Combined Light and Electron Microscopy Using Diaminobenzidine Photooxidation to Monitor Trafficking of Lipids Derived from Lipoprotein Particles

Clemens      Rohrl; Claudia      Meisslitzer-Ruppitsch; Robert      Bittman; Zaiguo      Li; Georg      Pabst; Ruth      Prassl; Witta      Strobl; Josef      Neumuller; Adolf      Ellinger; Margit      Pavelka; Herbert      Stangl

doi:10.2174/138920112799095338

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.

Keywords: Bodipy, cholesterol, cholesteryl ester, diaminobenzidine (DAB) photooxidation, HepG2, high density lipoprotein (HDL), reconstituted HDL particles, transmission electron microscopy, HDL-associated sterol, Bodipy-cholesterol, multivesicular bodies (MVBs), Bodipy-cholesteryl oleate, intracellular pathways in HepG2 cells, lipoprotein particles, Golgi apparatus