Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol‐auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE‐containing cells were exposed to membrane‐impermeant collisional quenchers (spin‐labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass‐bead lysis or repeated freeze‐thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.      

Selective Visualization of Fluorescent Sterols in Caenorhabditis elegans by Bleach‐Rate‐Based Image Segmentation

The nematode Caenorhabditis elegans is a genetically tractable model organism to investigate sterol transport. In vivo imaging of the fluorescent sterol, dehydroergosterol (DHE), is challenged by C. elegans’ high autofluorescence in the same spectral region as emission of DHE. We present a method to detect DHE selectively, based on its rapid bleaching kinetics compared to cellular autofluorescence. Worms were repeatedly imaged on an ultraviolet‐sensitive wide field (UV‐WF) microscope, and bleaching kinetics of DHE were fitted on a pixel‐basis to mathematical models describing the intensity decay. Bleach‐rate constants were determined for DHE in vivo and confirmed in model membranes. Using this method, we could detect enrichment of DHE in specific tissues like the nerve ring, the spermateca and oocytes. We confirm these results in C. elegans gut‐granule‐loss (glo) mutants with reduced autofluorescence and compare our method with three‐photon excitation microscopy of sterol in selected tissues. Bleach‐rate‐based UV‐WF imaging is a useful tool for genetic screening experiments on sterol transport, as exemplified by RNA interference against the rme‐2 gene coding for the yolk receptor and for worm homologues of Niemann‐Pick C disease proteins. Our approach is generally useful for identifying fluorescent probes in the presence of high cellular autofluorescence.