ESCRT-0 mutations lead to accumulation of ubiquitylated cargoes, as well as of Notch and Dome in endosomes.

<p>(A–F) High magnification of a region of mosaic eye imaginal discs (A–D), or of FE (E–F) shows accumulation of ubiquitylated cargoes in mutant cells (GFP-negative), as revealed by an antibody against mono- and poly- ubiquitin chains (Ubi). High magnification of the boxed areas is shown in insets. (G–H) Mutant FE cells (GFP-negative) show accumulation of the Notch receptor. Notch receptor has been revealed using anti-NICD specific to the intracellular domain of Notch. Apical as well as intracellular accumulations of Notch ICD epitope is seen in <i>Hrs</i> and <i>Stam</i> FE mutant cells. High magnification of the boxed areas is shown in insets. (I–K) Co-localization with anti Notch ECD (NECD) or Notch ICD (NICD) and Avl, marking early endosomes, in mosaic eye imaginal discs. Notch ECD is mainly accumulated in early endosomes in GFP-negative mutant tissue. (L–L’) Mosaic eye imaginal discs were stained with Ubi and anti-Domeless (Dome). <i>Hrs</i>, <i>Stam</i> mutant cells (GFP-negative) accumulate ubiquitylated cargoes and moderate levels of Dome, compared to WT. (M–O) Endocytic trafficking assay with anti-Notch ECD to label Notch at the surface of living imaginal discs. In WT tissue, after labeling (0 hrs), Notch is present mostly at the apical surface of the cell. After a 5-hour chase (5 hrs) Notch is completely degraded in WT but still present in endosomes in <i>Stam</i> mutant discs, indicating that Notch is internalized but it is not degraded.</p

<i>Hrs</i>, <i>Stam</i> or <i>Hrs</i>, <i>Stam</i> double mutant tissue do not display altered tissue architecture.

<p>(A–H) Epithelial morphology of mosaic FE cells (A–D) and eye discs (E–H) revealed by phalloidin staining to detect F-actin. Follicle cells of 5–7 stage egg chambers homozygous for the mutations (GFP-negative) show normal epithelial architecture compared to WT (GFP-positive). Eye disc cells homozygous for the mutations (GFP-negative) do not show any disruption of tissue architecture. (I–L) High magnification of a region of mosaic eye imaginal discs. Homozygous cells are marked by the absence of GFP. Apoptotic Caspase-3 (magenta) is activated cell autonomously in a subset of <i>Hrs</i> and <i>Stam</i> as well as <i>Hrs</i>, <i>Stam</i> mutant cells, compared to WT. (M–P) WT and predominantly mutant eye-antennal discs for the indicated gene stained with phalloidin revealed that <i>Hrs</i>, <i>Stam</i> mutant discs form morphologically normal eye-antennal discs. (Q–T) Adult eyes deriving from mosaic discs of the indicated genotype. Clones or WT (Q) or mutant cells (R–T) are marked by the absence of red pigment in bright field images indicating that mutant tissue can form photoreceptors.</p

Ultrastructural visualization of uPAR endocytic vesicles.

<p>HEK293-uPAR cells were incubated with anti-uPAR monoclonal antibody R3 (that recognizes the D1 extracellular domain) at 4°C for 20 minutes, washed and then probed with protein A gold 10 nm in the absence and in the presence of 200 nM RAP. After extensive washing, cells were warmed in culture medium for 5 to 20 minutes at 37°C to reveal the identity of uPAR primary endocytic vesicles. Samples were then fixed in 2.5% Glutaraldehyde in 0.1 M Sodium Cacodylate buffer and processed for standard EM plastic embedding. At t = 0, uPAR labelling was observed along the plasma membrane, especially in membrane ruffles (R) but excluded from clathrin coated pits (CP). Upon warming at 37°C, uPAR labelling was observed in ruffling regions of the membrane (R), and in clearly large uncoated vesicular profiles of similar morphology to macropinosomes (MP) at t = 5 and t = 20 minutes as well as in early endocytic elements at 20 minutes. uPAR is visible in endocytic vescicles upon 20 minutes internalization in the presence of 200 nM RAP. Note the full macropinosome (MP) and the empty clathrin-coated pit (CP).</p

Amiloride inhibits uPAR constitutive uptake in a dose-dependent manner.

<p>Panel A. Biotinylation experiments in HEK293-uPAR cells incubated in the absence or presence of amiloride. Lane 1: background, Lane 2: constitutively internalized uPAR, Lane 3: inhibition by amiloride. Panel B: Amiloride inhibits dose-dependently constitutive uPAR endocytosis measured by the cell cytometry assay. Panel C: 5 mM Amiloride specifically inhibits uPAR and 70kDa-Dextran endocytosis, but not Transferrin receptor endocytosis, as observed by the cell cytometry assay. Panel D: Amiloride specifically inhibits uPAR and CD59 but not LRP-1 endocytosis, as observed by the cell cytometry assay. Panel E: Amiloride inhibits uPAR but not uPA:PAI-1 dependent endocytosis, measured by the cell cytometry assay.</p

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

uPAR is constitutively internalized in the absence of ligands.

<p>In all cases, the images presented are representative of 3–5 independent experiments. Panel A shows a biotinylation assay performed in HT1080 cells. Cells were acid-washed in order to remove pre-bound ligands, surface biotinylated and then internalization was allowed for 15 minutes at 37°C. Western Blots analysis of cell extracts revealed that uPAR is being internalized in the absence of uPA:PAI-1 in HT1080 (lane 1: background, lane 2: internalized uPAR in the absence of uPA:PAI-1, lane 3: internalized uPAR in the presence of uPA:PAI-1, lane 4: total biotinylated uPAR). Panel B shows the same experiment in HEK293-uPAR cells (Lane 1: background, Lane 2: internalized uPAR in the absence of uPA:PAI-1, Lane 3: total biotinylated uPAR). Panel C shows the biotinylation assay performed in HEK293-uPAR cells, where internalization was allowed for different times, as indicated (total: total biotinylated uPAR). Panel D shows uPAR endocytosis measured by cell cytometry assay in HEK293-uPAR cells. HEK293-uPAR cells were first incubated with anti uPAR polyclonal antibodies bound to Neutravidin-PE for 30 min at 0°C, and then the temperature was raised for 15 min to 37°C. Samples were also incubated at 0°C to account for unspecific binding. After this period, the remaining antibodies at the cell surface were removed by acid washing at 0°C. By cell cytometry we obtained fluorescent intensity values that allow us to calculate the amount of internalized receptor as % of total cell surface uPAR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003730#s4" target="_blank">materials and methods</a>). White bar: amount of receptor internalized at 4°C. Grey bar: amount of receptor internalized at 37°C. Black bar: actual amount of receptor internalized at 37°C after 15 min, corresponding to 17.5% of cell surface uPAR. Panel E shows the biotinylation assay performed to follow uPAR recycling (see description of the experiment in the text). Lane 1: background. Lane 2: total biotinylated uPAR. Lane 3: internalized uPAR at 37°C (first step). Lane 4 and 6: internalized uPAR (second step) for 10 and 20 minutes 37°C, respectively, without GSH treatment. Lane 5 and 7: internalized uPAR (second step) for 10 and 20 minutes at 37°C, respectively, with GSH treatment. Panel F shows the time-course of uPAR recycling to the cell surface and of its degradation or shedding. The data are expressed in percent of receptors internalized after 20 minutes that is recycled to the plasma membrane (continuous line) or that is lost dring the second incubation (dotted line).</p

uPAR is present in early endosomes and late endosomes/lysosomes in the presence of endogenous ligands (HT1080 cells).

<p>Immunofluorescence of HT1080 cells (left panel) co-stained with anti-uPAR antibody (green) and monoclonal antibody anti-EEA1 (red, top) and monoclonal antibody anti-LAMP1 (red, bottom), respectively, and visualized with confocal laser microscopy. Overlays (merge) show the co-localization of uPAR with EEA1 and LAMP-1 positive membranes (yellow). Scale bars 10 µm. On the right side is shown a representative electron micrograph of uPAR (protein A gold 10 nm) present in early endocytic (full arrow) and lysosomal (arrowhead) compartments previously loaded with BSA 5 nm gold for 2 hours as fluid phase tracer. Scale bar 200 nm.</p

uPAR constitutive endocytosis does not require PI3K activity.

<p>Panel A: Biotinylation experiments in HEK293-uPAR cells incubated in the absence or presence of Wortmannin and RAP. Lane 1: no treatment, Lane 2: 200 nM RAP, Lane 3: 300 nM Wortmannin, Lane 4: Wortmannin and RAP. Wortmannin and RAP do not inhibit constitutive uPAR endocytosis after 15 min at 37°C. Panel B: densitometric analysis of the immunoblots of Panel A.</p

uPAR is found in endocytic compartments in the absence of ligands in HEK293-uPAR cells.

<p>Immunofluorescence of HEK293-uPAR cells labeled with a polyclonal anti-uPAR antibody and imaged by confocal microscopy. Image 2 (left), shows the predominant location of uPAR in lamellipodia-like protrusions and in intracellular compartments, including large vesicles (arrowheads). Image 2 (right), shows a different z-axis of the same cell where large putative macropinosomes are evident (arrowheads, inset 3× magnification). Scale bar 10 µm. Image 2B shows HEK293-uPAR co-stained with the early endocytic marker EEA-1 and the late endocytic/lysosomal marker LAMP-1. In these cells, internal uPAR (green) is not present in the lysosomal compartment as shown by the lack of overlap with LAMP-1 (red). On the other hand, intracellular uPAR (green) completely overlapped with EEA-1 positive puncta (arrowheads). Note the large lamellipodia-like protrusions induced by uPAR expression. Scale bar 10 µm. Insets are magnifications of the relevant areas.</p

Role of Lipid Rafts in uPA:PAI-1 dependent and ligand-independent uPAR endocytosis.

<p>Panel A: Flotation assay of TX-100 extracts of HEK293-uPAR analyzed by immunoblotting. uPAR is located in both detergent-resistant (fraction 2 and 3) but mostly in detergent-soluble fractions of the gradient (lanes 6–8). Caveolin-1 displays a similar distribution, while TfR, LRP-1, β1 integrin and EEA1 are exclusively found in the detergent-soluble fractions. P: pellet. Panel B: uPAR flotation in control and beta-methyl-cyclodextrin (10 mM, 30 minutes at 37°C) treated cells. Under these conditions, uPAR is found only in detergent-soluble fractions (Lanes 6–8). Notice that the flotation in untreated cells is the same as in Panel A. Panel C: flotation analysis of uPAR internalization with cell surface biotinylated HEK293-uPAR cells. UPAR is located in lipid rafts but it is internalized outside of these microdomains. Upper row: cells treated with biotin but not subjected to reduction with GSH, showing the distribution of uPAR present at the cell surface. Second row: uPAR internalized in the absence of ligands (15 min 37°C). Third row: uPA:PAI-1-dependent uPAR internalization. Fourth row: uPAR constitutively internalized in beta-methyl-cyclodextrin treated cells. All images are representative of 3–4 independent experiments.</p