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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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Nocodazole treatment reveals different focal adhesion dynamics in WT and DKO cells. (A) DKO and WT cells were plated for 24 h and treated with or without 10 μM nocodazole for 2 h. Nocodazole was washed out, and microtubules were allowed to regrow for 30, 60, or 120 min. Paxillin and microtubules were visualized with respective antibodies. Scale bar, 10 μm. (B, C) Means ± SD number of FAs in 40–50 cells/condition. Data were analyzed by one-way ANOVA with treatment as the main factor, followed by Bonferroni/Dunn posthoc test with correction for multiple comparisons. (B) DKO cells: ***p < 0.001, 30-min washout compared with untreated cells; #p < 0.05 compared with treated cells; **p < 0.01, 120-min washout compared with untreated cells. (C) WT cells: ***p < 0.001, treated cells compared with all other conditions.
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Figure 5: Nocodazole treatment reveals different focal adhesion dynamics in WT and DKO cells. (A) DKO and WT cells were plated for 24 h and treated with or without 10 μM nocodazole for 2 h. Nocodazole was washed out, and microtubules were allowed to regrow for 30, 60, or 120 min. Paxillin and microtubules were visualized with respective antibodies. Scale bar, 10 μm. (B, C) Means ± SD number of FAs in 40–50 cells/condition. Data were analyzed by one-way ANOVA with treatment as the main factor, followed by Bonferroni/Dunn posthoc test with correction for multiple comparisons. (B) DKO cells: ***p < 0.001, 30-min washout compared with untreated cells; #p < 0.05 compared with treated cells; **p < 0.01, 120-min washout compared with untreated cells. (C) WT cells: ***p < 0.001, treated cells compared with all other conditions.

Mentions: The dramatic defect in FA disassembly in DKO cells suggests that arrestins regulate FA turnover. Microtubule targeting of FAs promotes disassembly (Kaverina et al., 1999; Small et al., 2002) in a RhoA-independent manner (Ezratty et al., 2005, 2009). Because arrestins bind clathrin (Goodman et al., 1996) and microtubules (Hanson et al., 2007), we hypothesized that microtubule-bound arrestins might recruit clathrin necessary for FA turnover (Ezratty et al., 2009). To test whether the absence of arrestins specifically affects microtubule-dependent FA disassembly, we treated WT and DKO cells with nocodazole to destabilize microtubules, and then monitored FAs as the microtubules regrew (Figure 5A). Consistent with previous reports, upon nocodazole treatment of WT cells, the number of FAs doubled. In agreement with FA lifetimes determined in live-cell imaging (Figure 4), after 30 min of nocodazole washout, the number of FAs in WT cells returned to baseline level, paralleling microtubule regrowth (Figure 5C). In contrast, DKO cells did not respond to either microtubule destabilization or regrowth, suggesting that microtubules have little effect on FAs in cells lacking arrestins (Figure 5B). Thus arrestins likely participate in microtubule-dependent rapid FA disassembly.


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)

Nocodazole treatment reveals different focal adhesion dynamics in WT and DKO cells. (A) DKO and WT cells were plated for 24 h and treated with or without 10 μM nocodazole for 2 h. Nocodazole was washed out, and microtubules were allowed to regrow for 30, 60, or 120 min. Paxillin and microtubules were visualized with respective antibodies. Scale bar, 10 μm. (B, C) Means ± SD number of FAs in 40–50 cells/condition. Data were analyzed by one-way ANOVA with treatment as the main factor, followed by Bonferroni/Dunn posthoc test with correction for multiple comparisons. (B) DKO cells: ***p < 0.001, 30-min washout compared with untreated cells; #p < 0.05 compared with treated cells; **p < 0.01, 120-min washout compared with untreated cells. (C) WT cells: ***p < 0.001, treated cells compared with all other conditions.
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Related In: Results  -  Collection

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Figure 5: Nocodazole treatment reveals different focal adhesion dynamics in WT and DKO cells. (A) DKO and WT cells were plated for 24 h and treated with or without 10 μM nocodazole for 2 h. Nocodazole was washed out, and microtubules were allowed to regrow for 30, 60, or 120 min. Paxillin and microtubules were visualized with respective antibodies. Scale bar, 10 μm. (B, C) Means ± SD number of FAs in 40–50 cells/condition. Data were analyzed by one-way ANOVA with treatment as the main factor, followed by Bonferroni/Dunn posthoc test with correction for multiple comparisons. (B) DKO cells: ***p < 0.001, 30-min washout compared with untreated cells; #p < 0.05 compared with treated cells; **p < 0.01, 120-min washout compared with untreated cells. (C) WT cells: ***p < 0.001, treated cells compared with all other conditions.
Mentions: The dramatic defect in FA disassembly in DKO cells suggests that arrestins regulate FA turnover. Microtubule targeting of FAs promotes disassembly (Kaverina et al., 1999; Small et al., 2002) in a RhoA-independent manner (Ezratty et al., 2005, 2009). Because arrestins bind clathrin (Goodman et al., 1996) and microtubules (Hanson et al., 2007), we hypothesized that microtubule-bound arrestins might recruit clathrin necessary for FA turnover (Ezratty et al., 2009). To test whether the absence of arrestins specifically affects microtubule-dependent FA disassembly, we treated WT and DKO cells with nocodazole to destabilize microtubules, and then monitored FAs as the microtubules regrew (Figure 5A). Consistent with previous reports, upon nocodazole treatment of WT cells, the number of FAs doubled. In agreement with FA lifetimes determined in live-cell imaging (Figure 4), after 30 min of nocodazole washout, the number of FAs in WT cells returned to baseline level, paralleling microtubule regrowth (Figure 5C). In contrast, DKO cells did not respond to either microtubule destabilization or regrowth, suggesting that microtubules have little effect on FAs in cells lacking arrestins (Figure 5B). Thus arrestins likely participate in microtubule-dependent rapid FA disassembly.

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