Figure 1.
VEC clustering at cell–cell contacts inhibits VEGFR-2 endocytosis. (A) The internalization of VEGFR-2 from the plasma membrane was analyzed in sparse and confluent HUVECs treated with VEGF for 5 min. To detect the internalized receptor, cells were treated with a recombinant single chain antibody to human VEGFR-2, scFvA7, and acid washed before fixation and processing for immunofluorescence microscopy. Internalized VEGFR-2 appears in a vesicular pattern that is more abundant in sparse than in confluent cultures. (bottom) The granular staining after different incubation lengths with VEGF was quantified using the ImageJ program (see Materials and methods). The results (referred to as events per cell) reported in the graph are means ± SD (error bars) of three independent experiments. At least seven random fields were analyzed for each time point in each experiment. (B) VEC-null and -positive confluent cultures were treated as described in A, but the anti–mouse VEGFR-2 clone Avas12α1 was used. The micrographs show a typical vesicular labeling pattern after a 10-min treatment with VEGF. The staining appears more abundant in VEC-null than in VEC-positive cells. (bottom) The time course of vesicular labeling in response to VEGF was analyzed as described in A. The binding of the antibody does not activate VEGFR-2 nor does it induce its internalization (for details see Fig. S5, available at http://www.jcb.org/cgi/content/full/jcb.200602080/DC1). In A and B, nuclei stained with DAPI appear blue. *, P ≤ 0.05; **, P ≤ 0.01 by comparing sparse versus confluent (A, bottom) and VEC-null versus -positive (B, bottom) cells by analysis of variance and the Duncan test. Bars (A), 15 μm; (B) 20 μm.