Constitutive endocytosis of uPAR is LRP-1-independent.

<p>Panel A: HEK293-uPAR were stained with R3 uPAR (green) and clathrin heavy chain antibodies (red) or with uPAR (red) and LRP-1 antibodies (green). Representative confocal images are displayed. Note that uPAR-rich lamellipodia-like protrusions of HEK293-uPAR cells were largely devoid of clathrin and LRP-1, indicating spatial segregation between uPAR and these endocytic markers. Scale bars 10 µm. Panel B shows acid washed HT1080 cells cultured with Hypertonic Medium to block clathrin-coated pits assembly (0.45 M Sucrose, 45 minutes at 37°C) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003730#pone.0003730-Heuser1" target="_blank">[13]</a>. Immunofluorescence of uPAR and EEA1 shows co-localization in untreated as well as Sucrose treated cells. The treatment did not change uPAR distribution, although a minimal effect on the organization of the early endosomal compartment was observed. As a control, Transferrin-cy3 was bound to the surface, washed and shifted for 30 minutes at 37°C in untreated and treated cells. As expected, Sucrose treatment significantly perturbed Transferrin entry into the endosomal system. Scale bars 10 µm. Panel C (left) shows a biotinylation assay of uPAR internalization. HEK293-uPAR cells were incubated with 200 nM RAP (Receptor Associated Protein) for 1 hour at 0°C in order to inhibit LRP-1association to uPAR, biotinylated, shifted at 37°C for 15 minutes and GSH-treated. (Lane 1: untreated cells, Lane 2: cells treated with 200 nM RAP, Lane 3: cells treated with 50 nM uPA:PAI-1), Lane 4: cells treated with both 50 nM uPA:PAI-1 and 200 nM RAP). Western Blot analysis indicated that constitutively internalized uPAR is not sensitive to RAP treatment. The right section shows the quantitation of the data obtained by densitometric analysis of the blots. Endocytosis of untreated cells is given the arbitrary value of 1. Panel D shows the uptake of uPAR, uPA-PAI1-cy3 and Transferrin-cy3 in untreated or RAP-treated cells by confocal imaging. HEK293-uPAR cells were incubated with a monoclonal Ab anti-uPAR (R3), uPA-PAI1-cy3 and Transferrin-cy3 for 15 minutes on ice, and then extensively washed before being shifted at 37°C for 15 minutes. Cells were fixed in 3% paraformaldehyde, permeabilized with 0.1% saponin and labelled with anti-mouse cy3 antibody for uPAR detection. Images show that anti-uPAR antibody internalization is not perturbed by RAP treatment (middle panel). On the contrary, uPA-PAI-cy3 endocytosis was strongly inhibited by RAP (right panel). As expected, Transferrin endocytosis was not perturbed by RAP treatment (left panel). Scale bars 10 µm. Panel E shows the effect of the GFP-tagged dominant-negative mutant of eps15 (Eps15 Δ95/295, green) on uPAR ligand-independent uptake by confocal imaging. HEK293-uPAR cells were incubated with anti-uPAR monoclonal antibody and Transferrin-cy3 on ice, washed and then shifted for 15 minutes at 37°C. As expected, cells transfected with the eps15 mutant completely abolished Transferrin uptake (upper panel). Internalization of anti-uPAR antibodies occurred in both cells transfected with the eps15 mutant (middle panel) as well as in cells treated with RAP (lower panel). Images are representative of three different experiments. Scale bars 10 µm.</p>

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