Figure 4.

From the plasma membrane rafts α2β1 integrin enters first simple tubulovesicular structures, which quickly mature to multivesicular bodies devoid of GPI-GFP and CTxB. (A) Expressed GPI-GFP (green; after 4-h cycloheximide treatment, 100 μg/ml) and exogenously added CTxB (green; 1 μg/ml) were internalized in the presence of clustered α2β1 integrin (red) for different times. The colocalization of α2β1 with these markers was quantified from 30 cells from three separate experiments by evaluating the number of colocalized voxels using BioImageXD. The results are expressed as percentages of α2β1 voxels colocalizing with marker voxels (±SE). Bars, 10 μm. (B) α2β1 integrin was treated on ice with a nonfunctional integrin antibody (HAS6), followed by a clustering secondary antibody and protein A gold (10 nm), and then internalized for different times at 37°C. Structures positive for α2β1 integrin were classified for being tubulovesicular (see image for 5-min time point) or multivesicular structures (images between 15 min and 2 h). Together, 150–200 structures were calculated from different times. α2β1 integrin associated with intraluminal membranes are pointed with arrows. The relative distribution of multivesicular structures of all integrin structures is plotted on the right. Bars, 200 nm.