Medizinische Universität Graz - Research portal
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SHR Neuro Cancer Cardio Lipid Metab Microb
Rohrl, C; Meisslitzer-Ruppitsch, C; Bittman, R; Li, ZG; Pabst, G; Prassl, R; Strobl, W; Neumuller, J; Ellinger, A; Pavelka, M; Stangl, H.
Combined Light and Electron Microscopy Using Diaminobenzidine Photooxidation to Monitor Trafficking of Lipids Derived from Lipoprotein Particles
CURR PHARM BIOTECHNO. 2012; 13(2): 331-340.
Doi: 10.2174/138920112799095338
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- Co-authors Med Uni Graz
- Prassl Ruth
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- 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.
- Find related publications in this database (using NLM MeSH Indexing)
- 3,3'-Diaminobenzidine - chemistry
- Biological Transport - physiology
- Boron Compounds - chemistry
- Cholesterol - metabolism
- Cholesterol Esters - metabolism
- Endosomes - metabolism
- Fluorescence -
- Hep G2 Cells -
- Humans -
- Light -
- Lipid Metabolism - physiology
- Lipoproteins, HDL - metabolism Lipoproteins, HDL - ultrastructure
- Lysosomes - metabolism
- Microscopy, Electron - methods
- Microscopy, Fluorescence - methods
- Multivesicular Bodies - metabolism
- Oxidation-Reduction -
- Photochemical Processes -
- Tumor Cells, Cultured -
- trans-Golgi Network - metabolism
- Find related publications in this database (Keywords)
- Bodipy
- cholesterol
- cholesteryl ester
- diaminobenzidine (DAB) photooxidation
- HepG2
- high density lipoprotein (HDL)
- reconstituted HDL particles
- transmission electron microscopy