Protein level of PV1 correlates with the number of structures capable of forming diaphragms <i>in vivo</i>.

<p>A) Electron micrographs of capillary ECs of the kidneys (<i>top panels</i>) and lungs (<i>middle and bottom panels</i>) of WT, Cav1−/− and cavin-1−/− mice, as indicated. TEC and fenestrae are present in the kidneys of WT, Cav1−/− and cavin-1−/− mice (<i>top panels</i>). (<i>Middle and bottom panels</i>) Caveolae with stomatal diaphragms are present in the lungs of WT and absent in Cav1−/− and cavin-1−/− mice (<i>middle panel</i>). Insets in <i>middle panels</i> are a 2-fold magnification of the noted stretches of ECs. Bottom panels are a 3-fold magnification of ECs of Cav1−/− (<i>left</i>) and cavin-1−/− (<i>right</i>). Bars −200 nm. B) Morphometric analysis of the number of lung endothelial caveolae in WT and Cav1−/− mice demonstrating the absence of caveolae in the latter. C) Morphometric analysis of the numbers of kidney endothelial TEC, fenestrae and caveolae in WT and Cav1−/− mice.</p

Protein level of PV1 is maintained by the presence of caveolae <i>in vivo</i>.

<p>A–B) Protein levels of PV1, Cav1 and CD31 in the lung (A) and kidney (B) total membranes of Cav1−/−, Cav1+/− and WT mice were detected by immunoblotting. C) Protein levels of PV1, cavin-1 and CD31 in the lung total membranes of cavin-1−/− and WT mice were detected by immunoblotting. D) PV1 mRNA levels in the lung (<i>left panel</i>) and kidney (<i>right panel</i>) of WT, Cav1−/− and cavin-1−/− mice.</p

PV1 is retained on the surface of lung endothelial cells by caveolae.

<p>A) Schematic of the timeline (<i>upper right</i>) and the principal steps of PV1 internalization flow cytometric assay (<i>right</i>). An example of data gating and fluorescence intensity histogram is given in the lower left panels. B) Amount of PV1 on the surface of MLEC-wt (<i>WT</i>) and MLEC-Cav1KO (<i>Cav1 KO)</i> at <i>t₀</i> expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 (<i>PV1</i>). Labeling of cells with isotype control non-immune antibodies showed the level of unspecific binding (<i>control</i>) (error bars correspond to stdev, n = 4, *p<0.01). C) Amount of internalized PV1 at different time points in MLEC-WT at 37°C (<i>solid line</i>) and 4°C (<i>dashed line</i>) expressed as median fluorescence intensity per cell from fluorophore-labeled anti-PV1 (<i>PV1</i>) (stdev, n = 6, *p<0.01). D) PV1 internalization in MLEC-WT (<i>WT, top panels</i>) and MLEC-Cav1KO (<i>Cav1 KO, bottom panels)</i> cells at different time points, as detected by confocal microscopy. Images are maximum projections of confocal stacks in green channel (PV1, <i>lower panels</i>) or green merged with blue (nuclei labeled with DAPI, <i>upper panels</i>). E) Internalization rate of PV1 in MLEC-WT (<i>solid line, solid circles</i>) and Cav1KO (<i>dashed line, open circles</i>) cells, expressed as a percentage from the total amount of PV1 on the cell surface. (stdev, n = 4 per time point, *p<0.01). F) Degradation curves of ³⁵S labeled PV1 in MLEC-Cav1KO (Cav1KO, <i>dashed line</i>, <i>open circles</i>) and MLEC-WT (WT, <i>solid circles</i>), isolated from Cav1−/− and wild type mice, respectively. Data is representative of three experiments carried out in duplicate. G, H) PV1 degradation rates were measured in MLEC-WT (WT) and MLEC-Cav1KO (Cav1KO) treated with lysosomal or proteasomal inhibitors. G) Western blots used for densitometric quantifications of PV1 protein level. H) Quantitation of protein levels of PV1.</p

Protein level of PV1 is maintained by the presence of caveolae <i>in vitro</i>.

<p>A) Protein levels of PV1 in MLEC-wt(<i>WT</i>), MLEC-Cav1KO (<i>Cav1KO</i>) and MLEC-Cav1-ECRC (<i>ECRC</i>) cells detected by immunoblotting with anti-PV1 antibodies. <i>M</i> - Corresponds to membrane proteins, <i>C</i> – cytosolic proteins. Equal amount of membrane protein was loaded whereas the cytosolic proteins were normalized to membrane extract volume. The membrane and cytosolic proteins were also partially deglycosylated with PNGase F (<i>+</i>), which resulted in the drop in PV1 molecular weight. Note very low PV1 level in Cav1KO cells and increased PV1 protein level in cells reconstituted with Cav1 (Cav1-ECRC). The top and bottom panels are different exposures of the same blot. B) PV1 is predominantly associated with caveolae on the surface of lung endothelial cells. PV1 (<i>red</i>) colocalizes with Cav1-EGFP (<i>green</i>) at the plasma membrane of live MLEC, as shown by TIRFM. Insets demonstrate the extensive colocalization of the two labels (<i>white arrowheads</i>). Scale bars −20 µm.</p

PV1 is internalized in clathrin and dynamin independent manner in WT and Cav1−/− cells.

<p>A) PV1 does not colocalize with clathrin-GFP on the cell surface. Confocal micrographs of MLEC-WT transfected with clathrin-GFP (<i>Clathrin, green</i>) and labeled with fluorescent anti-PV1 antibodies (<i>PV1, red</i>). The insets represent a low power field with two transfected cells. The areas in shaded in grey are magnified in lowed panels. B–G) PV1 and transferrin internalization rates in MLECs were quantified by flow cytometry. Error bars correspond to StDev. B–D) Percentage of fluorescent antibody labeled PV1 internalized from the cell surface. B) PV1 internalization at 15 and 60 min in presence and absence of the clathrin pathway inhibitor PitStop2 (<i>PS2</i>) or the inactive PitStop2 negative control (<i>NC</i>) (n = 4, <i>p</i>>0.05). C,D) PV1 internalization at 15 and 60 min in presence of dynamin inhibitors Dyngo-4a (C) (n = 4, <i>p</i>>0.05) or Dynasore (<i>D</i>) (n = 4, p>0.05). E) Median fluorescent intensity of transferrin-Alexa647 internalized within 10 min in the presence and absence of PitStop2, Dynasore or Dyngo4a (n = 4, *<i>p</i><0.01). D–G) Internalization of PV1 (F) and transferrin (G) at 15 min in untransfected MLECs (mock) and MLECs transfected with eGFP-encoding plasmid (GFP), dynamin 2-eGFP fusion (Dyn2 wt) or dominant-negative form of dynamin 2 fused to eGFP (Dyn2 K44A MLECs (n = 4, *<i>p</i><0.01). H) Schematic of PV1 (<i>green</i>) trafficking in ECs. <i>De novo</i> formed PV1 enters the secretory pathway and arrives at the cell surface by exocytosis (<i>green arrow</i>) using secretory vesicles (Step 1). On the plasma membrane PV1 is targeted to caveolae, fenestrae or TEC (Step 2) where it forms diaphragms. PV1 is internalized via clathrin- and dynamin-independent endocytic mechanism (<i>Step 3 and 4</i>) followed by degradation in the lysosomes (<i>Step 5</i>).</p

Absence of caveolae in lung ECs does not affect transcription and translation levels of PV1.

<p>A) PV1 mRNA levels in MLEC-wt (<i>WT</i>) and MLEC-Cav1KO (<i>Cav1KO</i>) cells measured by real time quantitative PCR. The data was obtained from quadruplicate samples and normalized to b-actin mRNA levels (ΔΔCt). Bars – SEM. B) Pulse ³⁵S metabolic labeling of MLEC-WT (<i>top panel</i>) and MLEC-Cav1KO (<i>bottom panel</i>) cells followed by PV1 immunoprecipitation at the indicated time points and ³⁵S fluorography. Duplicate samples are shown for each time point assessed. PV1 has four active N-glycosylation sites and therefore shows five bands, the lowest representing the non-glycosylated form and the four higher bands representing various degrees of N-glycosylation. C) Densitometric quantitation of the amount of PV1 translated after 10 min pulse with ³⁵S-methionine and cysteine in MLEC-WT and MLEC-Cav1KO cells. Error bars correspond to SEM (n = 3).</p