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

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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 microm. After 5 min, FM dyes labeled cortical, trembling structures up to 500 nm in size. After 15 min, FM dyes localized to endoplasmic organelles up to 1 microm 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-labeled organelles were also stained by neutral red and lysotracker yellow, indicative of acidic compartments. Filipin, a sterol-specific marker, likewise labeled 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-labeled 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 labeled plasma membrane areas and the significance of actin-independent FM internalization (via endocytosis or energy-dependent flippases) are discussed.