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 degrees C, FM1-43 stained the plasma membrane, and after 30 to 120 min of incubation at 26 degrees C, FM1-43 labeled cytoplasmic vesicles and then the vacuole. Fluorimetric quantitation demonstrated dye uptake temperature sensitivity (approximately 65% reduction at 16 degrees C, >90% at 4 degrees C). FM1-43 uptake in suspension cells was stimulated more than twofold by brefeldin A and inhibited approximately 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.
Emans
Neil
N
Institute for Molecular Biotechnology, Biology VII, Rheinisch-Westfälische Technische Hochschule Aachen, Worringerweg 1, D-52074 Aachen, Germany. emans@molbiotech.rwth-aachen.de
Zimmermann
Sabine
S
Fischer
Rainer
R
D000730:Androstadienes / Q000494:pharmacology*; D020126:Brefeldin A / Q000494:pharmacology*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis / Q000187:drug effects; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D059828:Plant Cells; D010944:Plants / Q000187:drug effects* / Q000378:metabolism; D011726:Pyridinium Compounds / Q000378:metabolism*; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; C073804:FM1 43; D005456:Fluorescent Dyes; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D020126:Brefeldin A; D000077191:Wortmannin