Microinjecting FM4–64 validates it as a marker of the endocytic pathway in plants

Authors

  • P.A.C. Van GISBERGEN,

    1. Laboratory of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
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  • A. ESSELING-OZDOBA,

    1. Laboratory of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
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    • *

      Current address: Department of Tumor Immunology, Nijmegen Centre for Molecular Life Sciences (NCMLS), P.O. Box 9101, 6500 HB Nijmegen, The Netherlands

  • J.W. VOS

    1. Laboratory of Plant Cell Biology, Wageningen University, Arboretumlaan 4, 6703 BD Wageningen, The Netherlands
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  • Submitted to the special issue of the Journal of Microscopy on the Royal Microscopical Society Meeting in Salzburg, Austria, 31 March to 5 April 2007.

J.W. Vos. Tel: +31-(0)317-484321; fax: +31-(0)317-485005; e-mail: janw.vos@wur.nl

Summary

The amphiphilic dye FM4–64 is used to investigate endocytosis and vesicle trafficking in living eukaryotic cells. The standing hypothesis is that it is inserted into the outer leaflet of the plasma membrane and, from there, is passed on to intracellular membrane compartments by endocytosis. We tested this hypothesis by microinjecting FM4–64 into the cytoplasm and vacuole of Nicotiana tabacum BY-2 suspension culture cells and Tradescantia virginiana stamen hair cells. We found that the dye did not label any membranes when injected into the cytoplasm, but clearly labelled the tonoplast when injected directly into the vacuole. However, because the dye is pH-sensitive, the fluorescence intensity between the plasma membrane and tonoplast varied. We conclude that FM4–64 is a specific marker for the endocytic pathway. Nevertheless, little is known about the molecular interactions of FM4–64 with these particular phospholipid membrane leaflets. We, therefore, appeal for biochemical research to determine which membrane lipids FM4–64 interacts with.

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