Sterol Dynamics During Endocytic Trafficking in Arabidopsis
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Sterols are lipids found in membranes of eukaryotic cells. Functions of sterols have been demonstrated for various cellular processes including endocytic trafficking in animal, fungal, and plant cells. The ability to visualize sterols at the subcellular level is crucial to understand sterol distribution and function during endocytic trafficking. In plant cells, the polyene antibiotic filipin is the most extensively used tool for the specific detection of fluorescently labeled 3-β-hydroxysterols in situ. Filipin can to some extent be used to track sterol internalization in live cells, but this application is limited, due to the inhibitory effects filipin exerts on sterol-dependent endocytosis. Nevertheless, filipin-sterol labeling can be performed on aldehyde-fixed cells which allows for sterol detection in endocytic compartments. This approach can combine studies correlating sterol distribution with experimental manipulations of endocytic trafficking pathways. Here, we describe step-by-step protocols and troubleshooting for procedures on live and fixed cells to visualize sterols during endocytic trafficking. We also provide a detailed discussion of advantages and limitations of both methods. Moreover, we illustrate the use of the endocytic recycling inhibitor brefeldin A and a genetically modified version of one of its target molecules for studying endocytic sterol trafficking.
Key wordsSterol labeling Endocytosis Protocols Immunofluorescence Confocal microscopy Arabidopsis root Endocytic trafficking mutants
We gratefully acknowledge David Ehrhardt (Stanford, USA) for making available LTI6a-GFP seeds; Gerd Jürgens (Tübingen, Germany) for providing anti-KNOLLE serum and GNOM-GFP, GNOMsens-myc, and GNOMres-myc seeds; Ben Scheres (Utrecht, the Netherlands) for supplying ARF1-EGFP seeds; Ian Moore (Oxford, UK) for sharing RAB-A2a-EYFP seeds; Karin Schumacher (Heidelberg, Germany) for making available VHA-a1-GFP seeds; and Takashi Ueda (Tokyo, Japan) for providing ARA6-GFP (RAB-F1-GFP) seeds.
- 14.Pan J, Fujioka S, Peng J et al (2009) The E3 ubiquitin ligase SCFTIR1/AFB and membrane sterols play key roles in auxin regulation of endocytosis, recycling, and plasma membrane accumulation of the auxin efflux transporter PIN2 in Arabidopsis thaliana. Plant Cell 21:568–580PubMedCentralPubMedCrossRefGoogle Scholar
- 27.Hartmann M-A (2004) Sterol metabolism and functions in higher plants. In: Daum G (ed) Lipid metabolism and membrane biogenesis. Springer, Berlin, pp 183–211Google Scholar