Showing papers by "Daniel Wüstner published in 2010"
TL;DR: In this article, a method to detect DHE selectively, based on its rapid bleaching kinetics compared to cellular autofluorescence, was presented. But this method was not suitable for the detection of specific tissues, such as the nerve ring, the spermateca and oocytes.
Abstract: 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.
40 citations
TL;DR: It is demonstrated 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).
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 l...
7 citations