Plant Cell Journal of Pharmacology and Experimental Therapeutics
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The Plant Cell, Vol. 14, 71-86, January 2002, Copyright © 2002,
American Society of Plant Biologists

Uptake of a Fluorescent Marker in Plant Cells Is Sensitive to Brefeldin A and Wortmannin

Neil Emans1,a, Sabine Zimmermanna and Rainer Fischera,b

a Institute for Molecular Biotechnology, Biology VII, Rheinisch-Westfälische Technische Hochschule Aachen, Worringerweg 1, D-52074 Aachen, Germany
b Fraunhofer Institute for Molecular Biology and Applied Ecology, Grafschaft, Auf dem Aberg 1, D-57392 Schmallenberg, Germany

1 To whom correspondence should be addressed. E-mail emans{at}molbiotech.rwth-aachen.de; fax 49-241-871062

We assessed FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-[dibutylamino]styryl)pyridinium dibromide] as a fluorescent endocytosis marker in intact, walled plant cells. At 4°C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26°C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (~65% reduction at 16°C, >90% at 4°C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited ~0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was stimulated, and brefeldin A treatment caused the accumulation of large prevacuolar endosomal vesicles heavily labeled with FM1-43. Three-dimensional time lapse imaging revealed that FM1-43–labeled vacuoles and vesicles are highly dynamic. Thus, FM1-43 serves as a fluorescent marker for imaging and quantifying membrane endocytosis in intact plant cells.




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