Fig. 1.

Internalization of β₁-integrin depends on the activity of dynamin GTPase. A: SK-CO15 cells grown on collagen-coated transwell filters were wounded and infected with adenoviral constructs encoding the hemagglutinin (HA)-tagged dominant-negative mutant of dynamin-1 (K44A) or wild-type dynamin-1. The β₁-integrin antibody internalization assay (10 min) was performed 20 h postinfection. Cells were fixed, and localization of internalized β₁-integrin antibody (red) and the expression tag (green) was determined by immunofluorescent labeling and confocal microscopy. Arrows point to the absence of β₁-integrin-containing vesicles in cells overexpressing D/N dynamin-1. Scale bar, 20 μm. B: surface-biotinylated β₁-integrin internalization assay. Spreading SK-CO15 cells were infected with the same adenoviral constructs as in A or with the control, transactivator-bearing virus, and 16 h postinfection cells were surface biotinylated. Surface proteins were internalized, and biotinylated β₁-integrin was captured on enzyme-linked immunosorbent assay plates and detected with anti-β₁-integrin antibody. Data are expressed as percentage of control (means ± SE, n = 8 experiments). *Statistically significant difference (P < 0.05).