The Abl and Arg non-receptor tyrosine kinases regulate different zones of stress fiber, focal adhesion, and contractile network localization in spreading fibroblasts

Abstract

Directed cell migration requires precise spatial control of F-actin-based leading edge protrusion, focal adhesion (FA) dynamics, and actomyosin contractility. In spreading fibroblasts, the Abl family kinases, Abl and Arg, primarily localize to the nucleus and cell periphery, respectively. Here we provide evidence that Abl and Arg exert different spatial regulation on cellular contractile and adhesive structures. Loss of Abl function reduces FA, F-actin, and phosphorylated myosin light chain (pMLC) staining at the cell periphery, shifting the distribution of these elements more to the center of the cell than in wild-type (WT) and arg^—/— cells. Conversely, loss of Arg function shifts the distribution of these contractile and adhesion elements more to the cell periphery relative to WT and abl^—/— cells. Abl/Arg-dependent phosphorylation of p190RhoGAP (p190) promotes its binding to p120RasGAP (p120) to form a functional RhoA GTPase inhibitory complex, which attenuates RhoA activity and downstream pMLC and FA formation. p120 and p190 colocalize both in the central region and at the cell periphery in WT cells. This p120:p190 colocalization redistributes to a more peripheral distribution in abl^—/— cells and to a more centralized distribution in arg^—/— cells, and these altered distributions can be restored to WT patterns via re-expression of Abl or Arg, respectively. Thus, the altered p120:p190 distribution in the mutant cells correlates inversely with the redistribution in adhesions, actin, and pMLC staining in these cells. Our studies suggest that Abl and Arg exert different spatial regulation on actomyosin contractility and focal adhesions within cells.

Introduction

Cell migration in response to chemotactic and haptotactic cues is essential for proper organismal development [Cantley, 1996; Gilbert, 2003; Ridley et al., 2003]. Dysregulation of cell migration can lead to developmental disorders and diseases, including cancer invasion and metastasis [Friedl et al., 1998; Gilbert, 2003; Hatten, 1999; Schmitz et al., 2000; Simons and Mlodzik, 2008; Watanabe et al., 2005; Yang and Weinberg, 2008]. Directed cell migration is regulated through the integrated function of cell surface receptors, numerous signaling proteins, the cytoskeleton, and cell adhesion systems [Broussard et al., 2008; Elbaum et al., 1999; Friedl and Gilmour, 2009; Guvakova and Surmacz, 1999; Ridley et al., 2003; Simons and Mlodzik, 2008].

Localized control of actomyosin contractility is essential for directed cell migration. The proper spatial regulation of contraction drives cell migration and determines migration direction [Beningo et al., 2006; Even-Ram et al., 2007; Totsukawa et al., 2004]. Non-muscle myosin II isoforms A and B localize to different cellular regions to regulate traction forces and directionality, respectively, during cell spreading and migration. Myosin activation depends on phosphorylation of myosin light chain (MLC) by Rho kinase (ROCK) and myosin light chain kinase (MLCK) [Ikebe, 2008; Kohama et al., 1996; Ridley et al., 2003; Yoneda et al., 2005]. Cell migration guidance cues coordinate myosin activity by stimulating the RhoA (Rho) GTPase to activate ROCK [Ridley et al., 2003]. Focal adhesion (FA) dynamics are also tightly regulated during directed cell migration [Broussard et al., 2008; Gupton and Waterman-Storer, 2006; Ridley et al., 2003; Totsukawa et al., 2000; Totsukawa et al., 2004]. FAs primarily assemble at the leading edge of the cell and disassemble at the trailing edge of the cell as it moves forward [Broussard et al., 2008]. The proper coordination of adhesion dynamics with actomyosin contractility through spatially-localized regulatory elements is critical to polarized cell migration.

The Abl family kinases, Abl and Arg, regulate actomyosin contractility, adhesion dynamics, and cytoskeletal dynamics [Lapetina et al., 2009; Miller et al., 2004; Peacock et al., 2007; Pendergast, 2002; Zandy et al., 2007]. During integrin-mediated fibroblast adhesion and spreading, Arg localizes to the cellular periphery to promote cellular protrusion, relax actomyosin contractility, and promote FA disassembly [Lapetina et al., 2009; Miller et al., 2004; Peacock et al., 2007]. In response to adhesion, Arg phosphorylates p190RhoGAP (p190) on two tyrosine residues, which promotes binding to p120RasGAP (p120). This activated p120:p190 complex localizes to the cell membrane, where it can inhibit active Rho [Bradley et al., 2006]. Several studies suggest that Abl may also regulate contractility and FA dynamics, possibly by targeting similar substrates as Arg [Antoku et al., 2008; Chen et al., 2009; Lewis and Schwartz, 1998; Salgia et al., 1995; Zandy et al., 2007; Zandy and Pendergast, 2008]. In contrast to Arg, Abl shuttles between the nucleus and cytoplasm [Hantschel et al., 2005; Lewis et al., 1996; Taagepera et al., 1998; Woodring et al., 2003; Yoshida and Miki, 2005]. Their distinct localization suggests that Abl and Arg may exert different spatial control over adhesive and contractile structures.

Here we show that Abl and Arg regulate different zones of contractile and adhesive structures in spreading fibroblasts. abl^—/— cells exhibit higher FA and stress fiber (SF) staining and lower p120:p190 colocalization in the center of cells relative to wild type (WT) and arg^—/— cells. In contrast, arg^—/— cells show higher peripheral FA and SF staining and lower peripheral p120:p190 colocalization relative to WT and abl^—/— cells. We find that re-expression of Abl-YFP or Arg-YFP in abl^—/— or arg^—/— cells, respectively, converts the biased central/peripheral localization patterns into more intermediate, WT-like distributions. The peripheral contractile distribution in arg^—/— cells leads to significantly stronger collagen gel contractility compared to WT and abl^—/— cells. Our results indicate that Abl and Arg differentially regulate the spatial distributions of FAs and SFs via the p120:p190-Rho signaling pathway.

Materials and Methods

Constructs

The Arg-YFP expression construct has been previously described [Miller et al., 2004]. The Abl-YFP expression construct was generated using an identical strategy inserting YPF in frame to murine c-Abl type IV via a six amino acid linker.

Cell culture and retroviral infection

The generation of WT and arg^—/— 3T3 fibroblast lines was described previously [Koleske et al., 1998; Miller et al., 2004]. abl^—/— 3T3 fibroblasts were generated in a manner similar to the other fibroblasts. Infection of these lines with retroviruses expressing Arg-YFP/Abl-YFP has been described previously [Miller et al., 2004].

Lysate preparation and immunoblot analysis

Lysates were prepared and analyzed by immunoblot as previously described [Bradley et al., 2006; Peacock et al., 2007].

Immunofluorescence microscopy

Cells were plated on glass coverslips coated with 10 μg/ml FN (Sigma-Aldrich) as indicated in the figure legends and blocked with 1% BSA (Gibco/BRL) for the times indicated in the figure legends. Cells were rinsed before fixation with PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EDTA, 2 mM MgCl₂, pH 6.9) that was pre-warmed to 37°C. Cells were fixed with 4% PFA (pre-warmed to 37°C) for 20 minutes at room temperature and permeabilized with 0.5% or 1% Triton-X100 for 10 minutes. Cells were stained with antibodies to paxillin (Transduction Labs, cat #610051), phospho-Myosin Light Chain 2 (Ser19) (pMLC) (Cell Signaling, cat #3675/3671), p190-A RhoGAP (C59F7 Rabbit mAb Cell Signaling, cat #2860), p120RasGAP (mAb clone B4F8, Upstate), Alexa 594–labeled or Alexa 488-labeled secondary antibodies (Molecular Probes), followed by Alexa 350-phalloidin (Molecular Probes) to visualize the F-actin cytoskeleton. Cells were imaged on a Zeiss Axioskop microscope equipped with differential interference contrast optics and a Nikon TE2000-S microscope.

Adhesion-dependent RhoA and contractility assays

Adhesion-dependent Rho activity levels were measured as previously described [Bradley et al., 2006] using an enzyme-linked immunosorbent assay (ELISA)-based kit (RhoA G-LISA; Cytoskeleton, Denver, CO). Contractility assays were performed as previously described [Arora and McCulloch, 1994; Peacock et al., 2007].

Quantitation of FAs and SFs

Radial intensity distributions were determined using CellProfiler software [Carpenter et al., 2006; Lamprecht et al., 2007; Vokes and Carpenter, 2008]. Images were taken of cells stained for paxillin, F-actin, p120, p190, or pMLC at 40× magnification. TIFF images were converted to 8-bit grayscale. p120 and p190 were analyzed using the modified NIH ImageJ program created by the McMaster Biophotonics group at McMaster University for colocalization, using the Colocalization highlighter function. Colocalization images were then inverted and used in CellProfiler analysis. CellProfiler analysis involved identifying a nuclear center, outlining the nucleus, and using F-actin brightness/contrast enhanced images to outline the cellular periphery. The radial distribution function was then used to obtain 10 equal bins for the distribution of mean fraction intensity at each bin's distance from the nucleus outwards to the cell periphery. At least 20 cells for each line were analyzed and the means for each bin distance were determined.

Statistical analysis

We used two-factor ANOVA with repeated measures and ANOVA (α = 0.05) to compare the multiple groups of data from cells of different genotypes. post hoc Student-Newman-Keuls tests were used to determine the statistical significance of the differences between these groups. This test is the most stringent and appropriate to compare two or more unpaired groups.

Results

Abl and Arg localize to different cellular regions in spreading fibroblasts

Previous work has shown that Abl prominently localizes to the nucleus, while Arg localizes to the cell periphery in several different cell types [Koleske et al., 1998; Taagepera et al., 1998; Van Etten et al., 1989; Wang and Kruh, 1996]. Upon retroviral expression of Arg-YFP in arg^—/— cells (arg^—/— + Arg-YFP) and Abl-YFP in abl^—/— cells (abl^—/— +Abl-YFP), the spatial distribution of Abl-YFP and Arg-YFP reflect these spatial distributions in spreading cells (Fig. 2B and 2C). Abl-YFP localizes to the nucleus, with some residual localization in the cytoplasm (Fig. 2B). In contrast, Arg-YFP localizes solely to the cytoplasm, where it is particularly abundant in a perinuclear region of the cell and at distinct points in the cellular periphery [Miller et al., 2004] (Fig. 2C).

Figure 2. Abl and Arg regulate the spatial localization of focal adhesions and F-actin bundles.

(A-C) WT + YFP (A), abl^—/— + Abl-YFP (B), and arg^—/— + Arg-YFP (C) fibroblasts were plated on 10 μg/mL FN-coated coverslips for one hour, fixed and stained for paxillin and F-actin. Images were obtained with a 40× objective lens. Merged image (merge) shows paxillin (green), F-actin (red), and YFP (blue). Bar = 10 μm.

(D-F) Cell lysates (60 μg) of WT + YFP, abl^—/— + Abl-YFP, and arg^—/— + Arg-YFP fibroblasts were probed for Abl, Arg and Hsp70 (control) (D). Abl-YFP and Arg-YFP levels are 5-fold higher than in WT +YFP cells for both re-expression lines, n = 3 experiments (E-F).

(G-H) Mean fractional paxillin (G) or F-actin (H) intensity distribution (the fraction of total intensity at a given radial distance) for WT + YFP, abl^—/— + Abl-YFP, and arg^—/— + Arg-YFP cells at 0-40% (“central”), 40-80% (“medial”), and 80-100% (“peripheral”) of the radius from the nucleus to the cell periphery. Mean ± S.E. At least 21 cells were analyzed for each genotype. Two-factor ANOVA with repeated measures indicated a significant interaction between FA staining distribution and genotype [F(4,126) = 16.7, p < 0.0001] and between F-actin staining distribution and genotype [F(4,126) = 7.5, p < 0.0001]. *p < 0.05, by post hoc Student-Newman-Keuls.

Loss of Abl or Arg function shifts the subcellular distribution of stress fibers and focal adhesions

Our previous work showed that peripheral FAs and SFs are increased in arg^—/— cells relative to WT cells (Figs. 1A and 1C) [Peacock et al., 2007]. In contrast, abl^—/— cells contain more and larger FAs and F-actin bundles in the center of the cell than WT or arg^—/— cells (Figs. 1A-C). We noted that generally the peripheral focal adhesions in arg^—/— cells are larger, but the peripheral focal adhesions in abl^—/— cells are smaller and more numerous (Figs. 1A-C). We also found that Abl levels in arg^—/— cells and Arg levels in abl^—/— cells are similar to those in control WT cells, indicating that the loss of function of one kinase does not affect levels of the other (Figs. 1D-F). We used CellProfiler image analysis software to quantify the mean intensity for paxillin and F-actin staining at different radial distributions outward from the nucleus toward the cell periphery (Figs. 1G-H) [Carpenter et al., 2006; Lamprecht et al., 2007; Vokes and Carpenter, 2008]. The intensity distributions were broken down into three cell regions: central (comprising the inner 0-40% of the cell's radius from the center of the nucleus out to the cell periphery), medial (comprising the middle 40-80% of the cell's radius from the center of the nucleus out to the cell periphery), and peripheral (comprising the outer 80-100% of the cell's radius from the center of the nucleus out to the cell periphery) (Figs. 1G-H). We utilized these cellular zones, because they corresponded to the general distribution of morphological features within spreading cells. The central (0-40%) region corresponded to the nucleus and peri-nuclear region, the medial (40-80%) region corresponded to the flat, intermediate region in the cell, and the peripheral (80-100%) region corresponded to the more irregular lamellar/lamellipodial region of the cell. Using this analysis, we found that abl^—/— cells had significantly higher central paxillin staining compared to arg^—/— cells and significantly higher central F-actin staining compared to WT and arg^—/— cells (Figs. 1A-C and 1G-H). In contrast, arg^—/— cells show higher peripheral paxillin and F-actin staining relative to both WT and abl^—/— cells, while the center of arg^—/— cells show significantly lower staining intensity for both structures relative to abl^—/— cells (Figs. 1A-C and 1G-H). WT cells form a ring of FAs and SFs at the border of the medial and peripheral domains, exhibiting significantly higher intensity in this region than either knockout line (Figs. 1A-C and 1G-H).

Figure 1. Abl and Arg regulate the localization of focal adhesions and F-actin bundles.

(A) WT, (B) abl^—/—, and (C) arg^—/— fibroblasts were plated on 10 μg/mL FN-coated coverslips, fixed and stained for paxillin and F-actin. Images were obtained with a 40× objective lens. Merged image (merge) shows F-actin (red) and paxillin (green). Bar = 10 μm.

(D-F) Cell lysates (60 μg) of WT, abl^—/—, and arg^—/— fibroblasts were immunoblotted for Abl, Arg, and Hsp70 (control) (D). Abl levels are similar in WT and arg^—/— cells, while Arg levels are similar in WT and abl^—/— cells, n = 4 experiments (E-F).

(G-H) Mean fractional paxillin (G) or F-actin (H) intensity distribution (the fraction of total intensity at a given radial distance) for WT, abl^—/—, and arg^—/— cells at 0-40% (“central”), 40-80% (“medial”), and 80-100% (“peripheral”) of the radius from the nucleus to the cell periphery. Mean ± S.E. At least 23 cells were analyzed for each genotype and the quantification. Two-factor ANOVA with repeated measures indicated a significant interaction between FA staining distribution and genotype [F(4,140) = 7.9, p < 0.0001] and between F-actin staining distribution and genotype [F(4,140) = 16.4, p < 0.0001]. *p < 0.05, by post hoc Student-Newman-Keuls.

We next assessed whether retroviral-mediated re-expression of Abl or Arg in abl^—/— or arg^—/— cells, respectively, could revert the altered localization of FAs and SFs in these cells (Figs. 2A-C). In each case, we found that Abl-YFP and Arg-YFP were re-expressed at 5-fold over normal WT endogenous levels in abl^—/— cells (abl^—/— + Abl-YFP cells) and arg^—/— cells (arg^—/— + Arg-YFP cells), respectively (Figs. 2D-F). As hypothesized, abl^—/— + Abl-YFP cells contained reduced FA and SF intensity in the central/nuclear region and more intensity in the peripheral region compared with abl^—/— cells (compare Figs. 2B and 2G-H with Figs. 1B and 1G-H). In fact, the FA and SF distribution of these cells exhibited a slightly more peripheral bias than WT + YFP cells, consistent with the modest Abl-YFP overexpression. Similarly, arg^—/— + Arg-YFP cells contained reduced FA and SF intensity in the peripheral region and more intensity in the internal region compared with arg^—/— cells (compare Figs. 2C and 2G-H with Figs. 1C and 1G-H). Here, the FA and SF distribution in the arg^—/— + Arg-YFP cells exhibited a slightly more central bias than in WT +YFP cells, again consistent with the 5-fold Arg-YFP overexpression. Combined with the analysis of the abl^—/— and arg^—/— cells reported above, these results suggest that Abl and Arg differentially regulate the spatial distribution of contractile and adhesive structures in spreading fibroblasts, with Abl and Arg attenuating these structures at the cell center and periphery, respectively.

Loss of Abl and Arg coordinately alters spatial localization of p120 and p190 in spreading fibroblasts

The localized assembly of the p120:p190 Rho-inhibitory complex could greatly affect the spatial regulation of both contractility and adhesion. We hypothesized that Abl and Arg may exert their differential effects on FAs and SFs via localization of the p120:p190 complex. In WT cells, p120 and p190 localized to the nuclear region, throughout the cytoplasm, and at the cell periphery, exhibiting a localization pattern intermediate to the two knockout extremes (Figs. 3A-C). We note that p190 is expressed at normal endogenous levels in arg^—/— and abl^—/— cells (Figs. 3D-E). We also note that p120 levels in WT and abl^—/— cells are similar, but that p120 levels are reduced by 20% in arg^—/— cells (Figs. 3D and 3F). abl^—/— cells show reduced p120 and p190 staining intensity in regions close to and including the nucleus but increased intensity for both p120 and p190 at the cellular periphery compared to WT and arg^—/— cells (Figs. 3A-C). In contrast, arg^—/— cells show almost exclusive nuclear/perinuclear staining for p120 and p190 and very little staining at the cell periphery compared to both WT and abl^—/— cells (Figs. 3A-C).

Figure 3. Abl and Arg regulate p120 and p190 colocalization.

(A) WT, (B) abl^—/—, and (C) arg^—/— fibroblasts were plated on 10 μg/mL FN-coated coverslips for one hour, fixed and stained for p120RasGAP, p190RhoGAP, and F-actin. Images were obtained with a 40× objective lens. Merged image (merge) shows p120 (red), p190 (green), and F-actin (blue). NIH ImageJ colocalization image (p120:p190 colocalization) for WT, abl^—/—, and arg^—/— fibroblasts. Pixels that were above computer-determined threshold values for p120 and p190 are coded white, while other pixels are coded black. Thresholds were set to display the maximal differences in colocalization staining between the cell genotypes. Bar = 10 μm.

(D-F) Cell lysates (30 μg) of WT, abl^—/—, and arg^—/— fibroblasts were probed for p120, p190, and Hsp70 (control) (D). p190 levels are similar to WT cells for both knockout lines (E). p120 levels are similar for WT and abl^—/— cells, but p120 levels in arg^—/— cells are 20% reduced relative to WT and abl^—/— cells, n = 4 experiments.

(G) Mean fractional colocalization intensity distribution (the fraction of total intensity at a given radial distance) for WT, abl^—/—, and arg^—/— cells at 0-40% (“central”), 40-80% (“medial”), and 80-100% (“peripheral”) of the radius from the nucleus to the cell periphery. Mean ± S.E. 21 cells were analyzed for each genotype. Two-factor ANOVA with repeated measures indicated a significant interaction between p120:p190 colocalization staining distribution and genotype [F(4,120) = 23.7, p < 0.0001]. *p < 0.05, by post hoc Student-Newman-Keuls.

(H) Rho activity levels were measured in WT and abl^—/— cells at various times after plating on fibronectin. Points represent mean RhoGTP levels ± S.E., n ≥ 3 for each genotype. Two-factor ANOVA does not show a significant effect of genotype on Rho activity levels [F(1,29) = 0.3241, p = 0.5755].

Our lab has previously shown that the localization of p190 to the cell periphery requires Arg function [Bradley et al., 2006]. We wanted to determine if p120 localization depended on Abl and/or Arg expression. abl^—/— + Abl-YFP cells showed a much tighter nuclear/perinuclear distribution of p120 staining compared to the widespread distribution in abl^—/— cells (compare Fig. S1B with Fig. 3B). arg^—/— + Arg-YFP cells showed a broader p120 distribution compared to the narrow nuclear p120 distribution of arg^—/— cells (compare Fig. S1C with Fig. 3C). Quantitative analysis reveals that p120 localization is restored to normal WT distribution in abl^—/— + Abl-YFP and arg^—/— + Arg-YFP cells (Fig. S1D). The combined results from Figures 3 and S1 suggest that Abl and Arg regulate spatial localization of both p120 and p190, which are important regulators of Rho signaling pathways.

While the spatial localization of the individual p120 and p190 proteins is important, it is p120:p190 complex formation that is required for Rho inhibition [Bradley et al., 2006]. Therefore, we used NIH ImageJ software to obtain colocalization data for p120 and p190 in the different cell lines (Fig. 3A-C). We again used CellProfiler image analysis software to quantify the mean intensity for p120 and p190 colocalization at different radial distributions outward from the nucleus toward the cell periphery (Fig. 3G). WT cells showed a more balanced p120:p190 colocalization pattern throughout the cell relative to the two knockout line extremes (Fig. 3G). WT cells had significantly higher p120:p190 colocalization in the central region relative to abl^—/— cells, but significantly less colocalization in this region compared to arg^—/— cells (Fig. 3G). At the periphery, WT cells had significantly more colocalization than arg^—/— cells, but significantly less colocalization than abl^—/— cells (Fig. 3G). abl^—/— cells exhibited half the p120:p190 colocalization intensity in the central cellular region and 3.5 times the amount of p120:p190 colocalization at the cell periphery compared with arg^—/— cells (Fig. 3G). These data suggest a potential role for Abl in promoting p190 and p120 colocalization to a nuclear/perinuclear region, while Arg leads to a broader co-localization pattern within the cytoplasm and at the cell periphery.

We have previously shown that Arg-dependent phosphorylation of p190 promotes its association with p120 and localization to the cell periphery, which is necessary for efficient Rho inhibition. Upon plating on fibronectin, arg^—/— cells fail to recruit p190 to the periphery and do not engage in proper adhesion-dependent Rho inhibition [Bradley et al., 2006]. In order to determine the role that Abl plays in adhesion-dependent Rho inhibition, we performed similar adhesion assays with abl^—/— cells. We found that the Rho activity profile of abl^—/— cells is not significantly different from that of WT cells plated on fibronectin (Fig. 3H). Thus, the redistribution of the p120:p190 complex observed in abl^—/— cells does not alter total Rho activity levels during initial adhesion and spreading on fibronectin.

Loss of Abl and Arg coordinately alters spatial localization of the contractile apparatus in spreading fibroblasts, leading to differences in cellular contractility

To determine whether the contractile apparatus distribution matches the distributions observed for the F-actin networks, we analyzed the pMLC staining distributions in WT, abl^—/—, and arg^—/— cells plated on fibronectin (Figs. 4A-D). As hypothesized, the pMLC distribution matches the F-actin distribution for the three cell types. abl^—/— cells show significant pMLC intensity in the central cellular region relative to arg^—/— cells, while arg^—/— cells exhibit lower, central pMLC intensity and higher, peripheral pMLC intensity relative to WT and abl^—/— cells (Figs. 4A-D). These results suggest that the increased F-actin bundle intensity in the central regions of abl^—/— cells and peripheral regions of arg^—/— cells correspond to increased actomyosin contractility in those regions.

Figure 4. Abl and Arg regulate different zones of the actomyosin contractile apparatus.

(A) WT, (B) abl^—/—, and (C) arg^—/— fibroblasts were plated on 10 μg/mL FN-coated coverslips for one hour, fixed and stained for paxillin and F-actin. Images were obtained with a 40× objective lens. Merged image (merge) shows pMLC (green) and F-actin (red). Bar = 10 μm.

(D) Mean fractional pMLC intensity distribution (the fraction of total intensity at a given radial distance) for WT, abl^—/—, and arg^—/— cells at 0-40% (“central”), 40-80% (“medial”), and 80-100% (“peripheral”) of the radius from the nucleus to the cell periphery. Mean ± S.E. At least 24 cells were analyzed for each genotype and the quantification. Two-factor ANOVA with repeated measures indicated a significant interaction between pMLC staining distribution and genotype [F(4,148) = 22.7, p < 0.0001]. *p < 0.05, by post hoc Student-Newman-Keuls.

(E) Contractility of WT, abl^—/—, and arg^—/— cells collagen/FN gels. The percent decrease in gel diameter is shown for each genotype. The control is a gel with no cells added. Mean ± S.E. 10 experiments for each genotype. ANOVA between data for all genotypes [F(3,36) = 103.1, p < 0.0001]. *p < 0.05, by post hoc Student-Newman-Keuls.

We wanted to determine if pMLC localization depended on Abl and/or Arg expression. abl^—/— + Abl-YFP cells exhibited a much more peripheral distribution of pMLC staining compared to the central distribution in abl^—/— cells (compare Fig. S2B with Fig. 4B). arg^—/— + Arg-YFP cells showed a much wider pMLC distribution throughout the cell compared to the peripheral distribution in arg^—/— cells (compare Fig. S2C with Fig. 4C). Quantitative analysis reveals a close matching of pMLC distributions in abl^—/— + Abl-YFP and arg^—/— + Arg-YFP cells relative to pMLC distribution in WT +YFP cells (Fig. S2D). The combined results from Figures 4 and S2 suggest that Abl and Arg regulate spatial localization of pMLC, which consequently regulates cellular contractility.

To test the functional output of the contractile structure distributions that we observed, we utilized collagen gel contraction assays to measure relative contractility among the three different cell lines [Arora and McCulloch, 1994]. We measured the decrease in size of the collagen/fibronectin gels as they were compressed by cell contraction (Fig. 4E). We found that arg^—/— cells contracted the gels to a greater extent than WT or abl^—/— cells. These results suggest that the peripheral FA and SF network in arg^—/— cells is either more primed or more effective at contraction than the analogous networks in WT or abl^—/— cells.

Discussion

In contrast to the WT cells, both knockouts exhibit significant perturbations of both the adhesion and contractile structures and contractility, which likely impacts their morphology and migration patterns (Figs. 1-4) [Peacock et al., 2007]. We have already observed that the large peripheral SFs of arg^—/— cells exert increased contractile force on these cells during migration, sliding adhesions along the surface and ripping the cell forward whether adhesions de-adhere or not [Peacock et al., 2007]. Peripheral contractile forces have also been shown to be critical for cell migration [Totsukawa et al., 2004], while internal contractile forces are important for traction force generation and ECM remodeling, but not for cell migration [Amano et al., 2000; Gaggioli et al., 2007; Totsukawa et al., 2004]. Arg counteracts these peripheral structures, cellular contraction, and this rapid migration, which potentially allows for careful, cue-guided cell migration (Fig. 1 and 4) [Peacock et al., 2007]. Here we show that Abl attenuates these same adhesive and contractile structures within the cell interior. The loss of Abl function has a more modest impact on whole-cell contractility than the loss of Arg, although there is a slight increase in cellular contractility relative to WT cells (Fig. 4E). Relaxation of central adhesion and contractility by Abl may promote cell spreading and cell migration. Regulation of these central adhesive and contractile structures may also regulate traction force generation and ECM remodeling.

We show here that the unique localization of Abl to the central, nuclear/perinuclear region and Arg to the cytoplasmic/peripheral region has important, direct consequences on the p120:p190 Rho-inhibitory complex (Figure S3). Abl restricts p120:p190 localization to the nuclear/perinuclear region, while Arg activity leads to a widespread distribution of the complex throughout the cell and at the cell periphery (Figure S3). Localization of the p120:p190 complex to particular regions has important effects on the adhesion and contractile structures in those regions. Abl suppresses internal FAs and SFs in favor of peripheral structures, while Arg helps attenuate these structures at the periphery in favor of a more central distribution. The combined effects of Abl and Arg lead to a tight coordination of p120:p190 complex distribution in WT cells, which controls the spatial localization of FAs and SFs. In migrating cells, careful control of these adhesive and contractile zones is critical for proper migration and directed migration towards cues (Figure S3). Our data suggest that Abl and Arg play important and complementary roles in the spatial regulation of these zones.

Abbreviations list

p120

120kD GTPase-activating protein for Ras

p190

190kD GTPase-activating protein for Rho

Abl

Abelson

Arg

Abelson-related gene

Arg-YFP

Arg-yellow fluorescent protein

F-

filamentous

FA

focal adhesion

ArgKI-YFP

kinase-inactive Arg point mutant

MT

microtubule

MLCK

myosin light chain kinase

MLC

regulatory myosin light chain

Rho

RhoA

ROCK

Rho-associated kinase

S.E.

Standard error

SF

stress fiber

pMLC

Ser-19 phosphorylated regulatory myosin light chain

WT

wild type