NBD-cholesterol probes to track cholesterol distribution in model membranes.
A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L(o)) and liquid-disordered (L(o)) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L(o) domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L(o) domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.
Ramirez
Daniel M Carter
DM
Steacie Institute for Molecular Sciences, National Research Council Canada, Ottawa, ON K1A 0R6, Canada.
Ogilvie
William W
WW
Johnston
Linda J
LJ
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001692:Biological Transport; D002518:Ceramides / Q000378:metabolism; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D004396:Coloring Agents / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D015335:Molecular Probes / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013108:Sphingomyelin Phosphodiesterase / Q000378:metabolism; D013194:Staining and Labeling
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D002518:Ceramides; D004396:Coloring Agents; D008051:Lipid Bilayers; D015335:Molecular Probes; D002784:Cholesterol; D013108:Sphingomyelin Phosphodiesterase; D009327:4-Chloro-7-nitrobenzofurazan