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Open Access Methodology

Methods of staining and visualization of sphingolipid enriched and non-enriched plasma membrane regions of Arabidopsis thaliana with fluorescent dyes and lipid analogues

Jörg O Blachutzik12, Fatih Demir13, Ines Kreuzer1, Rainer Hedrich1 and Gregory S Harms24*

Author Affiliations

1 Institute for Molecular Plant Physiology and Biophysics, University Würzburg, Julius-von-Sachs Platz 2, D-97082, Würzburg, Germany

2 Microscopy Group, Rudolf Virchow Center, University of Würzburg, Josef Schneider Str. 2, D15, D-97080, Würzburg, Germany

3 Present address: Institute of Neuro- and Sensory Physiology, Düsseldorf University Hospital, Universitätsstr. 1, D-40225, Düsseldorf, Germany

4 Departments of Biology and Physics, Wilkes University, 84 W. South St., Wilkes-Barre, PA 18766, USA

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Plant Methods 2012, 8:28  doi:10.1186/1746-4811-8-28

Published: 6 August 2012

Abstract

Background

Sterols and Sphingolipids form lipid clusters in the plasma membranes of cell types throughout the animal and plant kingdoms. These lipid domains provide a medium for protein signaling complexes at the plasma membrane and are also observed to be principal regions of membrane contact at the inception of infection. We visualized different specific fluorescent lipophilic stains of the both sphingolipid enriched and non-sphingolipid enriched regions in the plasma membranes of live protoplasts of Arabidopsis thaliana.

Results

Lipid staining protocols for several fluorescent lipid analogues in plants are presented. The most emphasis was placed on successful protocols for the single and dual staining of sphingolipid enriched regions and exclusion of sphingolipid enriched regions on the plasma membrane of Arabidopsis thaliana protoplasts. A secondary focus was placed to ensure that these staining protocols presented still maintain cell viability. Furthermore, the protocols were successfully tested with the spectrally sensitive dye Laurdan.

Conclusion

Almost all existing staining procedures of the plasma membrane with fluorescent lipid analogues are specified for animal cells and tissues. In order to develop lipid staining protocols for plants, procedures were established with critical steps for the plasma membrane staining of Arabidopsis leaf tissue and protoplasts. The success of the plasma membrane staining protocols was additionally verified by measurements of lipid dynamics by the fluorescence recovery after photobleaching technique and by the observation of new phenomena such as time dependent lipid polarization events in living protoplasts, for which a putative physiological relevance is suggested.

Keywords:
Protoplasts; Lipid polarization; Lipophilic fluorescent dyes; Laurdan; Sphingolipid; Liquid (dis-) ordered phase; Plasma membrane; Fluorescence microscopy