Correlated fluorescence-atomic force microscopy of membrane domains: structure of fluorescence probes determines lipid localization.
Coupling atomic force microscopy (AFM) with high-resolution fluorescence microscopy is an attractive means of identifying membrane domains by both physical topography and fluorescence. We have used this approach to study the ability of a suite of fluorescent molecules to probe domain structures in supported planar bilayers. These included BODIPY-labeled ganglioside, sphingomyelin, and three new cholesterol derivatives, as well as NBD-labeled phosphatidylcholine, sphingomyelin, and cholesterol. Interestingly, many fluorescent lipid probes, including derivatives of known raft-associated lipids, preferentially partitioned into topographical features consistent with nonraft domains. This suggests that the covalent attachment of a small fluorophore to a lipid molecule can abolish its ability to associate with rafts. In addition, the localization of one of the BODIPY-cholesterol derivatives was dependent on the lipid composition of the bilayer. These data suggest that conclusions about the identification of membrane domains in supported planar bilayers on the basis of fluorescent lipid probes alone must be interpreted with caution. The combination of AFM with fluorescence microscopy represents a more rigorous means of identifying lipid domains in supported bilayers.
Shaw
James E
JE
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada M5S 3G9.
Epand
Raquel F
RF
Epand
Richard M
RM
Li
Zaiguo
Z
Bittman
Robert
R
Yip
Christopher M
CM
D016477:Artifacts; D005456:Fluorescent Dyes / Q000737:chemistry*; D008051:Lipid Bilayers / Q000737:chemistry*; D008560:Membrane Fluidity; D021962:Membrane Microdomains / Q000737:chemistry* / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008968:Molecular Conformation; D044367:Phase Transition
D005456:Fluorescent Dyes; D008051:Lipid Bilayers