Holo-high density lipoprotein (HDL) particle uptake is a controversially discussed pathway to regulate cholesterol homeostasis. The uptake of holo-HDL particles was investigated in HepG2 cells by combined light and electron microscopical methods using diaminobenzidine (DAB) photooxidation to convert fluorescent signals into electron-dense precipitates that are visible in the electron microscope (EM).
HDL fluorescently linked to Alexa 568 was used in fluorescence microscopy (FM) and after photooxidation correlatively by EM. HDL was rapidly internalized, accumulated in endosomal compartments with a plateau after 12 h and was cleared 12 h upon replacement by unlabeled HDL. After DAB photooxidation, the endosomal compartments were identified as multivesicular bodies (MVBs) in the EM; their luminal contents, but not the internal vesicles were stained. Differently shaped and labeled surface domains and appendices, revealed by electron tomography, indicate the dynamics of HDL-positive MVBs. HDL was very rarely found in lysosomes and hardly colocalized with LIMP-II, indicating that not degradation, but different mechanisms - including resecretion - account for HDL clearance.
Subsequently we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids. It became possible for the first time to visualize intracellular pathways of lipoprotein particle-derived lipids, i.e. free (FC) and esterified cholesterol (CE), by EM. Reconstituted HDL particles containing the fluorescent cholesterol probes Bodipy-cholesterol, Bodipy-cholesteryl oleate or cholesteryl Bodipy-ester, in which the cholesterol or the fatty acid moiety is labeled, respectively, were used. Bodipy-cholesterol was found in tubular endosomes and MVBs, the trans-Golgi network and stacked Golgi cisternae. In contrast, incubation with HDL containing labeled cholesteryl ester gave an uptake pattern comparable to holo-HDL particles. Additionally, Bodipy-cholesteryl oleate was found in lysosomes. This indicates different pathways for FC and CE in HepG2 cells. Thus, DAB photooxidation enables the analysis of intracellular transport of lipoproteins and lipoprotein-derived lipids at the light and ultrastructural level. Especially, the established DAB photooxidation to visualize free and esterified cholesterol surrogates is expected to enable future studies in lipid trafficking that require high resolution.