Mechanical forces facilitate actin polymerization at focal adhesions in a zyxin-dependent manner.

We examined the effects of mechanical forces on actin polymerization at focal adhesions (FAs). Actin polymerization at FAs was assessed by introducing fluorescence-labeled actin molecules into permeabilized fibroblasts cultured on fibronectin. When cell contractility was inhibited by the myosin-II inhibitor blebbistatin, actin polymerization at FAs was diminished, whereas α5β1 integrin remained accumulated at FAs. This suggests that actin polymerization at FAs depends on mechanical forces. To examine the action of mechanical forces more directly, the blebbistatin-treated cells were subjected to a sustained uniaxial stretch, which induced actin polymerization at FAs. These results demonstrate the novel role of mechanical forces in inducing actin polymerization at FAs. To reveal the molecular mechanism underlying the force-induced actin polymerization at FAs, we examined the distribution of zyxin, a postulated actin-regulatory protein. Actin-polymerizing activity was strong at zyxin-rich FAs. Accumulation of zyxin at FAs was diminished by blebbistatin, whereas uniaxial stretching of the cells induced zyxin accumulation. Displacing endogenous zyxin from FAs by expressing the FA-targeting region of zyxin decreased the force-induced actin polymerization at FAs. These results suggest that zyxin is involved in mechanical-force-dependent facilitation of actin polymerization at FAs.


Introduction
α-actinin and Ena/VASP, serving as a scaffold at FAs (Beckerle, 1997). Recently, several studies have shown that the dynamics of zyxin at FAs are affected by mechanical forces acting on FAs. Inhibition of actomyosin-based cell contractility induces the dislocation of zyxin from FAs (Rottner et al., 2001). Cyclic stretching and relaxing of cells results in the translocation of zyxin from FAs to nuclei (Cattaruzza et al., 2004) and actin stress fibers (Yoshigi et al., 2005). Lele et al. have demonstrated that the unbinding rate constant of zyxin at FAs increases with decreasing mechanical load on FAs (Lele et al., 2006). These results lead to the hypothesis that mechanical forces acting on FAs facilitate the recruitment of zyxin and Ena/VASP, inducing actin polymerization at FAs. However, it remains unknown whether zyxin is, in fact, involved in the regulation of actin polymerization at FAs.
In the present study, we examine the effect of mechanical forces on actin polymerization at FAs, and demonstrate that mechanical forces facilitate actin polymerization at FAs in a zyxin-dependent manner.

Actin polymerization at zyxin-rich FAs
Human skin fibroblasts grown on fibronectin (FN) developed many FAs, which contained α 5 integrin (Fig. 1A). To assess actin polymerization at FAs, Alexa-Fluor-568-conjugated actin (Alexa568-actin) was applied to the cells in the presence of digitonin. Alexa568-actin was incorporated at FAs located in the peripheral region, with much less being incorporated in the central region of cells (Fig. 1B). The difference in the amount of actin that was incorporated in different regions did not seem to arise from the difference in accessibility of artificially introduced molecules to FAs depending on their location; when the mixture of Alexa568actin (ca. 42 kDa) and anti-α 5 -integrin cytoplasmic-domain antibody (ca. 150 kDa) was applied, the antibody was associated with FAs in both peripheral and central regions, whereas Alexa568-actin was incorporated preferentially at peripheral FAs (supplementary material Fig. S1). Alexa568-actin that was microinjected into living cells was also incorporated preferentially at peripheral FAs (supplementary material Fig. S1). The incorporation of exogenous actin at FAs in permeabilized cells was markedly reduced in the presence of 0.1 μM cytochalasin D, a potent inhibitor of actin polymerization at barbed ends of actin filaments , indicating that the incorporation is primarily caused by actin polymerization from pre-existing free barbed ends, as shown previously (Chan et al., 1998).
The difference in the level of actin polymerization between peripheral and central FAs might depend on the molecular composition of the FAs. We examined the distribution of the FA protein zyxin, which has a capability to induce actin assembly in an Ena/VASP-dependent manner . Zyxin was accumulated at peripheral FAs, but less so at central FAs (Fig. 1G,H). VASP was colocalized with zyxin to peripheral FAs (Fig. 1I-K and supplementary material Fig. S2). Sites of zyxin accumulation corresponded to the sites at which Alexa568-actin was incorporated ( Fig. 1L-N). These results indicate that actin-polymerizing activity is strong at zyxin-rich FAs.

Accumulation of zyxin is involved in actin polymerization at FAs
We examined whether zyxin is involved in the actin polymerization at peripheral FAs. For this purpose, the green fluorescent protein (GFP)-tagged LIM region of human zyxin (ZYX LIM -GFP) was expressed, because this region is responsible for recruiting zyxin to FAs and expression of the isolated LIM region causes the displacement of endogenous zyxin from FAs (Nix et al., 2001). When expressed in human skin fibroblasts, ZYX LIM -GFP localized to peripheral FAs ( Fig. 2A,G,I). Less endogenous zyxin was accumulated at FAs in cells expressing ZYX LIM -GFP at a higher level (Fig. 2B,E-M). To quantitatively analyze the effect of the expression of ZYX LIM -GFP on the accumulation of endogenous zyxin at FAs, the fluorescence intensity of endogenous zyxin at FAs was averaged and plotted against that of ZYX LIM -GFP for each cell ( Fig. 2N; see Materials and Methods). The fluorescence intensity of endogenous zyxin at FAs was negatively correlated with that of ZYX LIM -GFP ( Fig. 2N,P). GFP alone neither localized to FAs ( (G,H) A cell that was double stained for α 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. (Nix et al., 2001). By contrast, the accumulation of α v integrin at FAs was not affected by the expression of ZYX LIM -GFP ( Fig. 2P and supplementary material Fig. S3), indicating that FAs themselves were not disassembled in cells expressing ZYX LIM -GFP. Accumulation of vinculin and palladin, FA proteins that, similar to zyxin, are capable of binding to VASP and α-actinin, was not affected by the expression of ZYX LIM -GFP (Fig. 2P and supplementary material Figs S4 and S5). Expression levels of the endogenous FA proteins zyxin, VASP and α-actinin in cells transfected with ZYX LIM -GFP did not significantly differ from those in cells transfected with GFP or zyxin-GFP (supplementary material Fig. S6).
We assessed actin incorporation at FAs in ZYX LIM -GFP-expressing cells. The incorporation of Alexa568actin at peripheral FAs was decreased in cells expressing ZYX LIM -GFP (Fig. 3A,B). The average fluorescence intensity of Alexa568-actin at FAs was negatively correlated with that of ZYX LIM -GFP ( Fig.  3G,I). Expression of GFP alone did not affect the incorporation of Alexa568-actin at FAs (Fig. 3C,D). A positive correlation between the expression of zyxin-GFP and actin incorporation at peripheral FAs was observed in three out of four cases (Fig. 3E,F,H,I). All these results suggest that zyxin is involved in actin polymerization at FAs.

Mechanical forces induce zyxin accumulation at FAs and facilitate local actin polymerization
Both peripheral and central FAs contained the α 5 β 1integrin heterodimer (supplementary material Fig.  S2), talin ( Fig. 4A-C) and β 1 integrin in a ligandbinding form (Fig. 4G,H), indicating that both sets of FAs were functional. However, zyxin was accumulated and actin was polymerized preferentially at peripheral FAs ( Fig. 1). Two major classes of FAs are found in fibroblasts: the α 5 β 1integrin-dominated fibrillar adhesion and the α v β 3integrin-dominated focal contact (Singer et al., 1988;Zamir et al., 1999;Katz et al., 2000). Because α v integrin was preferentially accumulated at peripheral FAs (supplementary material Fig. S2) (Katz et al., 2000) and colocalized with zyxin (supplementary material Fig. S2), zyxin seems to be a constituent of α v β 3 -integrin-mediated adhesion structures (i.e. focal contacts). Consistently, α-actinin, a constituent of focal contacts (Katz et al., 2000), was also colocalized with zyxin at peripheral FAs (supplementary material Fig. S2). To examine the role of α v β 3 integrin in zyxin accumulation and actin polymerization, cells were grown on vitronectin (VN), a ligand for α v β 3 integrin. FAs containing α v integrin were distributed in both peripheral and central regions of cells grown on VN (Fig.  5A,C). However, zyxin was accumulated (Fig. 5B) and actin was incorporated (Fig. 5D) preferentially at peripheral FAs in these cells, indicating that the accumulated α v integrin alone is not sufficient to induce zyxin accumulation and actin polymerization.
The cytoplasmic molecular composition of FAs is affected not only by integrin species but also by mechanical loads at FAs (Katz et al., 2000). To assess the effect of mechanical forces on zyxin accumulation and actin polymerization at FAs, cells were treated with a myosin-II-specific inhibitor, blebbistatin (Straight et al., 2003), to inhibit actomyosin-based cellular contractile forces. Blebbistatin treatment diminished both zyxin accumulation ( Fig.  6A-D) and actin polymerization ( Fig. 6E-H) at FAs; however, α 5 integrin was accumulated (Fig. 6C,G) with talin ( Fig. 4D-F) and β 1 integrin in a ligand-binding form (Fig. 4I,J). In these cells, a small fraction of α v -integrin clusters remained, but these clusters did not contain zyxin ( Fig. 6I-N), indicating again that accumulation of α v integrin is not enough to induce zyxin accumulation. Because blebbistatin might affect myosin-II-independent processes (Shu et al., 2005), we also examined the effect of inhibiting Rho kinase, which regulates myosin-II activity (Fukata et al., 2001), with the Rho-kinase-specific inhibitor Y-27632 (Uehata et al., 1997), and obtained similar results (supplementary material Fig. S7). Therefore, zyxin accumulation and actin polymerization at peripheral FAs presumably depend on actomyosin-based mechanical forces.
To examine the action of mechanical forces on zyxin accumulation and actin polymerization more directly, mechanical forces were applied to blebbistatin-treated cells by stretching the elastic silicone substratum to which the cells adhered. Uniaxial stretching of the substratum (50% stretch for 3 minutes) induced both zyxin accumulation ( Fig. 8J). All these results strongly suggest that mechanical forces are responsible for zyxin accumulation and actin polymerization at FAs.
The role of mechanical forces in zyxin accumulation was also examined without pharmacological treatments. Cells expressing zyxin-GFP were grown on a flexible polyacrylamide substratum coated with FN. When the substratum was locally deformed towards the cell, the fluorescence intensity of zyxin-GFP at FAs decreased (supplementary material Fig. S8 and Movie 1). When the substratum was stretched again, the fluorescence intensity recovered (supplementary material Fig. S8 and Movie 1). These results confirm that mechanical forces regulate zyxin accumulation at FAs.
Zyxin was shown to be involved in actin polymerization at FAs (Fig. 3). We examined the role of zyxin in the stretch-induced actin polymerization observed in blebbistatin-treated cells. When cells expressing ZYX LIM -GFP were treated with blebbistatin, ZYX LIM -GFP was dislocated from peripheral FAs (Fig. 9). When the Journal of Cell Science 121 (17)   substratum was stretched, ZYX LIM -GFP was again accumulated at peripheral FAs in these cells (Fig.  10A), suggesting that the localization of the isolated LIM region to FAs is also force dependent. Stretchinduced accumulation of endogenous zyxin at FAs was decreased by expressing ZYX LIM -GFP (Fig. 10A,B) but not GFP (Fig. 10C,D). Thus, stretch-induced zyxin accumulation was inhibited by ZYX LIM -GFP. Inhibition of zyxin accumulation by expressing ZYX LIM -GFP suppressed stretch-induced actin polymerization at peripheral FAs (Fig. 10E,F). Expression of GFP alone had no effect on stretchinduced actin polymerization (Fig. 10G,H). These results suggest that mechanical force induces the accumulation of zyxin at peripheral FAs, leading to actin polymerization in blebbistatin-treated cells.

Discussion
It has repeatedly been shown that mechanical forces play a crucial role in the molecular assembly of FAs. In the current study, we have demonstrated a novel aspect of mechanical forces: that they facilitate actin polymerization at FAs. Furthermore, we have revealed that the force-dependent accumulation of zyxin at FAs is crucial for this process.

Mechanical-force-induced accumulation of zyxin at FAs
Recently, it has been revealed that mechanical cues affect the distribution of zyxin to FAs. Zyxin dissociated from FAs when the mechanical load on the FAs was reduced by inhibiting the actomyosin interaction, by ablating individual stress fibers with a focused laser or by softening the substratum (Rottner et al., 2001;Lele et al., 2006) (this study). These results suggest that mechanical loads on FAs are required for the recruitment of zyxin to FAs. In the present study, we showed, by stretching the elastic silicone substratum and by deforming locally the polyacrylamide gel substratum, that externally applied mechanical forces induce zyxin accumulation at FAs, thus demonstrating the role of mechanical forces in localizing zyxin to FAs. It has previously been reported that cyclic stretching and relaxing the substrata resulted in the dislocation of zyxin from FAs (Cattaruzza et al., 2004;Yoshigi et al., 2005). Combined with our results, this suggests that sustained mechanical loads are necessary for zyxin accumulation and that a transient decrease in the mechanical force causes dislocation of zyxin from FAs.
We found that zyxin is accumulated preferentially at peripheral FAs. On FN, α v β 3 integrin was preferentially accumulated at peripheral FAs, suggesting that α v β 3 integrin is involved in recruiting zyxin to FAs. However, accumulation of α v β 3 integrin is not sufficient to induce zyxin accumulation, because α v -integrin clusters in central regions of cells on VN (Fig. 5) or in cells treated with blebbistatin ( Fig. 6) were not associated with zyxin. Larger traction forces are presumably exerted at peripheral FAs than at central FAs (Tan et al., 2003). Therefore, the large mechanical loads on peripheral FAs might facilitate the accumulation of zyxin.
Uniaxial stretching of the substratum induced zyxin accumulation and actin polymerization in blebbistatin-treated cells. When F-actin bundles were oriented at larger angles to the stretch axis, zyxin was less accumulated and actin was less polymerized upon the stretch.
This suggests that zyxin accumulation is dependent on the amplitude of the stress in the actin-cytoskeleton-FA complexes, because the stress in the actin bundles that are oriented at larger angles to the stretch axis should be smaller. This could be a part of the mechanism in which FAs and the actin cytoskeleton sense and respond to the direction of tension.
The isolated LIM region of zyxin accumulated at FAs in a forcedependent manner, and expression of the LIM region inhibited the force-induced accumulation of endogenous zyxin at FAs. These results indicate that the LIM region of zyxin is crucial for the forcedependent recruitment of zyxin to FAs. The LIM region of zyxin interacts with the FA-associated adapter protein p130Cas (Yi et al., 2002). However, zyxin is distributed to FAs in p130Cas-deficient cells (Yi et al., 2002), indicating that the interaction between zyxin and p130Cas is not necessary for the force-induced recruitment of zyxin to FAs. The zyxin LIM region also binds to cysteine-rich protein (CRP) (Schmeichel and Beckerle, 1994). Because CRP is located at FAs (Sadler et al., 1992), the interaction of zyxin with CRP might contribute to the localization of zyxin. The N-terminal domain of zyxin binds to α-actinin, and this interaction is involved in the recruitment of zyxin to FAs (Drees et al., 1999;Reinhard et al., 1999). However, it is not clear whether the zyxin LIM region affects the interaction between zyxin and α-actinin. CRP and αactinin could be potential candidates for the molecule responsible for the force-induced recruitment of zyxin to FAs. Future study is needed to resolve this issue.
Little zyxin exists at focal complexes (FXs) -small and shortlived adhesive structures formed just behind the leading edge of a cell (Zaidel-Bar et al., 2003). FXs are formed independently of Rhokinase-mediated myosin activation (Rottner et al., 1999). When Rho kinase is activated, FXs mature into FAs (Rottner et al., 1999;Totsukawa et al., 2004). This maturation accompanies recruitment of zyxin to the adhesion sites (Zaidel- Bar et al., 2003). These results suggest that the myosin activity mediated by Rho kinase affects zyxin localization to adhesion sites, supporting the notion of the force dependence of zyxin accumulation.

Role of zyxin in actin polymerization at FAs
Zyxin is related in sequence and structure to the bacterial protein ActA (Golsteyn et al., 1997), which is a bacterial factor required for actin polymerization on the bacterial surface within the cytoplasm of the infected host (Domann et al., 1992;Kocks et al., 1992). Therefore, it has been implied that zyxin contributes to the regulation of actin polymerization in mammalian cells. Consistent with this, zyxin mutants with a plasma-membrane-or mitochondria-targeting sequence induced a local actin assembly (Golsteyn et al., 1997;Fradelizi et al., 2001). However, the role of endogenous zyxin in the regulation of actin polymerization remained to be elucidated. In this study, we showed that actinpolymerizing activity is strong at zyxin-rich FAs, and that displacing zyxin from FAs impaired the polymerization, suggesting that accumulation of endogenous zyxin at FAs is crucial for actin polymerization at these sites.
Similar to zyxin, two FA proteins -vinculin and palladin -are capable of binding to Ena/VASP and α-actinin (Critchley 2000;Otey et al., 2005). Displacing zyxin from FAs or zyxin knockout causes dislocation of Ena/VASP from FAs without affecting accumulation of vinculin (Drees et al., 1999;Drees et al., 2000;Nix et al., 2001;Hoffman et al., 2006) (this study) and palladin (this study). These results suggest that vinculin or palladin cannot be an alternative to zyxin in Ena/VASP recruitment and actin incorporation at FAs. The accumulation of VASP at FAs was also dependent on the actomyosin interaction (our unpublished result). Ena/VASP would enhance actin polymerization through its anti-capping effect on the barbed end of F-actin and/or through its interaction with the monomeric actinbinding protein profilin (Krause et al., 2003). Thus, the forceinduced accumulation of zyxin would recruit Ena/VASP to FAs and facilitate the local actin polymerization.
FA is an active site for actin polymerization (Glacy, 1983;Turnacioglu et al., 1998;Fradelizi et al., 2001). The actin regulatory protein mDia1 (DIAPH1) is involved in actin polymerization at FAs (Butler et al., 2006;Hotulainen and Lappalainen, 2006). However, some polymerization is still observed at FAs in mDia1-depleted cells, suggesting that mDia1-independent mechanisms also exist (Hotulainen and Lappalainen, 2006). In the present study, we revealed that the accumulation of zyxin enhances actin polymerization at FAs. It is likely that both zyxin-Ena/VASPdependent and mDia-dependent processes are involved in actin polymerization at FAs. It should be noted that the actin-polymerizing activity at FAs almost disappeared when the cells were treated with blebbistatin or Y-27632. This suggests that, irrespective of the regulatory pathway, actin-polymerizing activity at FAs depends on actomyosin-based mechanical forces. A theoretical study predicted that mDia-induced actin polymerization would be facilitated by mechanical forces (Kozlov and Bershadsky, 2004). However, the exact roles of forces in mDia-dependent actin polymerization at FAs have not been examined experimentally. To date, the zyxindependent process presented here is the only experimentally evidenced force-dependent regulatory mechanism of actin polymerization at FAs.
Mechanical forces acting on FAs induce enlargement of FAs Riveline et al., 2001;Wang et al., 2001;Galbraith et al., 2002;Kaverina et al., 2002). However, it is unlikely that force-dependent localization of zyxin to FAs plays a crucial role in the size control of FAs, because the size of accumulations of α v integrin and vinculin was not apparently changed in cells expressing the isolated LIM region of zyxin (supplementary material Figs S3 and S4), and the morphology of FAs is not altered in zyxin-Journal of Cell Science 121 (17) 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. null fibroblasts (Hoffman et al., 2006). The molecular mechanism regulating the size of FAs in response to forces is still unknown.
Mechanical-force-induced actin polymerization at FAs increases the local amount of F-actin at FAs. Because FAs contain many actinbinding proteins , changes in the amount of Factin at FAs might affect the structure and function of FAs. Consistent with this idea, an FA protein with actin-binding capability, α-actinin, accumulates preferentially at peripheral FAs (Katz et al., 2000), which are the active sites for actin polymerization. α-actinin exhibits correlated motion with F-actin within FAs (Brown et al., 2006;Hu et al., 2007), suggesting its association with F-actin. The accumulation of F-actin at FAs would strengthen the integrin-actin-cytoskeleton linkage (Glogauer et al., 1998). The possible regulation of FAs through F-actin accumulation is based on a balance between actin polymerization at FAs (this study) and retrograde flux of F-actin from FAs (Guo and Wang, 2007;Endlich et al., 2007). We have demonstrated here that mechanical forces induce actin polymerization at FAs and that the polymerization is dependent on the force-induced accumulation of zyxin at FAs. Further studies are needed to reveal the underlying mechanisms of this process, including which molecule is the mechanosensor, how it senses mechanical forces, how the sensed forces are used for zyxin accumulation, and how zyxin acts on other molecules and facilitates actin polymerization at FAs.

Plasmid constructs and transfection
The GFP-tagged human zyxin construct was kindly provided by Jürgen Wehland and Klemens Rottner (German Research Centre for Biotechnology, Germany). Using this construct as a template, a fragment of zyxin encoding the LIM region (amino acids 338-572) was amplified by polymerase chain reaction with primers 5Ј-ATCAAGAATTCACCATGGAGAACCAAAACCAGGTGCGCTCCCC-3Ј and 5Ј-TATCTGGATCCTTGGTCTGGGCTCTAGCAGTGTGGCAC-3Ј. The zyxin fragment was subcloned into EcoRI-BamHI restriction sites of the pEGFP-N1 vector (Clontech Laboratories, Mountain View, CA). The construct was verified by DNA sequencing. The GFP-tagged α-actinin construct was kindly provided by Carol A. Otey (University of North Carolina at Chapel Hill, NC).
Hs-68 cells on glass coverslips, silicone chambers or polyacrylamide substrata were transiently transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's instruction. Cells were analyzed 40-48 hours after transfection.
The immunofluorescence intensity of zyxin was higher at peripheral FAs than central FAs (see Results); the heterogeneity of fluorescence intensity was not due to an artifact of immunostaining, because zyxin-GFP also accumulated preferentially at peripheral FAs (supplementary material Fig. S1).

Stretching-cell assay
Cells grown on an elastic silicone chamber were treated with 100 μM blebbistatin for 30 minutes and then the chamber was uniaxially stretched to 150% of its original length (50% stretch) for 3 minutes in the presence of blebbistatin. The stretched cells were used for the actin-polymerization assay and/or immunofluorescence staining.

Polyacrylamide substratum
Polyacrylamide-gel substrata coated with FN were prepared as described previously (Dembo and Wang, 1999); concentrations of acrylamide and bisacrylamide were 5% and 0.1%, respectively. The polyacrylamide substratum was deformed with a glass microneedle. The microneedles were prepared from glass capillaries with a diameter of 1 mm (G-1, Narishige, Tokyo) using a Flaming/Brown micropipette puller (P-97, Sutter Instrument, Novato, CA). The tip of the microneedle was removed before use to increase bending rigidity. The microneedle was inserted into the polyacrylamide substratum and displaced with a micromanipulator (MC-35A, Narishige). Experiments were carried out in the standard external solution (140 mM NaCl, 5 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgCl 2 , 10 mM glucose and 10 mM HEPES, pH 7.4).

Fluorescence microscopy and image analysis
The cells were observed with an epifluorescence microscope (IX70, Olympus, Tokyo) equipped with an oil-immersion objective (NA 1.40, 100ϫ; PlanApo, Olympus) and a charge-coupled device camera (Micromax, Princeton Instruments, Trenton, NJ). Acquired images were analyzed off line with the public domain Object-Image program (version 2.08).The average fluorescence intensity of a particular FA protein or incorporated fluorescent actin at FAs in a cell was calculated as follows: the five FAs with the highest immunofluorescent intensity (in immunofluorescence experiments) or EGFP intensity (in actin-polymerization experiments) were chosen in each cell for analyses. The mean fluorescence intensity of the FA protein or incorporated actin at the five FAs was calculated in each cell. The correlation analysis of fluorescence intensities of two different proteins at FAs was carried out by plotting the mean value of one protein against the mean value of the other. When the seven brightest FAs were chosen in each cell and used for the analyses, we obtained essentially the same results (data not Journal of Cell Science 121 (17) Arrows in F and H indicate actin incorporated at peripheral FAs. Stretchinduced accumulation of ZYX LIM -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 ZYX LIM -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.
shown). We could not choose the ten brightest FAs for analyses, because ten typical FAs were not always found in single cells.