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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 are required for normal clathrin dynamics at FA. (A, B) Frames from representative live-cell TIRF imaging sequences of MEFs coexpressing FA marker GFP-paxillin and mCherry-clathrin. (A) WT MEFs. (B) DKO MEFs. Cell overviews are shown on the left. Boxed regions from overviews are enlarged on the right over the time period of 2 min, 45 s. Chevrons indicate clathrin pits, which appear and disappear in WT but are stationary in DKO cell. (C) Velocity of clathrin pit movement at FA is significantly higher in WT than in DKO cells. *p < 0.05 for the track velocity, ★p < 0.001 for instant velocity, according to unpaired Student's t test. (D) DKO and WT MEFs were fractionated as described in Materials and Methods. Aliquots of lysate (L), supernatant (S), and microtubule pellet (P) were analyzed by Western blot using clathrin (top blot) and tubulin (bottom blot) antibodies. (E) The distribution of clathrin between supernatant and microtubule pellet was calculated for two experiments. **p < 0.01, unpaired Student's t test.
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Figure 7: Arrestins are required for normal clathrin dynamics at FA. (A, B) Frames from representative live-cell TIRF imaging sequences of MEFs coexpressing FA marker GFP-paxillin and mCherry-clathrin. (A) WT MEFs. (B) DKO MEFs. Cell overviews are shown on the left. Boxed regions from overviews are enlarged on the right over the time period of 2 min, 45 s. Chevrons indicate clathrin pits, which appear and disappear in WT but are stationary in DKO cell. (C) Velocity of clathrin pit movement at FA is significantly higher in WT than in DKO cells. *p < 0.05 for the track velocity, ★p < 0.001 for instant velocity, according to unpaired Student's t test. (D) DKO and WT MEFs were fractionated as described in Materials and Methods. Aliquots of lysate (L), supernatant (S), and microtubule pellet (P) were analyzed by Western blot using clathrin (top blot) and tubulin (bottom blot) antibodies. (E) The distribution of clathrin between supernatant and microtubule pellet was calculated for two experiments. **p < 0.01, unpaired Student's t test.

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.


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 are required for normal clathrin dynamics at FA. (A, B) Frames from representative live-cell TIRF imaging sequences of MEFs coexpressing FA marker GFP-paxillin and mCherry-clathrin. (A) WT MEFs. (B) DKO MEFs. Cell overviews are shown on the left. Boxed regions from overviews are enlarged on the right over the time period of 2 min, 45 s. Chevrons indicate clathrin pits, which appear and disappear in WT but are stationary in DKO cell. (C) Velocity of clathrin pit movement at FA is significantly higher in WT than in DKO cells. *p < 0.05 for the track velocity, ★p < 0.001 for instant velocity, according to unpaired Student's t test. (D) DKO and WT MEFs were fractionated as described in Materials and Methods. Aliquots of lysate (L), supernatant (S), and microtubule pellet (P) were analyzed by Western blot using clathrin (top blot) and tubulin (bottom blot) antibodies. (E) The distribution of clathrin between supernatant and microtubule pellet was calculated for two experiments. **p < 0.01, unpaired Student's t test.
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Figure 7: Arrestins are required for normal clathrin dynamics at FA. (A, B) Frames from representative live-cell TIRF imaging sequences of MEFs coexpressing FA marker GFP-paxillin and mCherry-clathrin. (A) WT MEFs. (B) DKO MEFs. Cell overviews are shown on the left. Boxed regions from overviews are enlarged on the right over the time period of 2 min, 45 s. Chevrons indicate clathrin pits, which appear and disappear in WT but are stationary in DKO cell. (C) Velocity of clathrin pit movement at FA is significantly higher in WT than in DKO cells. *p < 0.05 for the track velocity, ★p < 0.001 for instant velocity, according to unpaired Student's t test. (D) DKO and WT MEFs were fractionated as described in Materials and Methods. Aliquots of lysate (L), supernatant (S), and microtubule pellet (P) were analyzed by Western blot using clathrin (top blot) and tubulin (bottom blot) antibodies. (E) The distribution of clathrin between supernatant and microtubule pellet was calculated for two experiments. **p < 0.01, unpaired Student's t test.
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.

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