Abstract
Eukaryotic cells contain membranes exhibiting different levels of lipid order mostly related to their relative amount of sterol-rich domains, thought to mediate temporal and spatial organization of cellular processes. We previously provided evidence in Arabidopsis thaliana that sterols are crucial for execution of cytokinesis, the last stage of cell division. Recently, we used di-4-ANEPPDHQ, a fluorescent probe sensitive to order of lipid phases, to quantify the level of membrane order of the cell plate, the membrane structure separating daughter cells during somatic cytokinesis of higher plant cells. By employing quantitative, ratiometric fluorescence microscopy for mapping localized lipid order levels, we revealed that the Arabidopsis cell plate represents a high-lipid-order domain of the plasma membrane. Here, we describe step-by-step protocols and troubleshooting for ratiometric live imaging procedures employing the di-4-ANEPPDHQ fluorescent probe for quantification of membrane lipid order during plant cell division in suspension cell cultures and roots of Arabidopsis thaliana.
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Acknowledgements
Work on di-4-ANEPPDHQ-based detection of membrane order by M.G. and T.S. was supported by grants from the Carl Tryggers Foundation and to M.G. a grant from the Swedish Kempe Foundations. We thank the Centre de Microscopie INRA/Université de Bourgogne, UMR1347 Agroécologie, Plateforme DImaCell, BP 86510, F-21000 Dijon, France, where the experiments on the Arabidopsis cell suspension culture were performed.
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Gerbeau-Pissot, P., Der, C., Grebe, M., Stanislas, T. (2016). Ratiometric Fluorescence Live Imaging Analysis of Membrane Lipid Order in Arabidopsis Mitotic Cells Using a Lipid Order-Sensitive Probe. In: Caillaud, MC. (eds) Plant Cell Division. Methods in Molecular Biology, vol 1370. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3142-2_17
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DOI: https://doi.org/10.1007/978-1-4939-3142-2_17
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