Figure 2.

α2β1 integrin clustering sorts fluid-phase cargo to caveosomes. (A) Clustered α2β1 integrin was internalized for 5 and 15 min in the presence of 10 mg/ml HRP (left). Protein A gold (10 nm) shows the location of α2β1 integrin in tubulovesicular and multivesicular structures colocalizing with HRP after 5 and 15 min, respectively (arrowheads). Percentage of α2β1 integrin-positive endosomes (±SE) containing HRP was quantified from >200 structures. (B) Colocalization of lysine-fixable dextran (pulsed for 1 h and chased for 1 h) with caveolin-1. The assay was performed with (α2 clustered) or without (α2 unclustered) α2β1 clustering, and colocalization was quantified as described in Materials and Methods by using BioImageXD (30 cells counted from three independent experiments). Examples of cells measured for this quantification are shown in C. Colocalized voxels are shown as white color (dextran Alexa 546, red; caveolin-1, green). (D) To verify that dextran was targeted to caveosomes due to integrin clustering, colocalization between dextran (1 mg/ml FITC-dextran) and internalized SV40 was measured (quantification of colocalization was from 30 cells from 3 independent experiments). Dextran was internalized for 2 h (1-h pulse followed by 1-h chase) in the presence of nocodazole and after SV40 pretreatment for 1.5 h. Bars, 200 nm (A), 10 μm (B–D).