HDL stimulates the exit of apoE from LRP1- containing endosomes.

<p>HuH7 cells were incubated with Cy5-apoE-TRL for 20 minutes. ApoE recycling was then induced by Cy3-HDL. After 5 min (A–C) and 15 min (D–F), LRP1 (arrows) did no longer co-localize with Cy5-apoE which was instead found associated with Cy3-HDL (arrowheads; nuclei in blue). This was confirmed by high-magnification video-microscopy of LRP1-EGFP expressing HEK293 cells (G–I; due to short time-lapse between acquisition of red and blue channels, a minor signal offset due to endosomal movements was observed). Bar is 5 (G–I) and 20 µm (A–F), respectively.</p

Internalized RAP but not TRL-derived apoE is sorted towards lysosomes.

<p>Incubation of HuH7 cells with Cy5-apoE-TRL or Cy3-RAP was performed for 30 min at 37°C. Subsequent confocal immunofluorescence analysis revealed colocalization of LRP1 with apoE (A–B, see arrows in merged image C). Internalized Cy3-apoE did not appear within lysosomes as indicated by a counterstain with LAMP-1 (D–F). In contrast, after 30 min Cy3-RAP co-localized with LAMP-1 (G–H, see arrows in the merged image I), indicating lysosomal targeting of the LRP1 ligand RAP. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

LRP1-dependent apoE recycling.

<p>HEK293 cells were transfected with pFB-LRP-EGFP, an shRNA vector against LRP1 or EGFP as control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029385#pone.0029385-Laatsch2" target="_blank">[31]</a>. Pulse-chase experiments were performed by incubating HEK293 cells with Cy3-apoE-TRL for 60 min at 37°C. Cells were washed with heparin and incubated for additional 60 min at 37°C with media in the presence or absence of 20 µg/ml HDL₃. Then cell culture media were harvested and the amount of re-secreted Cy3-apoE was determined by SDS-PAGE and subsequent in-gel fluorescence detection. The position of apoE is indicated. Representative gels with the corresponding apoE bands are shown for LRP1-EGFP overexpression (A) and reduced LRP1 expression (B). The highest and lowest intensity in each gel is represented as maximum black and white, respectively; therefore only intra-gel comparisons were performed. The quantification of these experiments is shown as percent recycling versus the corresponding mock transfected cells, which was set to 100% in order to ensure a valid comparison of the changes in apoE recycling between the different experiments (C). *: p<0.05, **: p<0.01 for Student's t-test (n≥4) ± S.E.M.</p

Internalized TRL-derived apoE co-localizes with LRP1 in EEA1-positive endosomes.

<p>HuH7 cells were incubated with Cy5-apoE-TRL for 10 min at 37°C. As indicated by the arrows, subsequent immunofluorescence analysis revealed co-localization of apoE (A) with LRP1 (B) in EEA1-positive endosomes (C). The merged image is shown in D. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

TRL-derived apoE is internalized into peripheral endosomal compartments.

<p>Cy5-apoE-TRL and Cy3-RAP were prepared and protein labelling was analyzed by SDS-PAGE and subsequent in-gel fluorescence detection (A). Incubation of HuH7 cells without (B) and with Cy5-apoE-TRL (C) for 30 min at 37°C resulted in a punctuated endosomal pattern indicating receptor-mediated endocytosis. Nuclei were visualized by DAPI and appear in blue. Bar is 20 µm.</p

ApoE recycling is reduced in LRP1^−/− primary hepatocytes.

<p>LRP1<i>flox</i> mice were infected with AdEGFP or AdCre and three days after infection primary hepatocytes were isolated. Sixteen hours after seeding, the infection with AdCre resulted in a dramatic loss of LRP1 protein expression as determined by Western blotting (A) and indirect immunofluorescence (B–C). Pulse-chase experiments were performed by incubating LRP1-positive (LRP^+/+) and LRP1–negative (LRP^−/−) hepatocytes with Cy3-apoE-TRL for 60 min at 37°C. Cells were washed with heparin and incubated for additional 60 min at 37°C with media in the presence or absence (w/o) of 20 µg/ml HDL₃. Then supernatants were harvested and the amount of re-secreted Cy3-apoE was determined by SDS-PAGE and subsequent quantification as described above. HDL-induced apoE recycling was reduced in LRP1^−/− cells as seen by in-gel fluorescence of chase media (D). Quantification of 4 independent experiments ± S.E.M. revealed a strong reduction of HDL-induced apoE recycling in LRP1^−/− hepatocytes ± HDL (E).</p

LRP1-dependent sorting of apoE and RAP.

<p>Cell lysates from EGFP (lane 1 in A, B and C) or LRP1-EGFP (lane 2 in A, B and C) transfected cells were subjected to SDS-PAGE, and Western blotting was performed with antibodies against the 85 kDa subunit of LRP1 (A), the 515 kDa subunit of LRP1 (B) and the LDLR (C). The endogenous LRP1 precursor protein is cleaved by furin into 515 kDa and 85 kDa fragments (shown in lane 1 of A and B). The recombinant LRP-EGFP can be detected at approximately 600 kDa (lane 2 in A and B) and for the cleaved 85 kDa fragment fused to EGFP at 115 kDa (LRP1₈₅-EGFP; lane 2 in A). The overexpression of LRP1-EGFP expression slightly reduced the expression of LDLR (C). Western blotting using an antibody against beta actin verified equal protein loading. LRP1-EGFP transfected HEK293 cells were incubated with Cy3-apoE-TRL for 30 min. Confocal microscopy revealed strong colocalization of LRP1-EGFP and Cy3-apoE in endosomal compartments (D–E; see arrows in panel F; nuclei are stained with DAPI). High-magnification confocal live-cell microscopy of LRP1-EGFP expressing cells incubated with Cy3-RAP (G) or Cy3-apoE-TRL (H) revealed that RAP is still attached to the inner leaflet of the vesicular membrane and does not diffuse freely within the lumen (G, arrows and inlet). Additionally, RAP was detected in small, dense vesicles not containing LRP1 (G, arrowheads). In contrast, apoE was only present within LRP1-EGFP endosomes and evenly distributed inside the lumen (H, arrows). Bar is 5 (G–H) and 20 µm (D–F), respectively.</p