Improved visualization and quantitative analysis of fluorescent membrane sterol in polarized hepatic cells.

Dehydroergosterol is a natural yeast sterol which has recently been employed for direct observation of intracellular sterol transport by UV microscopy. Here, methods are described for improved visualization and quantification of dehydroergosterol in the membranes of polarized HepG2 cells. Using a new online assay, it is shown that dehydroergosterol derived from a cyclodextrin complex inserted into the plasma membrane with a half time of t(1/2) approximately 34 s. Based on a detailed bleaching analysis of dehydroergosterol, slightly different bleaching rates for dehydroergosterol in the basolateral and canalicular membrane were found, indicating different fluorophore environments. Bleaching correction in concert with 3D imaging allows for detection of dehydroergosterol enrichment in microvilli of the canalicular membrane forming the biliary canaliculus. Evidence is provided that some dehydroergosterol accumulating in a subapical compartment or apical recycling compartment can rapidly (t(1/2) approximately 2 min) exchange in vesicles towards the biliary canaliculus while the majority of dehydroergosterol does not redistribute from this compartment. The rapidly exchanging pool resembles only a small portion of the total subapical compartment or apical recycling compartment-associated dehydroergosterol (about 15-30%). Kinetic modelling supports the theory that the subapical compartment or apical recycling compartment to biliary canaliculus transport pathway for sterol is unidirectional. This pathway might be important for rapid biliary transport of free sterol produced by hydrolysis of cholesteryl esters derived from high density lipoprotein.