2 Department of Molecular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, 38 Nishigonaka, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan
3 Department of Physiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya, Aichi 466-8550, Japan


Figure 1
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  		Fig. 1. Actin polymerization and zyxin accumulation at peripheral FAs in cells grown on FN. (A,B) Cells were incubated with Alexa568-actin in 0.003% digitonin for 1 minute. After fixation, the cells were stained for {alpha}5 integrin. (A) {alpha}5 integrin; (B) Alexa568-actin. (C-F) Cells were incubated with Alexa568-actin (D,F) in digitonin supplemented with (E,F) or without (C,D) 0.1 µM cytochalasin D. The cells were fixed and stained for zyxin (C,E). (G,H) A cell that was double stained for {alpha}5 integrin (G) and zyxin (H). (I-K) A cell that was double stained for zyxin (I, red in K) and VASP (J, green in K). (L-N) Cells to which Alexa568-actin was introduced (M, red in N) were stained for zyxin (L, green in N). Scale bar: 20 µm.

 

Figure 2
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  		Fig. 2. Displacement of zyxin from FAs by expressing the isolated LIM region (338-572 aa) of human zyxin. (A-D) Cells were grown on FN and transfected with ZYXLIM-GFP (A) or GFP (C). These cells were stained for endogenous zyxin (B and D, respectively). Endogenous zyxin was detected with an antibody against aa 134-150 of human zyxin. (E-J) Magnified images of cells numbered 1 (E,F), 2 (G,H) and 3 (I,J) in B. ZYXLIM-GFP (E,G,I); endogenous zyxin (F,H,J). Scale bars: 20 µm. (K-M) Intensity profiles of the fluorescence images of ZYXLIM-GFP (light-gray lines) and of endogenous zyxin (black lines) along the yellow lines in F,H and J are shown in K,L and M, respectively. (N,O) The averaged fluorescence intensity of endogenous zyxin at FAs was plotted against that of ZYXLIM-GFP (N) or GFP (O) for each cell. Values were normalized with respect to the maximum value on each axis. CC, correlation coefficient. (P) Correlation coefficients of average fluorescence-intensity plots of the FA protein (zyxin, VASP, {alpha}v integrin or vinculin) vs ZYXLIM-GFP (black bars) or GFP (gray bars). Fluorescence images and intensity plots for VASP, {alpha}v integrin and vinculin are shown in supplementary material Figs S3 and S4. Each bar represents the mean ± s.d. of three independent experiments. *P<0.05; **P<0.005 (t-test).

 

Figure 3
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  		Fig. 3. Actin polymerization at FAs is decreased by displacing zyxin from FAs. Endogenous zyxin was displaced from FAs by expressing the isolated LIM region of human zyxin. (A-F) Alexa568-actin (B,D,F) was introduced into cells transfected with ZYXLIM-GFP (A), GFP (C) or zyxin-GFP (E). Asterisks indicate the cells expressing the exogenous molecules. Large amounts of the exogenous molecules in the cytoplasm were extracted from the cells during the introduction of Alexa568-actin in the presence of digitonin, but ZYXLIM-GFP and GFP in nuclei (# in A,C) were retained. Scale bar: 20 µm. (G,H) The averaged fluorescence intensity of Alexa568-actin at FAs was plotted against that of ZYXLIM-GFP (G) or zyxin-GFP (H) for each cell. Values were normalized with respect to the maximum value on each axis. CC, correlation coefficient. (I) Correlation coefficients of average fluorescence-intensity plots of Alexa568-actin vs ZYXLIM-GFP (black bars) or zyxin-GFP (gray bars). Each bar represents the mean ± s.d. of four independent experiments. *P<0.05 (t-test).

 

Figure 4
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  		Fig. 4. Talin and β1 integrin in a ligand-binding form accumulate at both peripheral and central FAs regardless of treatment with blebbistatin. (A-F) Cells grown on FN were treated with (D-F) or without (A-C) 100 µM blebbistatin for 30 minutes, and then fixed and double stained for {alpha}5 integrin (A,D, green in C,F) and talin (B,E, red in C,F). (G-J) Cells treated with (I,J) or without (G,H) 100 µM blebbistatin for 30 minutes were fixed and double stained with anti-{alpha}5-integrin antibody (G,I) and mAb B44, which recognizes the ligand-induced binding site (LIBS) on β1 integrin (H,J). Scale bars: 20 µm.

 

Figure 5
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  		Fig. 5. Accumulation of {alpha}v integrin alone is not sufficient to induce zyxin accumulation and actin polymerization. (A,B) A cell grown on VN was double stained for {alpha}v integrin (A) and zyxin (B). (C,D) Alexa568-actin was introduced into a cell grown on VN (D) and the cell was stained for {alpha}v integrin (C). Scale bar: 20 µm.

 

Figure 6
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  		Fig. 6. Inhibition of myosin-II activity diminishes zyxin accumulation and actin polymerization at peripheral FAs. Cells were grown on FN. (A-D) Cells were treated with (C,D) or without (A,B) 100 µM blebbistatin for 30 minutes, and were then double stained for {alpha}5 integrin (A,C) and zyxin (B,D). (E-H) Alexa568-actin (F,H) was introduced into cells treated with (G,H) or without (E,F) 100 µM blebbistatin for 30 minutes. The cells were fixed and stained for {alpha}5 integrin (E,G). (I-N) Cells were treated with (L-N) or without (I-K) 100 µM blebbistatin for 30 minutes, and were then double-stained for {alpha}v integrin (I,L, red in K,N) and zyxin (J,M, green in K,N). Scale bars: 20 µm.

 

Figure 7
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  		Fig. 7. Stretching of substrata induces zyxin accumulation at peripheral FAs in cells in which myosin II is inhibited. Cells grown on an FN-coated elastic substratum were treated with 100 µM blebbistatin for 30 minutes and then the substratum was uniaxially stretched (50% for 3 minutes) in the presence of blebbistatin. (A-D) Cells with (C,D) or without (A,B) stretching of the substratum were double stained for {alpha}5 integrin (A,C) and zyxin (B,D). (D) Arrows indicate accumulated zyxin at peripheral FAs. Double-headed arrow indicates the direction of the stretch axis. (C) Red arrows indicate the central {alpha}5-integrin clusters that are not associated with zyxin accumulation. (E-I) Stretched cells were double stained for F-actin (E,G, red in I) and zyxin (F,H, green in I). The boxed area in E is shown at higher magnification in G-I: zyxin was accumulated along F-actin bundles near their ends. (E) Zyxin was accumulated along F-actin bundles that were oriented parallel to the stretch axis (cell 1) but was not found along the bundles perpendicular to the axis (cell 2). Folds perpendicular to the stretch axis (arrowheads in E,F) were generated by relaxing the stretched substrata for observations. Scale bars: 20 µm (A-F); 10 µm (G-I). (J) The fluorescence intensity of zyxin near the end of an F-actin bundle was plotted against the angle of the bundle to the stretch axis. The fluorescence intensities within 6 µm along the F-actin bundle from its tip were measured and normalized with respect to the maximum value. A total of 104 F-actin bundles in 27 cells were plotted. The red line represents the linear fitting. CC, correlation coefficient.

 

Figure 8
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  		Fig. 8. Stretching of substrata induces actin polymerization at peripheral FAs in cells in which myosin II is inhibited. Cells grown on an FN-coated elastic substratum were treated with 100 µM blebbistatin for 30 minutes and then the substratum was uniaxially stretched, as in Fig. 7. (A-D) Alexa568-actin (B,D) was introduced into cells with (C,D) or without (A,B) stretching of the substratum. The cells were stained for {alpha}5 integrin (A,C). (D) Arrows indicate actin incorporated at peripheral FAs. Double-headed arrow indicates the direction of the stretch axis. (C) Red arrows indicate the central {alpha}5-integrin clusters that are not associated with actin incorporation. (E-I) Alexa568-actin was introduced into cells with stretching of the substratum (F,H, green in I). The cells were stained for F-actin (E,G, red in I). The boxed area in E is shown at higher magnification in G-I: actin was incorporated along F-actin bundles near their ends. (E) Actin was incorporated along F-actin bundles oriented parallel to the stretch axis (cell 1) but was not along the bundles oriented with large angles to the axis (cell 2). Folds perpendicular to the stretch axis were generated as in Fig. 7. Scale bars: 20 µm (A-F); 10 µm (G-I). (J) The fluorescence intensity of Alexa568-actin near the end of an F-actin bundle was plotted against the angle of the bundle to the stretch axis. The fluorescence intensities within 6 µm along the F-actin bundle from its tip were measured and normalized with respect to the maximum value. A total of 123 F-actin bundles in 34 cells were plotted. The red line represents the linear fitting. CC, correlation coefficient.

 

Figure 9
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  		Fig. 9. Inhibition of myosin-II activity diminishes the accumulation of ZYXLIM-GFP at peripheral FAs. Cells expressing ZYXLIM-GFP (B,D) were treated with (C,D) or without (A,B) 100 µM blebbistatin for 30 minutes and were then stained for {alpha}5β1 integrin (A,C). Scale bar: 20 µm.

 

Figure 10
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  		Fig. 10. Expressing ZYXLIM-GFP suppresses stretch-induced zyxin accumulation and actin polymerization at FAs. Cells transfected with either ZYXLIM-GFP or GFP were grown on FN-coated elastic substrata and were treated with 100 µM blebbistatin for 30 minutes. The substrata were then uniaxially stretched as in Fig. 7. (A-D) Cells transfected with ZYXLIM-GFP (A) or GFP (C) were stained for zyxin (B,D) after stretching of the substrata. Arrows in A indicate accumulated ZYXLIM-GFP, and arrows in B and D indicate accumulated zyxin. Asterisks indicate the cells expressing the exogenous molecules. (E-H) Alexa568-actin (F,H) was introduced into cells transfected with ZYXLIM-GFP (E) or GFP (G) after the stretching of substrata. Arrows in F and H indicate actin incorporated at peripheral FAs. Stretch-induced accumulation of ZYXLIM-GFP at FAs could not be observed after introducing exogenous actin molecules, probably because the extent of the stretch-induced accumulation is low and the accumulated ZYXLIM-GFP would be extracted from the cells during the introduction of actin molecules in the presence of digitonin. The double-headed arrow (bottom) indicates the direction of the stretch axis. Folds perpendicular to the stretch axis were generated as in Fig. 7. Scale bar: 20 µm.

 

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