FIGURE 1.
Overexpression of ACAP4 inhibits EGF-stimulated integrin β1 recycling. A, ARF6T27N overexpression inhibits integrin β1 recycling from endocytic recycling compartment to the plasma membrane. HeLa cells grown on coverslips were transiently transfected with GFP-ARF6T27N. After 24 h of expression, HeLa cells were deprived of serum overnight and allowed to internalize antibody-bound integrin β1 for 2 h. Subsequently, HeLa cells were treated with 0.1 μg/ml EGF for 5 min, fixed, permeabilized, and stained for GFP-ARF6T27N (green) and integrin β1 (red). Overexpression of GFP-ARF6T27N suppresses the normal recycling of integrin β1 shown in nontransfected HeLa cells. In GFP-ARF6T27N-transfected cells, integrin β1 is mainly internally accumulated, which co-localizes with GFP-ARF6T27N. Bar, 10 μm. B, ACAP4 overexpression similarly suppresses the EGF-stimulated integrin β1 recycling. This set of optical images was collected from GFP-ACAP4 overexpressed HeLa cells that were subjected to integrin β1 endocytic recycling assay as indicated in A and stained for GFP-ACAP4 (green) and integrin β1 (red). In transfected cells, integrin β1 is focally accumulated in the pericentriolar endosomes together with GFP-ACAP4. However, integrin β1 is recycled and has a diffuse surface distribution in nontransfected cells within 5 min of EGF addition. Bars, 10 μm. C, ACAP4 co-localizes with ARF6 to the tubular endosomes. HeLa cells were co-transfected with GFP-ARF6 and mCherry-ACAP4. After transfection for 24 h, the subcellular distributions of ACAP4 (red) and ARF6 (green) were visualized with real time imaging. Enlarged images are also shown in panel b. Bar, 10 μm. D, schematic drawing of ACAP4 structure features and its truncation mutants. ACAP4NT, residues 1–550 of ACAP4; ACAP4M residues 560–660 of ACAP4; ACAP4CT, residues 660–903 of ACAP4; Full-length, full-length ACAP4. E, this montage represents images collected from GFP-ACAP4 and its deletion mutant (green)-transfected HeLa cells. After 24 h of transfection, the integrin β1 (red) recycling of transfected cells was tested as indicated in A. Bars, 10 μm. F, quantitative analyses of the effect of ACAP4 and its deletion mutants on EGF-stimulated integrin β1 recycling. The number of transfected cells whose EGF-stimulated integrin β1 recycling was decreased was compared with that of nontransfected cells shown in E. Values represent the means ± S.E. of 100 cells from three different preparations. The error bars represent S.E. * indicates significant difference from that of GFP-ACAP4 (p < 0.05 by t test). G, this set of optical images was collected from HeLa cells transfected with GFP-ACAP1, GFP-ACAP2, and GFP-ACAP4, respectively. After 24 h of transfection, cells were starved, labeled with integrin β1 antibody, and subjected to the EGF-stimulated integrin β1 recycling assay. Cells were then fixed and stained for ACAPs (green) and integrin β1 (red). Bars, 10 μm. H, quantitation of the influence of ACAP1, ACAP2, and ACAP4 overexpression on EGF-stimulated integrin β1 recycling in G. Values represent the means ± S.E. of 100 cells from three different preparations. The error bars represent S.E. * indicates significant difference from that of GFP-ACAP4 (p < 0.05 by t test).