title: Solvatochromic Nile Red probes with FRET quencher reveal lipid order heterogeneity in living and apoptotic cells.
abstract: Detecting and imaging lipid microdomains (rafts) in cell membranes remain a challenge despite intensive research in the field. Two types of fluorescent probes are used for this purpose: one specifically labels a given phase (liquid ordered, Lo, or liquid disordered, Ld), while the other, being environment-sensitive (solvatochromic), stains the two phases in different emission colors. Here, we combined the two approaches by designing a phase-sensitive probe of the Ld phase and a quencher of the Ld phase. The former is an analogue of the recently developed Nile Red-based probe NR12S, bearing a bulky hydrophobic chain (bNR10S), while the latter is based on Black Hole Quencher-2 designed as bNR10S (bQ10S). Fluorescence spectroscopy of large unilamellar vesicles and microscopy of giant vesicles showed that the bNR10S probe can partition specifically into the Ld phase, while bQ10S can specifically quench the NR12S probe in the Ld phase so that only its fraction in the Lo phase remains fluorescent. Thus, the toolkit of two probes with quencher can specifically target Ld and Lo phases and identify their lipid order from the emission color. Application of this toolkit in living cells (HeLa, CHO, and 293T cell lines) revealed heterogeneity in the cell plasma membranes, observed as distinct probe environments close to the Lo and Ld phases of model membranes. In HeLa cells undergoing apoptosis, our toolkit showed the formation of separate domains of the Ld-like phase in the form of blebs. The developed tools open new possibilities in lipid raft research.
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
authors.forename: Rémy
authors.initials: R
authors.lastname: Kreder
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.|‡Laboratory of Nanobiotechnologies, Department of Molecular Immunology, Palladin Institute of Biochemistry of the NASU, Kiev 01601, Ukraine.
authors.forename: Kyrylo A
authors.initials: KA
authors.lastname: Pyrshev
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
authors.forename: Zeinab
authors.initials: Z
authors.lastname: Darwich
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
authors.forename: Oleksandr A
authors.initials: OA
authors.lastname: Kucherak
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Mély
authors.affiliation: †Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch, France.
authors.forename: Andrey S
authors.initials: AS
authors.lastname: Klymchenko
mesh_terms: D000818:Animals; D017209:Apoptosis; D016466:CHO Cells; D002470:Cell Survival; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D021962:Membrane Microdomains / Q000378:metabolism / Q000648:ultrastructure*; D010078:Oxazines / Q000138:chemical synthesis* / Q000737:chemistry; D044367:Phase Transition; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D010078:Oxazines; D053835:Unilamellar Liposomes; C044808:nile red