Figure 5.
Figure 5. Clathrin adaptors Dab2 and ARH localize to focal adhesions and are required for MT-induced focal adhesion disassembly and recruitment of clathrin to focal adhesions. (a) TIRF images of NIH3T3 fibroblasts treated with 10 µM nocodazole for 4 h and immunostained with antibodies against either pY397 FAK or vinculin and the indicated clathrin adaptors. Boxed regions are shown at higher magnification in the merged images in the panels on the right. (b) Quantification of adaptor protein colocalization with focal adhesion (FA) area in nocodazole-treated cells. The histogram represents data from two independent experiments in which colocalization was measured in 10–20 individual cells. (c) TIRF images of NIH3T3 fibroblasts treated with 10 µM nocodazole for 4 h and immunostained with antibodies against pY397 FAK, Dab2, and clathrin. The bottom row shows merged images as indicated. Arrowheads point to individual puncta of Dab2 and clathrin that colocalize at the focal adhesion. Data in the histogram show the percentage of focal adhesions with clathrin, Dab2, or clathrin and Dab2 colocalization. (d) Quantification of focal adhesion disassembly in NIH3T3 fibroblasts treated with the indicated siRNAs or short hairpin RNAs. The histogram represents data from three independent experiments in which several hundred cells were analyzed per condition. For ARH/Dab2: *, P = 0.004 by two-tailed Student’s t test compared with GAPDH. For individual ARH, Dab2, or numb: P > 0.2 (two-tailed Student’s t test compared with GAPDH). (e) Western blot of pY397 FAK during MT regrowth after nocodazole washout in an shDab2 cell line depleted of either ARH or GAPDH with siRNAs. Cells were treated for 4 h with nocodazole, and then the drug was washed out, and MTs were allowed to regrow for the indicated time. Vinculin is shown as a loading control. (f) Immunofluorescence of vinculin and ARH in an shDab2 cell line depleted of GAPDH or ARH by siRNA and stimulated for focal adhesion disassembly by MT regrowth for 60 min. (g) Levels of surface α5 integrin in NIH3T3 cells depleted of either Dab2 and ARH or GAPDH and stimulated for MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity normalized to the value at 0 min of MT regrowth. Data are averaged from five independent experiments. *, P = 0.003 by Student’s t test. (h) TIRF images of shDab2 cell line depleted of ARH and immunostained for pY397 FAK, clathrin heavy chain, or CD44 to visualize the plasma membrane. (i) Quantification of clathrin colocalization with focal adhesion area in shDab2 cells depleted of either GAPDH or ARH by siRNA. The histogram represents two experiments in which 15 cells were analyzed for each condition. (b–d, g, and i) Error bars are SEM. Bars: (a, f, and h) 15 µm; (c) 1 µm.

Clathrin adaptors Dab2 and ARH localize to focal adhesions and are required for MT-induced focal adhesion disassembly and recruitment of clathrin to focal adhesions. (a) TIRF images of NIH3T3 fibroblasts treated with 10 µM nocodazole for 4 h and immunostained with antibodies against either pY397 FAK or vinculin and the indicated clathrin adaptors. Boxed regions are shown at higher magnification in the merged images in the panels on the right. (b) Quantification of adaptor protein colocalization with focal adhesion (FA) area in nocodazole-treated cells. The histogram represents data from two independent experiments in which colocalization was measured in 10–20 individual cells. (c) TIRF images of NIH3T3 fibroblasts treated with 10 µM nocodazole for 4 h and immunostained with antibodies against pY397 FAK, Dab2, and clathrin. The bottom row shows merged images as indicated. Arrowheads point to individual puncta of Dab2 and clathrin that colocalize at the focal adhesion. Data in the histogram show the percentage of focal adhesions with clathrin, Dab2, or clathrin and Dab2 colocalization. (d) Quantification of focal adhesion disassembly in NIH3T3 fibroblasts treated with the indicated siRNAs or short hairpin RNAs. The histogram represents data from three independent experiments in which several hundred cells were analyzed per condition. For ARH/Dab2: *, P = 0.004 by two-tailed Student’s t test compared with GAPDH. For individual ARH, Dab2, or numb: P > 0.2 (two-tailed Student’s t test compared with GAPDH). (e) Western blot of pY397 FAK during MT regrowth after nocodazole washout in an shDab2 cell line depleted of either ARH or GAPDH with siRNAs. Cells were treated for 4 h with nocodazole, and then the drug was washed out, and MTs were allowed to regrow for the indicated time. Vinculin is shown as a loading control. (f) Immunofluorescence of vinculin and ARH in an shDab2 cell line depleted of GAPDH or ARH by siRNA and stimulated for focal adhesion disassembly by MT regrowth for 60 min. (g) Levels of surface α5 integrin in NIH3T3 cells depleted of either Dab2 and ARH or GAPDH and stimulated for MT-induced focal adhesion disassembly. The histogram represents the mean fluorescent intensity normalized to the value at 0 min of MT regrowth. Data are averaged from five independent experiments. *, P = 0.003 by Student’s t test. (h) TIRF images of shDab2 cell line depleted of ARH and immunostained for pY397 FAK, clathrin heavy chain, or CD44 to visualize the plasma membrane. (i) Quantification of clathrin colocalization with focal adhesion area in shDab2 cells depleted of either GAPDH or ARH by siRNA. The histogram represents two experiments in which 15 cells were analyzed for each condition. (b–d, g, and i) Error bars are SEM. Bars: (a, f, and h) 15 µm; (c) 1 µm.

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