A novel fluorescent ceramide analogue for studying membrane traffic in animal cells: accumulation at the Golgi apparatus results in altered spectral properties of the sphingolipid precursor.
A series of ceramide analogues bearing the fluorophore boron dipyrromethene difluoride (BODIPY) were synthesized and evaluated as vital stains for the Golgi apparatus, and as tools for studying lipid traffic between the Golgi apparatus and the plasma membrane of living cells. Studies of the spectral properties of several of the BODIPY-labeled ceramides in lipid vesicles demonstrated that the fluorescence emission maxima were strongly dependent upon the molar density of the probes in the membrane. This was especially evident using N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]-D-erythro-sphingosine (C5-DMB-Cer), which exhibited a shift in its emission maximum from green (integral of 515 nm) to red (integral of 620 nm) wavelengths with increasing concentrations. When C5-DMB-Cer was used to label living cells, this property allowed us to differentiate membranes containing high concentrations of the fluorescent lipid and its metabolites (the corresponding analogues of sphingomyelin and glucosylceramide) from other regions of the cell where smaller amounts of the probe were present. Using this approach, prominent red fluorescent labeling of the Golgi apparatus, Golgi apparatus-associated tubulovesicular processes, and putative Golgi apparatus transport vesicles was seen in living human skin fibroblasts, as well as in other cell types. Based on fluorescence ratio imaging microscopy, we estimate that C5-DMB-Cer and its metabolites were present in Golgi apparatus membranes at concentrations up to 5-10 mol %. In addition, the concentration-dependent spectral properties of C5-DMB-Cer were used to monitor the transport of C5-DMB-lipids to the cell surface at 37 degrees C.
Pagano
R E
RE
Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210.
Martin
O C
OC
Kang
H C
HC
Haugland
R P
RP
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002518:Ceramides / Q000737:chemistry / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D051379:Mice; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D013107:Sphingolipids / Q000737:chemistry*
D002518:Ceramides; D013107:Sphingolipids; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan