Uptake of a fluorescent marker in plant cells is sensitive to brefeldin A and wortmannin

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 deg C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 deg C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (about 65 per cent reduction at 16 deg C, > 90 per cent at 4 deg C). FM1-43 uptake in suspension cells was simulated more than twofold by brefeldin A and inhibited about 0.4-fold by wortmannin. FM1-43 delivery to the vacuole was largely inhibited by brefeldin A, although overall uptake was simulated, 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