Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles

Clemens Röhrl, Claudia Meisslitzer-Ruppitsch, Robert Bittman, Zaiguo Li, Georg Pabst, Ruth Prassl, Witta Strobl, Josef Neumüller, Adolf Ellinger, Margit Pavelka, Herbert Stangl

Research output: Contribution to journalArticlepeer-review

27 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)331-340
Number of pages10
JournalCurrent Pharmaceutical Biotechnology
Volume13
Issue number2
DOIs
Publication statusPublished - Feb 2012
Externally publishedYes

Keywords

  • Bodipy
  • Cholesterol
  • Cholesteryl ester
  • Diaminobenzidine (DAB) photooxidation
  • HepG2
  • High density lipoprotein (HDL)
  • Reconstituted HDL particles
  • Transmission electron microscopy
  • trans-Golgi Network/metabolism
  • Humans
  • Fluorescence
  • Multivesicular Bodies/metabolism
  • Photochemical Processes
  • Boron Compounds/chemistry
  • Light
  • Lipid Metabolism/physiology
  • 3,3'-Diaminobenzidine/chemistry
  • Biological Transport/physiology
  • Microscopy, Fluorescence/methods
  • Tumor Cells, Cultured
  • Microscopy, Electron/methods
  • Oxidation-Reduction
  • Cholesterol/metabolism
  • Cholesterol Esters/metabolism
  • Lysosomes/metabolism
  • Endosomes/metabolism
  • Hep G2 Cells
  • Lipoproteins, HDL/metabolism

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