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Fluorescent in situ visualization of sterols in Arabidopsis roots

Abstract

Sterols are eukaryotic membrane components with crucial roles in diverse cellular processes. Elucidation of sterol function relies on development of tools for in situ sterol visualization. Here we describe protocols for in situ sterol localization in Arabidopsis thaliana root cells, using filipin as a specific probe for detection of fluorescent filipin-sterol complexes. Currently, filipin is the only established tool for sterol visualization in plants. Filipin labeling can be performed on aldehyde-fixed samples, largely preserving fluorescent proteins and being compatible with immunocytochemistry. Filipin can also be applied for probing live cells, taking into account the fact that it inhibits sterol-dependent endocytosis. The experimental procedures described are designed for fluorescence detection by confocal laser-scanning microscopy with excitation of filipin-sterol complexes at 364 nm. The protocols require 1 d for sterol covisualization with fluorescent proteins in fixed or live roots and 2 d for immunocytochemistry on whole-mount roots.

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Figure 1: Filipin III as a probe for fluorescent detection of 3-β-hydroxysterols in Arabidopsis.
Figure 2: Filipin-sterol fluorescence displays fast photobleaching.
Figure 3: Filipin can be used in live-cell imaging, but it inhibits sterol-mediated endocytosis.
Figure 4: Filipin-sterol fluorescence can be co-detected with various proteins in multicolor colocalization experiments.

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Acknowledgements

We gratefully acknowledge G. Jürgens, W. Michalke, I. Moore and B. Scheres for sharing published research materials used in this paper. We thank E. Pesquet and S. Robert for helpful comments on the manuscript. Work on sterol visualization and function in the authors' laboratory was supported by grants from the Swedish Foundation for Strategic Research (SSF) and the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (Formas) to M.G., as well as a postdoctoral stipend from the Carl Tryggers Foundation to Y.B.

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Y.B., S.M. and M.G. performed the experiments. Y.B and M.G. designed the experiments and wrote the paper. All authors edited the manuscript before submission.

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Correspondence to Markus Grebe.

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Boutté, Y., Men, S. & Grebe, M. Fluorescent in situ visualization of sterols in Arabidopsis roots. Nat Protoc 6, 446–456 (2011). https://doi.org/10.1038/nprot.2011.323

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