TY - JOUR
T1 - Combined light and electron microscopy using diaminobenzidine photooxidation to monitor trafficking of lipids derived from lipoprotein particles
AU - Röhrl, Clemens
AU - Meisslitzer-Ruppitsch, Claudia
AU - Bittman, Robert
AU - Li, Zaiguo
AU - Pabst, Georg
AU - Prassl, Ruth
AU - Strobl, Witta
AU - Neumüller, Josef
AU - Ellinger, Adolf
AU - Pavelka, Margit
AU - Stangl, Herbert
PY - 2012/2
Y1 - 2012/2
N2 - 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.
AB - 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.
KW - Bodipy
KW - Cholesterol
KW - Cholesteryl ester
KW - Diaminobenzidine (DAB) photooxidation
KW - HepG2
KW - High density lipoprotein (HDL)
KW - Reconstituted HDL particles
KW - Transmission electron microscopy
KW - trans-Golgi Network/metabolism
KW - Humans
KW - Fluorescence
KW - Multivesicular Bodies/metabolism
KW - Photochemical Processes
KW - Boron Compounds/chemistry
KW - Light
KW - Lipid Metabolism/physiology
KW - 3,3'-Diaminobenzidine/chemistry
KW - Biological Transport/physiology
KW - Microscopy, Fluorescence/methods
KW - Tumor Cells, Cultured
KW - Microscopy, Electron/methods
KW - Oxidation-Reduction
KW - Cholesterol/metabolism
KW - Cholesterol Esters/metabolism
KW - Lysosomes/metabolism
KW - Endosomes/metabolism
KW - Hep G2 Cells
KW - Lipoproteins, HDL/metabolism
UR - http://www.scopus.com/inward/record.url?scp=84863034220&partnerID=8YFLogxK
U2 - 10.2174/138920112799095338
DO - 10.2174/138920112799095338
M3 - Article
C2 - 21470121
AN - SCOPUS:84863034220
SN - 1389-2010
VL - 13
SP - 331
EP - 340
JO - Current Pharmaceutical Biotechnology
JF - Current Pharmaceutical Biotechnology
IS - 2
ER -