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

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Abstract
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.