Quantitative imaging of lipid metabolism in yeast: from 4D analysis to high content screens of mutant libraries.
Due to their central role in cellular fat storage and lipid homeostasis, lipid droplets (LD) have attracted great interest in biomedical research. The integration of both biochemical and genetic tools and the use of model organisms have greatly contributed to the understanding of LD metabolism and its regulation. However, many important aspects such as LD biogenesis, intracellular dynamics, or their potential degradation by autophagy are still poorly understood. Microscopic techniques, in particular fluorescence microscopy using LD specific dyes or fluorescent protein tagging, represent excellent experimental tools to study the dynamic nature of both the protein and lipid content of LD. Single cell systems in culture are particularly suited to identify and characterize proteins required for LD formation and turnover, using genetic knock-down or gene deletion strategies. Here we describe experimental setups to investigate LD dynamics and turnover in yeast, using various labeling techniques suitable for three-dimensional imaging over time (4D imaging), quantitative microscopy and imaging-based screens of mutant libraries. Also, implementation of coherent anti-Stokes Raman scattering (CARS) microscopy as an emerging tool for label-free lipid imaging in living cells will be discussed.
Institute of Molecular Biosciences, University of Graz, Graz, Austria.
Heimo
H
Wolinski
Kristian
K
Bredies
Sepp D
SD
Kohlwein
D003627:Data Interpretation, Statistical; D005456:Fluorescent Dyes / Q000737:chemistry; D015698:Genomic Library; D007091:Image Processing, Computer-Assisted; D050356:Lipid Metabolism; D018613:Microscopy, Confocal / Q000379:methods; D053140:Organelle Shape; D015388:Organelles / Q000378:metabolism; D059010:Single-Cell Analysis; D013194:Staining and Labeling; D059008:Time-Lapse Imaging; D015003:Yeasts / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
D005456:Fluorescent Dyes