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Plant and Cell Physiology Advance Access published online on August 29, 2008

Plant and Cell Physiology, doi:10.1093/pcp/pcn122
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© The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

FM-dyes label Sterol-Rich Plasma Membrane Domains and are Internalized Independently of the Cytoskeleton in Characean Internodal Cells

Andreas Klima and Ilse Foissner*

Dept. Cell Biology, Div. Plant Physiology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria

*Corresponding author:Ilse Foissner, Dept. Cell Biology, Div. Plant Physiology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria, Tel. 0043 662 8044 5553, Fax 0043 662 8044 619, E-mail: ilse.foissner{at}sbg.ac.at


   Abstract

We applied the endocytic markers FM1-43, FM4-64 and filipin to internodal cells of the green alga Chara corallina. Both FM-dyes stained stable, long-living plasma membrane patches with a diameter of up to 1 µm. After 5 min, FM-dyes labelled cortical, trembling structures up to 500 nm in size. After 15 min, FM-dyes localized to endoplasmic organelles up to one µm in diameter, which migrated actively along actin bundles or participated in cytoplasmic mass streaming. After 30-60 min, FM-fluorescence appeared in the membrane of small, endoplasmic vacuoles but not in that of the central vacuole. Some of the FM-labelled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labelled plasma membrane domains which co-localized with the FM-patches. However, internalization of filipin could not be observed. KCN, cytochalasin D, latrunculin B and oryzalin had no effect on size, shape and distribution of FM- and filipin-labelled plasma membrane domains. Internalization of FM-dyes was inhibited by KCN but not by drugs which interfere with the actin or microtubule cytoskeleton. Our data indicate that the plasma membrane of characean internodal cells contains discrete domains which are enriched in sterols and probably correspond to clusters of lipid rafts. The inhibitor experiments suggest that FM-uptake is active but independent of actin filaments, actin polymerization and microtubules. The possible function of the sterol-rich, FM-labelled plasma membrane areas and the significance of actin-independent FM-internalization (via endocytosis or energy-dependent flippases) are discussed.

Keywords: Chara - Cytoskeleton - Endocytosis - Lipid raft - Plasma membrane domain - Sterol

(Received July 21, 2008; Accepted August 14, 2008)
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