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Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Cleghorn WM, Branch KM, Kook S, Arnette C, Bulus N, Zent R, Kaverina I, Gurevich EV, Weaver AM, Gurevich VV - Mol. Biol. Cell (2014)

Bottom Line: Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells.In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype.Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.

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Related in: MedlinePlus

Arrestin knockout increases number and size of focal adhesions. (A) Focal adhesions were detected in DKO and WT cells after 2 h on FN or PDL with anti-paxillin antibody. (B) Cells were plated on FN for 2 or 24 h, and focal adhesions were visualized by paxillin staining. (C) The focal adhesions in DKO and WT cells plated on FN for 2 or 24 h were quantified and analyzed by two-way ANOVA with genotype and time as main factors. ***p < 0.001 compared with WT, @@@p < 0.001 DKO 24 h compared with DKO 2 h, and ##p < 0.01 WT 24 h compared with WT 2h according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Means ± SD from three experiments (45–67 cells in each). (D) Distribution of focal adhesion size shown by scatter-plot. Focal adhesion size distributions were analyzed by nonparametric Kolmogorov–Smirnov test. Revealed differences: DKO 2 h, p = 0.0023; DKO 24 h, p < 0.0001; WT 24 h, p < 0.0001, as compared with WT 2 h. Data for 600–5000 focal adhesions. (E) Confocal images of DKO and WT cells expressing HA-tagged arrestins and stained for paxillin. Inset, arrestin-2-Δ7 colocalization with paxillin. (F) Focal adhesion number was calculated in arrestin-expressing DKO cells (25–50 cells/condition). Data were analyzed by one-way ANOVA with arrestin type as the main factor. **p < 0.01 DKO-arrestin-2, DKO-arrestin-3, DKO-arrestin-3Δ7 compared with WT; ***p < 0.001 DKO-arrestin-2-Δ7 compared with WT; ###p < 0.001 compared with DKO-GFP according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Scale bar, 10 μm (A, B, and E). (G) Colocalization of paxillin (red) and arrestins or GFP control (green) was determined using Coloc2 function in ImageJ after background correction in cells expressing HA-arrestins and stained for HA and endogenous paxillin, as described in Materials and Methods.
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Figure 3: Arrestin knockout increases number and size of focal adhesions. (A) Focal adhesions were detected in DKO and WT cells after 2 h on FN or PDL with anti-paxillin antibody. (B) Cells were plated on FN for 2 or 24 h, and focal adhesions were visualized by paxillin staining. (C) The focal adhesions in DKO and WT cells plated on FN for 2 or 24 h were quantified and analyzed by two-way ANOVA with genotype and time as main factors. ***p < 0.001 compared with WT, @@@p < 0.001 DKO 24 h compared with DKO 2 h, and ##p < 0.01 WT 24 h compared with WT 2h according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Means ± SD from three experiments (45–67 cells in each). (D) Distribution of focal adhesion size shown by scatter-plot. Focal adhesion size distributions were analyzed by nonparametric Kolmogorov–Smirnov test. Revealed differences: DKO 2 h, p = 0.0023; DKO 24 h, p < 0.0001; WT 24 h, p < 0.0001, as compared with WT 2 h. Data for 600–5000 focal adhesions. (E) Confocal images of DKO and WT cells expressing HA-tagged arrestins and stained for paxillin. Inset, arrestin-2-Δ7 colocalization with paxillin. (F) Focal adhesion number was calculated in arrestin-expressing DKO cells (25–50 cells/condition). Data were analyzed by one-way ANOVA with arrestin type as the main factor. **p < 0.01 DKO-arrestin-2, DKO-arrestin-3, DKO-arrestin-3Δ7 compared with WT; ***p < 0.001 DKO-arrestin-2-Δ7 compared with WT; ###p < 0.001 compared with DKO-GFP according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Scale bar, 10 μm (A, B, and E). (G) Colocalization of paxillin (red) and arrestins or GFP control (green) was determined using Coloc2 function in ImageJ after background correction in cells expressing HA-arrestins and stained for HA and endogenous paxillin, as described in Materials and Methods.

Mentions: FAs are key signaling hubs that recruit many proteins to the site of integrin activation (Sastry and Burridge, 2000; Gieger et al., 2009). Arrestins bind Src, ERK1/2, and JNK3 (Gurevich and Gurevich, 2006b), all of which regulate FAs (Webb et al., 2004; Huveneers and Danen, 2009). Rapid assembly and disassembly of these complexes plays a central role in cell adhesion and migration. On the basis of the decreased migration and increased adhesion of DKO cells, we hypothesized that FA dynamics is likely affected. To test this idea, we stained cells with rhodamine–phalloidin and an anti-paxillin antibody to visualize actin cytoskeleton and FAs, respectively. In WT MEFs, we observed a small number of FAs located primarily at the edges of cells plated on FN and virtually none in cells plated on PDL (Figure 3A). Strikingly, in DKO cells, the number of FAs was dramatically increased. In addition, FAs in DKO MEFs were present not only at the periphery but also throughout the cell on both FN and PDL (Figure 3A). Immunostaining for active phospho-paxillin (P-Y118) and phospho-FAK (P-Y397) also revealed similar differences in FA number and localization between WT and DKO cells (Supplemental Figure S2, A–C). Of importance, in single-knockout cells, the FA pattern was similar to WT on PDL, but cells lacking either arrestin had more FAs on FN, although not as many as DKO MEFs (Supplemental Figure S3, A–C). These data suggest that both arrestin-2 and -3 participate in the regulation of FAs and cell size, and the magnitude of DKO phenotype reflects the absence of both arrestins.


Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Cleghorn WM, Branch KM, Kook S, Arnette C, Bulus N, Zent R, Kaverina I, Gurevich EV, Weaver AM, Gurevich VV - Mol. Biol. Cell (2014)

Arrestin knockout increases number and size of focal adhesions. (A) Focal adhesions were detected in DKO and WT cells after 2 h on FN or PDL with anti-paxillin antibody. (B) Cells were plated on FN for 2 or 24 h, and focal adhesions were visualized by paxillin staining. (C) The focal adhesions in DKO and WT cells plated on FN for 2 or 24 h were quantified and analyzed by two-way ANOVA with genotype and time as main factors. ***p < 0.001 compared with WT, @@@p < 0.001 DKO 24 h compared with DKO 2 h, and ##p < 0.01 WT 24 h compared with WT 2h according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Means ± SD from three experiments (45–67 cells in each). (D) Distribution of focal adhesion size shown by scatter-plot. Focal adhesion size distributions were analyzed by nonparametric Kolmogorov–Smirnov test. Revealed differences: DKO 2 h, p = 0.0023; DKO 24 h, p < 0.0001; WT 24 h, p < 0.0001, as compared with WT 2 h. Data for 600–5000 focal adhesions. (E) Confocal images of DKO and WT cells expressing HA-tagged arrestins and stained for paxillin. Inset, arrestin-2-Δ7 colocalization with paxillin. (F) Focal adhesion number was calculated in arrestin-expressing DKO cells (25–50 cells/condition). Data were analyzed by one-way ANOVA with arrestin type as the main factor. **p < 0.01 DKO-arrestin-2, DKO-arrestin-3, DKO-arrestin-3Δ7 compared with WT; ***p < 0.001 DKO-arrestin-2-Δ7 compared with WT; ###p < 0.001 compared with DKO-GFP according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Scale bar, 10 μm (A, B, and E). (G) Colocalization of paxillin (red) and arrestins or GFP control (green) was determined using Coloc2 function in ImageJ after background correction in cells expressing HA-arrestins and stained for HA and endogenous paxillin, as described in Materials and Methods.
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Figure 3: Arrestin knockout increases number and size of focal adhesions. (A) Focal adhesions were detected in DKO and WT cells after 2 h on FN or PDL with anti-paxillin antibody. (B) Cells were plated on FN for 2 or 24 h, and focal adhesions were visualized by paxillin staining. (C) The focal adhesions in DKO and WT cells plated on FN for 2 or 24 h were quantified and analyzed by two-way ANOVA with genotype and time as main factors. ***p < 0.001 compared with WT, @@@p < 0.001 DKO 24 h compared with DKO 2 h, and ##p < 0.01 WT 24 h compared with WT 2h according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Means ± SD from three experiments (45–67 cells in each). (D) Distribution of focal adhesion size shown by scatter-plot. Focal adhesion size distributions were analyzed by nonparametric Kolmogorov–Smirnov test. Revealed differences: DKO 2 h, p = 0.0023; DKO 24 h, p < 0.0001; WT 24 h, p < 0.0001, as compared with WT 2 h. Data for 600–5000 focal adhesions. (E) Confocal images of DKO and WT cells expressing HA-tagged arrestins and stained for paxillin. Inset, arrestin-2-Δ7 colocalization with paxillin. (F) Focal adhesion number was calculated in arrestin-expressing DKO cells (25–50 cells/condition). Data were analyzed by one-way ANOVA with arrestin type as the main factor. **p < 0.01 DKO-arrestin-2, DKO-arrestin-3, DKO-arrestin-3Δ7 compared with WT; ***p < 0.001 DKO-arrestin-2-Δ7 compared with WT; ###p < 0.001 compared with DKO-GFP according to Bonferroni/Dunn posthoc test with correction for multiple comparisons. Scale bar, 10 μm (A, B, and E). (G) Colocalization of paxillin (red) and arrestins or GFP control (green) was determined using Coloc2 function in ImageJ after background correction in cells expressing HA-arrestins and stained for HA and endogenous paxillin, as described in Materials and Methods.
Mentions: FAs are key signaling hubs that recruit many proteins to the site of integrin activation (Sastry and Burridge, 2000; Gieger et al., 2009). Arrestins bind Src, ERK1/2, and JNK3 (Gurevich and Gurevich, 2006b), all of which regulate FAs (Webb et al., 2004; Huveneers and Danen, 2009). Rapid assembly and disassembly of these complexes plays a central role in cell adhesion and migration. On the basis of the decreased migration and increased adhesion of DKO cells, we hypothesized that FA dynamics is likely affected. To test this idea, we stained cells with rhodamine–phalloidin and an anti-paxillin antibody to visualize actin cytoskeleton and FAs, respectively. In WT MEFs, we observed a small number of FAs located primarily at the edges of cells plated on FN and virtually none in cells plated on PDL (Figure 3A). Strikingly, in DKO cells, the number of FAs was dramatically increased. In addition, FAs in DKO MEFs were present not only at the periphery but also throughout the cell on both FN and PDL (Figure 3A). Immunostaining for active phospho-paxillin (P-Y118) and phospho-FAK (P-Y397) also revealed similar differences in FA number and localization between WT and DKO cells (Supplemental Figure S2, A–C). Of importance, in single-knockout cells, the FA pattern was similar to WT on PDL, but cells lacking either arrestin had more FAs on FN, although not as many as DKO MEFs (Supplemental Figure S3, A–C). These data suggest that both arrestin-2 and -3 participate in the regulation of FAs and cell size, and the magnitude of DKO phenotype reflects the absence of both arrestins.

Bottom Line: Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells.In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype.Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.

Show MeSH
Related in: MedlinePlus