Sterol trafficking in yeast studied by one- and two-photon live-cell imaging of an intrinsically fluorescent ergosterol analog.

Ergosterol is the main sterol in yeast and an important lipid constituent of the yeast plasma membrane (PM). Methods for analysis of ergosterol trafficking between PM and subcellular compartments often rely on fluorescence microscopy, but existing sterol probes either mimic ergosterol poorly or have inconvenient fluorescence properties. Here, we present a novel intrinsically fluorescent probe that differs from ergosterol only by having a 3'-keto group and two additional conjugated double bonds in the ring system. We show that this analog, named Erg-Tetraene, can order fatty acyl chains of phospholipids and partitions partially into the liquid-ordered phase in model membranes containing cholesterol. The Erg-Tetraene has a red-shifted emission and a much stronger two-photon absorption than the widely used analog dehydroergosterol, allowing for its convenient imaging on commercial microscope systems. Using multi-color confocal and two-photon microscopy, we show that uptake of Erg-Tetraene into yeast depends on the sterol transporters Aus1/Pdr11 and is followed by rapid transport to the vacuole and to lipid droplets. Together, we present a novel analogue of ergosterol with improved fluorescence properties for sterol trafficking studies in yeast and other model organisms.