title: Membrane orientation and lateral diffusion of BODIPY-cholesterol as a function of probe structure.
abstract: Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.
authors.forename: Lukasz M
authors.initials: LM
authors.lastname: Solanko
authors.forename: Alf
authors.initials: A
authors.lastname: Honigmann
authors.forename: Henrik Skov
authors.initials: HS
authors.lastname: Midtiby
authors.forename: Frederik W
authors.initials: FW
authors.lastname: Lund
authors.forename: Jonathan R
authors.initials: JR
authors.lastname: Brewer
authors.forename: Vjekoslav
authors.initials: V
authors.lastname: Dekaris
authors.forename: Robert
authors.initials: R
authors.lastname: Bittman
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
authors.forename: Daniel
authors.initials: D
authors.lastname: Wüstner
mesh_terms: D001896:Boron Compounds / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism*; D004058:Diffusion; D005456:Fluorescent Dyes / Q000737:chemistry*; D008853:Microscopy
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D005456:Fluorescent Dyes; D002784:Cholesterol