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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

Arrestins regulate focal adhesion dynamics. (A) DKO and WT cells expressing GFP-paxillin were viewed with DeltaVision Core microscope, and images were captured at 1-min intervals. Representative images at 0, 10, 20, 30, 60, and 90 min. Arrowheads indicate representative focal adhesions. Scale bar, 10 μm. FA lifetimes were determined by counting the number of sequential frames where individual FA (GFP-paxillin) is visible. (B) Histogram distributions of FA lifetimes in 20-min intervals. Data from two or three experiments (150 FAs in 15 cells for each cell type). All distributions are significantly different from each other (p < 0.0001), except for DKO-Arr2 and DKO-Arr3 FA lifetimes, according to nonparametric Kolmogorov–Smirnov test. (C) The distribution of FA lifetimes in indicated cells. *p < 0.001 to DKO; bp < 0.01, #p < 0.001 to WT according to Kruskal–Wallis nonparametric test (H = 170.637, p < 0.0001; H corrected for ties = 170.679, tied p < 0.001). The data were also analyzed with Mann–Whitney test for means (pairwise comparisons: WT–DKO p < 0.0001; DKO–DKO-Arr2 p < 0.0001; DKO–DKO-Arr3 p < 0.0001). (D) Expression of arrestins and tagged paxillin determined by Western blot with bovine arrestin-2 and arrestin-3 (0.1 ng/lane) as standards (Std).
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Figure 4: Arrestins regulate focal adhesion dynamics. (A) DKO and WT cells expressing GFP-paxillin were viewed with DeltaVision Core microscope, and images were captured at 1-min intervals. Representative images at 0, 10, 20, 30, 60, and 90 min. Arrowheads indicate representative focal adhesions. Scale bar, 10 μm. FA lifetimes were determined by counting the number of sequential frames where individual FA (GFP-paxillin) is visible. (B) Histogram distributions of FA lifetimes in 20-min intervals. Data from two or three experiments (150 FAs in 15 cells for each cell type). All distributions are significantly different from each other (p < 0.0001), except for DKO-Arr2 and DKO-Arr3 FA lifetimes, according to nonparametric Kolmogorov–Smirnov test. (C) The distribution of FA lifetimes in indicated cells. *p < 0.001 to DKO; bp < 0.01, #p < 0.001 to WT according to Kruskal–Wallis nonparametric test (H = 170.637, p < 0.0001; H corrected for ties = 170.679, tied p < 0.001). The data were also analyzed with Mann–Whitney test for means (pairwise comparisons: WT–DKO p < 0.0001; DKO–DKO-Arr2 p < 0.0001; DKO–DKO-Arr3 p < 0.0001). (D) Expression of arrestins and tagged paxillin determined by Western blot with bovine arrestin-2 and arrestin-3 (0.1 ng/lane) as standards (Std).

Mentions: The accumulation and enlargement of FAs in DKO cells (Figure 3, C and D) suggest that the rate of FA disassembly might be reduced. To test this idea, we expressed GFP-paxillin in DKO and WT cells (Figure 4) and measured FA lifetimes using live-cell imaging (Figure 4, A and B). Individual FAs at the leading edge of the cell were tracked from formation to disassembly (representative FAs are indicated by red arrows in Figure 4A). All FAs in WT cells formed and disassembled within 20–40 min (Figure 4B), with a median lifetime of 23 min (Figure 4C). In contrast, lifetimes of FAs in DKO cells showed much broader distribution, with median ∼59 min. Of note, some FAs in DKO cells persisted >3 h (Figure 4B). DKO cells also demonstrated a defect in leading edge formation and loss of polarity (Supplemental Movies S1–S4). To test whether the defect in FA disassembly is a result of the lack of arrestins, we transfected red mCherry-paxillin into cells coexpressing GFP with arrestins (Figure 4D). Live-cell imaging revealed a shift in FA lifetimes toward WT (Figure 4, B and C), with median values reduced to 42 and 40.5 min in cells expressing arrestin-2 and arrestin-3, respectively (Figure 4C). Thus arrestins regulate FA turnover, and normal dynamics requires the presence of both subtypes.


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)

Arrestins regulate focal adhesion dynamics. (A) DKO and WT cells expressing GFP-paxillin were viewed with DeltaVision Core microscope, and images were captured at 1-min intervals. Representative images at 0, 10, 20, 30, 60, and 90 min. Arrowheads indicate representative focal adhesions. Scale bar, 10 μm. FA lifetimes were determined by counting the number of sequential frames where individual FA (GFP-paxillin) is visible. (B) Histogram distributions of FA lifetimes in 20-min intervals. Data from two or three experiments (150 FAs in 15 cells for each cell type). All distributions are significantly different from each other (p < 0.0001), except for DKO-Arr2 and DKO-Arr3 FA lifetimes, according to nonparametric Kolmogorov–Smirnov test. (C) The distribution of FA lifetimes in indicated cells. *p < 0.001 to DKO; bp < 0.01, #p < 0.001 to WT according to Kruskal–Wallis nonparametric test (H = 170.637, p < 0.0001; H corrected for ties = 170.679, tied p < 0.001). The data were also analyzed with Mann–Whitney test for means (pairwise comparisons: WT–DKO p < 0.0001; DKO–DKO-Arr2 p < 0.0001; DKO–DKO-Arr3 p < 0.0001). (D) Expression of arrestins and tagged paxillin determined by Western blot with bovine arrestin-2 and arrestin-3 (0.1 ng/lane) as standards (Std).
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Figure 4: Arrestins regulate focal adhesion dynamics. (A) DKO and WT cells expressing GFP-paxillin were viewed with DeltaVision Core microscope, and images were captured at 1-min intervals. Representative images at 0, 10, 20, 30, 60, and 90 min. Arrowheads indicate representative focal adhesions. Scale bar, 10 μm. FA lifetimes were determined by counting the number of sequential frames where individual FA (GFP-paxillin) is visible. (B) Histogram distributions of FA lifetimes in 20-min intervals. Data from two or three experiments (150 FAs in 15 cells for each cell type). All distributions are significantly different from each other (p < 0.0001), except for DKO-Arr2 and DKO-Arr3 FA lifetimes, according to nonparametric Kolmogorov–Smirnov test. (C) The distribution of FA lifetimes in indicated cells. *p < 0.001 to DKO; bp < 0.01, #p < 0.001 to WT according to Kruskal–Wallis nonparametric test (H = 170.637, p < 0.0001; H corrected for ties = 170.679, tied p < 0.001). The data were also analyzed with Mann–Whitney test for means (pairwise comparisons: WT–DKO p < 0.0001; DKO–DKO-Arr2 p < 0.0001; DKO–DKO-Arr3 p < 0.0001). (D) Expression of arrestins and tagged paxillin determined by Western blot with bovine arrestin-2 and arrestin-3 (0.1 ng/lane) as standards (Std).
Mentions: The accumulation and enlargement of FAs in DKO cells (Figure 3, C and D) suggest that the rate of FA disassembly might be reduced. To test this idea, we expressed GFP-paxillin in DKO and WT cells (Figure 4) and measured FA lifetimes using live-cell imaging (Figure 4, A and B). Individual FAs at the leading edge of the cell were tracked from formation to disassembly (representative FAs are indicated by red arrows in Figure 4A). All FAs in WT cells formed and disassembled within 20–40 min (Figure 4B), with a median lifetime of 23 min (Figure 4C). In contrast, lifetimes of FAs in DKO cells showed much broader distribution, with median ∼59 min. Of note, some FAs in DKO cells persisted >3 h (Figure 4B). DKO cells also demonstrated a defect in leading edge formation and loss of polarity (Supplemental Movies S1–S4). To test whether the defect in FA disassembly is a result of the lack of arrestins, we transfected red mCherry-paxillin into cells coexpressing GFP with arrestins (Figure 4D). Live-cell imaging revealed a shift in FA lifetimes toward WT (Figure 4, B and C), with median values reduced to 42 and 40.5 min in cells expressing arrestin-2 and arrestin-3, respectively (Figure 4C). Thus arrestins regulate FA turnover, and normal dynamics requires the presence of both subtypes.

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