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.

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.

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.

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].

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.