Multicolor bleach-rate imaging enlightens in vivo sterol transport

Abstract

Elucidation of in vivo cholesterol transport and its aberrations in cardiovascular diseases requires suitable model organisms and the development of appropriate monitoring technology. We presented recently a new approach to visualize transport of the intrinsically fluorescent sterol, dehydroergosterol (DHE) in the genetically tractable model organism Caenorhabditis elegans (C. elegans). DHE is structurally very similar to cholesterol and ergosterol, two sterols used by the sterol-auxotroph nematode. We developed a new computational method measuring fluorophore bleaching kinetics at every pixel position, which can be used as a fingerprint to distinguish rapidly bleaching DHE from slowly bleaching autofluorescence in the animals. Here, we introduce multicolour bleach-rate sterol imaging. By this method, we demonstrate that some DHE is targeted to a population of basolateral recycling endosomes (RE) labelled with GFP-tagged RME-1 (GFP-RME- 1) in the intestine of both, wild-type nematodes and mutant animals lacking intestinal gut granules (glo1-mutants). DHE-enriched intestinal organelles of glo1-mutants were decorated with GFPrme8, a marker for early endosomes. No co-localization was found with a lysosomal marker, GFPLMP1. Our new methods hold great promise for further studies on endosomal sterol transport in C. elegans.