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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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Arrestins regulate cell migration and adhesion. (A, B) Adhesion was measured by plating cells on serial dilutions of FN (0.01–1.25 μg/ml) for 15 (A) or 30 (B) min. The data were analyzed by one-way analysis of variance (ANOVA) with arrestin type as the main factor, which was highly significant at 30 min. DKO cells showed a dramatic increase in their ability to adhere compared with WT cells, ***p < 0.001, **p < 0.01. Means ± SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 + GFP, or GFP alone (controls). Cells were plated on 0.32 μg/ml FN. Means ± SD from 24 data points in three experiments. ***p < 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 μg/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four independent experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, ***p <0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means ± SD from 5 fields/chamber from three independent experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. ***p < 0.001 compared with WT. DKO-Arr2, ##p < 0.01, and DKO-Arr3, #p < 0.05, compared with DKO. (F) Arrestin expression in DKO cells was determined using arrestin-2– or arrestin-3–specific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards.
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Figure 2: Arrestins regulate cell migration and adhesion. (A, B) Adhesion was measured by plating cells on serial dilutions of FN (0.01–1.25 μg/ml) for 15 (A) or 30 (B) min. The data were analyzed by one-way analysis of variance (ANOVA) with arrestin type as the main factor, which was highly significant at 30 min. DKO cells showed a dramatic increase in their ability to adhere compared with WT cells, ***p < 0.001, **p < 0.01. Means ± SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 + GFP, or GFP alone (controls). Cells were plated on 0.32 μg/ml FN. Means ± SD from 24 data points in three experiments. ***p < 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 μg/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four independent experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, ***p <0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means ± SD from 5 fields/chamber from three independent experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. ***p < 0.001 compared with WT. DKO-Arr2, ##p < 0.01, and DKO-Arr3, #p < 0.05, compared with DKO. (F) Arrestin expression in DKO cells was determined using arrestin-2– or arrestin-3–specific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards.

Mentions: Cytoskeletal rearrangements drive cell movement and adhesion. Therefore we tested whether the lack of arrestins affects adhesion and migration. The adhesion of DKO cells was similar to WT after initial attachment (15 min; Figure 2A), but they adhered significantly better than WT as cells began to spread (30 min; Figure 2B). Thus DKO cells form initial attachments similar to WT but later demonstrate enhanced adhesion. Moreover, DKO cells demonstrated 3.8-fold- reduced migration toward the FN substrate in a Transwell assay (Figure 2D).


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 cell migration and adhesion. (A, B) Adhesion was measured by plating cells on serial dilutions of FN (0.01–1.25 μg/ml) for 15 (A) or 30 (B) min. The data were analyzed by one-way analysis of variance (ANOVA) with arrestin type as the main factor, which was highly significant at 30 min. DKO cells showed a dramatic increase in their ability to adhere compared with WT cells, ***p < 0.001, **p < 0.01. Means ± SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 + GFP, or GFP alone (controls). Cells were plated on 0.32 μg/ml FN. Means ± SD from 24 data points in three experiments. ***p < 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 μg/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four independent experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, ***p <0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means ± SD from 5 fields/chamber from three independent experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. ***p < 0.001 compared with WT. DKO-Arr2, ##p < 0.01, and DKO-Arr3, #p < 0.05, compared with DKO. (F) Arrestin expression in DKO cells was determined using arrestin-2– or arrestin-3–specific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards.
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Figure 2: Arrestins regulate cell migration and adhesion. (A, B) Adhesion was measured by plating cells on serial dilutions of FN (0.01–1.25 μg/ml) for 15 (A) or 30 (B) min. The data were analyzed by one-way analysis of variance (ANOVA) with arrestin type as the main factor, which was highly significant at 30 min. DKO cells showed a dramatic increase in their ability to adhere compared with WT cells, ***p < 0.001, **p < 0.01. Means ± SD from three experiments. (C) Adhesion of DKO cells expressing arrestin-2 + GFP, arrestin-3 + GFP, or GFP alone (controls). Cells were plated on 0.32 μg/ml FN. Means ± SD from 24 data points in three experiments. ***p < 0.001 compared with DKO. (D) Cells were plated in Transwell chambers coated with 0.32 μg/ml FN and allowed to migrate for 4 h. Cells were counted in six fields/chamber in each of four independent experiments. The data were analyzed by one-way ANOVA with cell type as the main factor, ***p <0.001. Insets, representative membranes postmigration. (E) Migration of DKO cells expressing arrestin-2 and GFP or arrestin-3 and GFP, or cells expressing GFP only (DKO and WT). Means ± SD from 5 fields/chamber from three independent experiments performed in duplicate analyzed by one-way ANOVA with cell type as the main factor. ***p < 0.001 compared with WT. DKO-Arr2, ##p < 0.01, and DKO-Arr3, #p < 0.05, compared with DKO. (F) Arrestin expression in DKO cells was determined using arrestin-2– or arrestin-3–specific antibodies, with corresponding purified bovine arrestins (0.1 ng/lane) run as standards.
Mentions: Cytoskeletal rearrangements drive cell movement and adhesion. Therefore we tested whether the lack of arrestins affects adhesion and migration. The adhesion of DKO cells was similar to WT after initial attachment (15 min; Figure 2A), but they adhered significantly better than WT as cells began to spread (30 min; Figure 2B). Thus DKO cells form initial attachments similar to WT but later demonstrate enhanced adhesion. Moreover, DKO cells demonstrated 3.8-fold- reduced migration toward the FN substrate in a Transwell assay (Figure 2D).

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