title: Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles.
abstract: Diaminobenzidine (DAB) photooxidation is a method for conversion of fluorescent signals into electron-dense precipitates that are visible in the electron microscope. Recently, we have applied this method to analyze organelles involved in holo-high density lipoprotein (HDL) particle uptake at the ultrastructural level. In the present work we extended the spectrum of molecules visualized via photooxidation to monitor the uptake of HDL-derived lipids in HepG2 cells. By the combined light-electron microscopic method and with the aid of the DAB photooxidation technique, it became possible for the first time to visualize different intracellular pathways of lipoprotein particle-derived lipids and analyze the compartments involved at the ultrastructural level. HDL-Alexa 568 was used to visualize holo-HDL particle uptake. Reconstituted HDL particles containing the fluorescent cholesterol analogues Bodipy-cholesterol, Bodipy-cholesteryl oleate, or cholesteryl Bodipy-ester were used to visualize uptake of the HDL-associated sterol. In Bodipy-cholesteryl oleate and cholesteryl Bodipy-ester, the cholesterol moiety or the fatty acid moiety is fluorescently labeled, respectively; in contrast, Bodipy-cholesterol is an analogue of free cholesterol. The cellular compartments involved in their intracellular routes after uptake were analyzed in the fluorescence and electron microscope after DAB photooxidation. Bodipy-cholesterol was found to be localized in tubular endosomes and multivesicular bodies (MVBs), in the trans-Golgi network, and in stacked Golgi cisternae. In contrast, HepG2 cells incubated with HDL containing Bodipy-cholesteryl oleate or cholesteryl Bodipyester gave an uptake pattern comparable to that of holo-HDL particles, with MVBs being involved. Bodipy-cholesteryl oleate was also found in lysosomes. These results indicate that HDL-derived cholesterol and cholesteryl ester are transported by different intracellular pathways in HepG2 cells. Thus, the DAB photooxidation method enables the analysis of intracellular transport of lipoprotein particle-derived lipids at the light and at the ultrastructural level.
authors.affiliation: Institute of Medical Chemistry, Center for Pathobiochemistry and Genetics, Medical University of Vienna, Wahringerstr. 10, 1090 Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Röhrl
authors.forename: Claudia
authors.initials: C
authors.lastname: Meisslitzer-Ruppitsch
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Zaiguo
authors.initials: Z
authors.lastname: Li
authors.forename: Georg
authors.initials: G
authors.lastname: Pabst
authors.forename: Ruth
authors.initials: R
authors.lastname: Prassl
authors.forename: Witta
authors.initials: W
authors.lastname: Strobl
authors.forename: Josef
authors.initials: J
authors.lastname: Neumüller
authors.forename: Adolf
authors.initials: A
authors.lastname: Ellinger
authors.forename: Margit
authors.initials: M
authors.lastname: Pavelka
authors.forename: Herbert
authors.initials: H
authors.lastname: Stangl
mesh_terms: D015100:3,3'-Diaminobenzidine / Q000737:chemistry*; D001692:Biological Transport / Q000502:physiology; D001896:Boron Compounds / Q000737:chemistry; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D005453:Fluorescence; D056945:Hep G2 Cells; D006801:Humans; D008027:Light; D050356:Lipid Metabolism / Q000502:physiology*; D008075:Lipoproteins, HDL / Q000378:metabolism* / Q000648:ultrastructure; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods; D057146:Multivesicular Bodies / Q000378:metabolism; D010084:Oxidation-Reduction; D055668:Photochemical Processes; D014407:Tumor Cells, Cultured; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D002788:Cholesterol Esters; D008075:Lipoproteins, HDL; D015100:3,3'-Diaminobenzidine; D002784:Cholesterol