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Figure S1. Internalization of β1-integrin and trasferrin in GFPRab5- and GFPRab21-expressing cells. (A) MDA-MB-231 cells transiently transfected with GFP-Rab5 or -Rab21 were immunostained for endogenous caveolin-1 (Cav1), Rab21, and EEA1. Intracellular β1-integrin is found associated with GFP-Rab5-, GFP-Rab21-, and endogenous Rab21-positive structures that displayed caveolin-1- positive domains on their limiting membranes. Shown are 1-µm confocal sections. In the left panels, arrows point to β1-integrin- and GFP-Rab5-positive structures and arrowheads to β1-integrin- and endogenous Rab21-positive structures. In the top right panel, arrows indicate vesicles with EEA1 in the vesicular lumen. In the bottom middle and right panels, arrows indicate Rab5- or Rab21- and caveolin-1-positive vesicles. (B-E) β1-Integrin antibody (P5D2) and Alexa 546-labeled transferrin (Tf) were bound to the surface of transiently transfected MDA-MB-231 cells on ice. The cells were warmed to 37°C for 5 or 15 min to allow internalization; remaining surface bound labels were removed with a brief acid wash; and the cells were fixed, permeabilized, and stained with Alexa 647-conjugated anti-mouse antibody to detect internalized β1-integrin. Shown are widefield epifluoresence images of representative cells. The fourth panels are a merge. Higher magnifications of representative vesicles are shown in the insets. After 5 min, there was a clear colocalization with internalized integrin and Tf, and both were seen on protruding domains on the GFP-Rab21 and -Rab5 endosomes (B and D, arrows). After 15 min, Tf had accumulated to GFP-Rab5-positive endosomes with little overlap with integrins (C, right, arrow). In addition, individual Tf-negative GFP-Rab5 endosomes showed accumulation of integrin cargo, and nontransfected cells (indicated by asterisks) internalized integrin efficiently as well (C). Also in GFP-Rab21-expressing cells the routes of the integrin and Tf diverged at 15 min. β1-Integrin was seen accumulating in small Rab21-positive vesicles and larger vesicles that contained GFP-Rab21 on the limiting membrane (E). In contrast, Tf (and a small subset of integrins) accumulated intracellularly to a Rab21-positive perinuclear area that may represent the recycling endosome. Together, these data are compatible with the idea that internalized β1-integrins traverse through the same compartment as transferrin but subsequently diverge into separate Rab21- (and Rab5-) positive vesicles that can also contain caveolin.