title: Interaction of cholesterol in ternary lipid mixtures investigated using single-molecule fluorescence.
abstract: Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.
authors.affiliation: Ralph N. Adams Institute for Bioanalytical Chemistry, University of Kansas , 2030 Becker Drive, Lawrence, Kansas 66047, United States.
authors.forename: Brittany N
authors.initials: BN
authors.lastname: DeWitt
authors.forename: Robert C
authors.initials: RC
authors.lastname: Dunn
mesh_terms: D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry*; D000818:Animals; D001896:Boron Compounds; D002645:Chickens; D002784:Cholesterol / Q000737:chemistry*; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D008859:Microscopy, Polarization; D010713:Phosphatidylcholines / Q000737:chemistry*; D010714:Phosphatidylethanolamines / Q000737:chemistry*; D013109:Sphingomyelins / Q000737:chemistry*; D014966:Xanthenes
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D013109:Sphingomyelins; D014966:Xanthenes; D015060:1,2-Dipalmitoylphosphatidylcholine; C007510:1,2-dipalmitoyl-3-phosphatidylethanolamine; C034657:Texas red; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine