Showing papers by "Daniel Wüstner published in 2017"
TL;DR: This protocol describes how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae, and explains in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE.
Abstract: Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In the second part, we explain in detail how to perform time-lapse experiments of commercially available BODIPY-tagged cholesterol (TopFluor-cholesterol®; TF-Chol) in comparison to DHE. Finally, using two-photon time-lapse imaging data of TF-Chol, we demonstrate how to use our imaging toolbox SpatTrack for tracking sterol rich vesicles in living cells over time.
17 citations
TL;DR: This work describes how to use SpatTrack, an imaging toolbox, which was developed for quantification of the distribution and dynamics of endo-lysosomal cargo from fluorescence images of living cells, and shows how to simulate realistic vesicle patterns in the cell geometry using Markov Chain Monte Carlo and suitable inter-vesicle and cell-vesicles interaction potentials.
Abstract: Late endosomes and lysosomes (LE/LYSs) play a central role in trafficking of endocytic cargo, secretion of exosomes, and hydrolysis of ingested proteins and lipids. Failure in such processes can lead to lysosomal storage disorders in which a particular metabolite accumulates within LE/LYSs. Analysis of endocytic trafficking relies heavily on quantitative fluorescence microscopy, but evaluation of the huge image data sets is challenging and demands computer-assisted statistical tools. Here, we describe how to use SpatTrack ( www.sdu.dk/bmb/spattrack ), an imaging toolbox, which we developed for quantification of the distribution and dynamics of endo-lysosomal cargo from fluorescence images of living cells. First, we explain how to analyze experimental images of endocytic processes in Niemann Pick C2 disease fibroblasts using SpatTrack. We demonstrate how to quantify the location of the sterol-binding protein NPC2 in LE/LYSs relative to cholesterol -rich lysosomal storage organelles (LSOs) stained with filipin. Second, we show how to simulate realistic vesicle patterns in the cell geometry using Markov Chain Monte Carlo and suitable inter-vesicle and cell-vesicle interaction potentials. Finally, we use such synthetic vesicle patterns as "ground truth" for validation of two-channel analysis tools in SpatTrack, revealing their high reliability. An improved version of SpatTrack for microscopy-based quantification of cargo transport through the endo-lysosomal system accompanies this protocol.