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Files in this Data Supplement:
Fig. S1. Preferential actin incorporation and zyxin staining at peripheral FAs are not due to experimental artifacts. (A,B) When the mixture of Alexa568-actin and anti-α5-integrin cytoplasmic-domain antibody molecules was introduced into the cells grown on FN, Alexa568-actin was incorporated preferentially at peripheral FAs (A), whereas antibody molecules were associated with both central and peripheral FAs (B). This indicates that the difference in the level of actin incorporated is not due to the difference in the accessibility of artificially introduced molecules to FAs. (C,D) Alexa568-actin (D) was microinjected into a living cell. One minute after the injection, the cell was fixed and stained for α5 integrin (C). White arrows indicate clusters of injected actin associated with peripheral FAs. Red arrows indicate central FAs, with which injected actin was not apparently associated. Asterisks indicate Alexa568-actin leaked from the micropipette and attached on the substratum. (E,F) Cells expressing zyxin-GFP (E) were stained for α5 integrin (F). Zyxin-GFP was accumulated preferentially at peripheral FAs, indicating that the strong immunofluorescence intensity of zyxin at peripheral FAs is not due to artifacts of immunostaining. Bars, 20 μm in (A,B,E,F), and 10 μm in (C,D).
Fig. S2. Difference in molecular composition between peripheral and central FAs. Cells were grown on FN. (A-F) Cells expressing α-actinin−GFP (B,E and green in C,F) were stained for α5 integrin (A and red in C) or zyxin (D and red in F). (G-I) A cell that was double stained for α5β1 integrin (G and red in I) and VASP (H and green in I). (J-L) A cell that was double stained for α5 integrin (J and red in L) and αv integrin (K and green in L). (M-O) A cell that was double stained for αv integrin (M and red in O) and zyxin (N and green in O). Bars, 20 μm.
Fig. S3. Effects of expressing the isolated zyxin LIM region on the accumulation of VASP and αv integrin at FAs. (A-H) Cells transfected with ZYXLIM-GFP (A,E) or GFP (C,G) were stained for VASP (B,D) or αv integrin (F,H). Bar, 20 μm. (I-L) The averaged fluorescence intensity of VASP (I,J) or αv integrin (K,L) at FAs was plotted against that of ZYXLIM-GFP (I,K) or GFP (J,L) for each cell. Values were normalized with respect to the maximum value on each axis. CC, correlation coefficient.
Fig. S4. Effects of expressing the isolated zyxin LIM region on the accumulation of vinculin at FAs. (A-D) Cells transfected with ZYXLIM-GFP (A) or GFP (C) were stained for vinculin (B,D). Bar, 20 μm. (E,F) The averaged fluorescence intensity of vinculin at FAs was plotted against that of ZYXLIM-GFP (E) or GFP (F) for each cell. Values were normalized with respect to the maximum value on each axis. CC, correlation coefficient.
Fig. S5. Characterizing the distribution of palladin in cells. (A-C) A cell that was double stained for zyxin (A and red in C) and palladin (B and green in C). Certain fraction of zyxin clusters was not apparently colocalized with palladin (arrows). (D-F) A cell, to which Alexa568-actin was introduced (D and red in F), was stained for palladin (E and green in F). Certain fraction of incorporated actin clusters was not apparently colocalized with palladin (arrows). (G-J) Cells transfected with ZYXLIM-GFP (G) or GFP (I) were stained for palladin (H,J). Asterisks indicate the cells expressing the exogenous molecules. Bars, 20 μm.
Fig. S6. Immunoblot analyses of cells transfected with GFP-tagged molecules. Cells transfected with zyxin-GFP (lane 1), ZYXLIM-GFP (lane 2) or GFP (lane 3) were lysed and analyzed by immunoblotting for GFP, zyxin, VASP, α-actinin and β-actin. Zyxin was probed with the anti-zyxin mAb 2C10-4A7, which recognized the LIM region. Asterisks indicate uncharacterized bands recognized by 2C10-4A7 but not by the anti-GFP antibody.
Fig. S7. Inhibition of Rho kinase diminishes zyxin accumulation and actin polymerization at peripheral FAs. (A-F) Cells grown on FN were treated with (D-F) or without (A-C) 40 μM Y-27632 for 60 minutes, and then double stained for α5 integrin (A,D and red in C,F) and zyxin (B,E and green in C,F). (G-L) Alexa568-actin (H,K and green in I,L) was introduced into cells treated with (J-L) or without (G-I) 40 μM Y-27632 for 60 minutes. The cells were fixed and stained for α5 integrin (G,J and red in I,L). Note that Alexa568-actin did not locate at the α5-integrin-positive FAs (L). Bar, 20 μm.
Fig. S8. Deformation of the substratum affects the zyxin accumulation at FAs. Cells expressing zyxin-GFP were grown on a polyacrylamide-gel substratum coated with FN. The substratum was locally deformed by manipulating a microneedle inserted into it. The needle was moved towards the cell to reduce mechanical stress caused by cellular contractile force, arrested there, and then moved away from the cell to increase mechanical stress. (A) Fluorescence intensity of zyxin-GFP at FAs (arrows in panel 0' 00'') decreased with deformation of the substratum towards the cell (arrows in panel 7' 24''), and returned to the control level when the deformation ceased (arrows in panel 9' 06''). Elapsed time is indicated in minutes and seconds. Bar, 5 μm. Supplementary material Movie 1 corresponds to these observations. (B) Fluorescence intensities of FA1 and FA2 in A were plotted against time. Fluorescence intensity was averaged for 2 μm from the tip of the FAs.
Movie 1. A transient decrease in the level of zyxin at FAs on deformation of the flexible polyacrylamide-gel substratum. The fluorescence intensity of zyxin-GFP at FAs (B, arrows) decreased with the deformation of the substratum, and returned to the control level when the deformation was ceased. Window B presents the higher magnification of the rectangular area in window A. The images were acquired at 12-second intervals for 567 seconds, and the speed of the movie is eight frames per second.
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