Fluorescent conjugates of brefeldin A selectively stain the endoplasmic reticulum and Golgi complex of living cells.
The fungal metabolite brefeldin A (BFA) interferes with vesicular trafficking in most animal cells. To gain insight into the mechanism of BFA action, we esterified it to the fluorophore, boron dipyromethene difluoride (BODIPY). BODIPY-BEA localized predominantly in the endoplasmic reticulum (ER) and Golgi complex of viable cells and was extracted by detergent treatment, suggesting it interacts primarily with lipid bilayers. The localization of the conjugate is conferred by BFA, since free BODIPY or BODIPY esterified to cyclopentanol did not specifically localize to internal membranes. BODIPY-BFA exhibited a similar biological activity to BFA, but only when used at higher concentrations and after a delay. HPLC analysis revealed that over this period, cells converted BODIPY-BFA to species co-eluting with free BODIPY and BFA. Therefore, BODIPY-BFA is probably inactive until BFA is released by cellular esterases. The specific localization of BODIPY-BFA to the ER and Golgi complex suggests that BFA might exert its effects on vesicular trafficking by perturbing the lipid bilayer of its target organelles. Because BODIPY-BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions, it should be useful for visualizing the ER in living cells.
Deng
Y
Y
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
Bennink
J R
JR
Kang
H C
HC
Haugland
R P
RP
Yewdell
J W
JW
D000818:Animals; D001896:Boron Compounds; D020126:Brefeldin A; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003517:Cyclopentanes / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000648:ultrastructure*; D007739:L Cells; D051379:Mice; D015394:Molecular Structure; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D013194:Staining and Labeling
D001896:Boron Compounds; D003517:Cyclopentanes; D005456:Fluorescent Dyes; D011500:Protein Synthesis Inhibitors; C095138:boron dipyromethenedifluoride-brefeldin A; D020126:Brefeldin A