Arrestins regulate cell spreading and motility via focal adhesion dynamics.
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.
Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.Show MeSH
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Mentions: Clathrin was shown to facilitate FA disassembly by actively promoting endocytosis and removal of FA components (Gurevich et al., 2002; Ezratty et al., 2009). To test whether arrestins regulate this function of clathrin, we examined the dynamics of clathrin-coated pits in the vicinity of FAs in WT and DKO cells coexpressing GFP-paxillin (to label FAs) and mCherry-clathrin, using total internal reflection fluorescence microscopy (TIRFM) live-cell analysis. We found significantly slower dynamics of clathrin-coated pits near FAs in DKO than with WT cells (Figure 7 and Supplemental Movies S5 and S6), suggesting that endocytic activity at these sites was strongly reduced. If clathrin is recruited to microtubules via arrestins, its association with the cytoskeleton should be reduced in DKO cells. To test this prediction experimentally, we pelleted Taxol-stabilized microtubules from WT and DKO cells and measured clathrin content in the pellet and supernatant by Western blot (Figure 7, D and E). Quantification showed that the fraction of microtubule-associated clathrin in DKO cells is dramatically lower than in WT MEFs, supporting arrestin dependence of clathrin recruitment to microtubules. Collectively our data implicate nonvisual arrestins in regulation of FA dynamics by modulating microtubule- and clathrin-dependent disassembly.
Affiliation: Department of Pharmacology, Vanderbilt University, Nashville, TN 37232.