Stable and sensitive probes for lysosomes: cell-penetrating fluorescent calix[4]arenes accumulate in acidic vesicles.
The uptake of a fluorescently labeled cationic calix[4] (NBDCalAm) in live, nonfixed cells has been investigated. The compound is taken into the cells rapidly and shows distinct endosomal distribution after 2 hours. This distribution pattern shows colocalization with lysosomal staining. The uptake is not altered by inhibition of clathrin or caveolae dependent pathways nor by depletion of the cellular ATP-pool. Immediately after uptake the probe is localized in the Golgi and brefeldin A treatment prevents transport to lysosomes. Pulse chase experiments with bafilomycin A1, monensin, and sodium azide showed that accumulation and retention of the probe in lysosomes is primarily driven by the activity of vacuolar ATPases. The NBD labeled calix[4]arene provides a very stable and sensitive marker for lysosomes, and has a considerable advantage over some commercially available lysosomal markers in so far that the fluorescent signal is stable even when the cells are incubated in dye-free medium after staining.
School of Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK. anja.mueller@uea.ac.uk
Anja
A
Mueller
Ruth
R
Lalor
Clara Moyano
CM
Cardaba
Susan E
SE
Matthews
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D047250:Calixarenes / Q000493:pharmacokinetics* / Q000494:pharmacology; D021941:Caveolae / Q000378:metabolism; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000493:pharmacokinetics* / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism*; D018942:Macrolides / Q000494:pharmacology; D008985:Monensin / Q000494:pharmacology; D010636:Phenols / Q000493:pharmacokinetics* / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D002966:Clathrin; D005456:Fluorescent Dyes; D018942:Macrolides; D010636:Phenols; C121325:calix(4)arene; D047250:Calixarenes; D020126:Brefeldin A; C040929:bafilomycin A1; D008985:Monensin; D019810:Sodium Azide; D025262:Vacuolar Proton-Translocating ATPases