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Fluorescent flippers for mechanosensitive membrane probes.

Dal Molin M, Verolet Q, Colom A, Letrun R, Derivery E, Gonzalez-Gaitan M, Vauthey E, Roux A, Sakai N, Matile S - J. Am. Chem. Soc. (2015)

Bottom Line: Twisted push-pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first "fluorescent flippers" are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns.Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET).These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, National Centre of Competence in Research (NCCR) Chemical Biology, University of Geneva , Geneva, Switzerland.

ABSTRACT
In this report, "fluorescent flippers" are introduced to create planarizable push-pull probes with the mechanosensitivity and fluorescence lifetime needed for practical use in biology. Twisted push-pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first "fluorescent flippers" are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns. Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET). These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy. Controls indicate that strong push-pull macrodipoles are important, operational probes do not relocate in response to lateral membrane reorganization, and two flippers are indeed needed to "really swim," i.e., achieve high mechanosensitivity.

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(A) Excitation spectra of 2 in DPPC LUVs(solid) andDOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (B) Same for emission (λex =420 nm). (C) Excitation spectra of 4 in DPPC LUVs (solid)and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (D) Time-resolved fluorescence decay of 2 (circles) and 1 (squares) in DPPC LUVs (empty)and DOPC LUVs (filled) at 25 °C. (E and F) Transcription of excitationshift to emission shift by FRET. (E) Excitation spectra of donor 3 (dashed, λem = 460 nm) and acceptor 2 (dotted, λem = 600 nm) in DPPC (blue) andDOPC (red). (F) Emission spectra of an equimolar mixture of donor 3 and acceptor 2 in DPPC (blue, solid) and DOPC(red, dashed, λex = 405 nm, blue arrow in E) withthe following controls: Emission spectra of donor 3 (cyan,dashed) and acceptor 2 (blue, dotted) in DPPC, excitationspectrum of acceptor 2 in DPPC (gray, dotted) and DOPC(black, dotted), all at 25 °C.
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fig2: (A) Excitation spectra of 2 in DPPC LUVs(solid) andDOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (B) Same for emission (λex =420 nm). (C) Excitation spectra of 4 in DPPC LUVs (solid)and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (D) Time-resolved fluorescence decay of 2 (circles) and 1 (squares) in DPPC LUVs (empty)and DOPC LUVs (filled) at 25 °C. (E and F) Transcription of excitationshift to emission shift by FRET. (E) Excitation spectra of donor 3 (dashed, λem = 460 nm) and acceptor 2 (dotted, λem = 600 nm) in DPPC (blue) andDOPC (red). (F) Emission spectra of an equimolar mixture of donor 3 and acceptor 2 in DPPC (blue, solid) and DOPC(red, dashed, λex = 405 nm, blue arrow in E) withthe following controls: Emission spectra of donor 3 (cyan,dashed) and acceptor 2 (blue, dotted) in DPPC, excitationspectrum of acceptor 2 in DPPC (gray, dotted) and DOPC(black, dotted), all at 25 °C.

Mentions: The mechanosensitivity of flipper probe 2 was evaluatedin large unilamellar vesicles (LUVs). LUVs composed of DPPC (dipalmitoyl-sn-glycero-3-phosphocholine) have a So–Ld transition at 41 °C. The excitation spectrum of 1.0μM 2 (1.3 mol %) added to Ld DPPC LUVsat 55 °C showed two maxima at λex = 453 nm andλex = 329 nm of equal intensity (ΔFex1/Fex2 = 0.97, Figure 2A, red, solid). The Δλex = +18 nm from chloroform suggested that flipper probe 2 could already be partially planarized in Ld DPPC.Cooled down to 25 °C, an intense peak with a flat maximum λex = 498–533 nm emerged, accompanied by a sharper butweaker band at λex = 352 nm (ΔFex1/Fex2 = 1.48, Figure 2A, blue, solid). A red shiftof up to Δλex = +80 nm in response to Ld–So transition, obtained with the firstunoptimized flippers, clearly exceeded Δλex = +44 nm of the best flipper-free probe 1. This findingsupported that increasing surface area in twisted push–pullprobes increases mechanosensitivity, as expected from “fluorescentflippers.”


Fluorescent flippers for mechanosensitive membrane probes.

Dal Molin M, Verolet Q, Colom A, Letrun R, Derivery E, Gonzalez-Gaitan M, Vauthey E, Roux A, Sakai N, Matile S - J. Am. Chem. Soc. (2015)

(A) Excitation spectra of 2 in DPPC LUVs(solid) andDOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (B) Same for emission (λex =420 nm). (C) Excitation spectra of 4 in DPPC LUVs (solid)and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (D) Time-resolved fluorescence decay of 2 (circles) and 1 (squares) in DPPC LUVs (empty)and DOPC LUVs (filled) at 25 °C. (E and F) Transcription of excitationshift to emission shift by FRET. (E) Excitation spectra of donor 3 (dashed, λem = 460 nm) and acceptor 2 (dotted, λem = 600 nm) in DPPC (blue) andDOPC (red). (F) Emission spectra of an equimolar mixture of donor 3 and acceptor 2 in DPPC (blue, solid) and DOPC(red, dashed, λex = 405 nm, blue arrow in E) withthe following controls: Emission spectra of donor 3 (cyan,dashed) and acceptor 2 (blue, dotted) in DPPC, excitationspectrum of acceptor 2 in DPPC (gray, dotted) and DOPC(black, dotted), all at 25 °C.
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fig2: (A) Excitation spectra of 2 in DPPC LUVs(solid) andDOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (B) Same for emission (λex =420 nm). (C) Excitation spectra of 4 in DPPC LUVs (solid)and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λem = 600 nm). (D) Time-resolved fluorescence decay of 2 (circles) and 1 (squares) in DPPC LUVs (empty)and DOPC LUVs (filled) at 25 °C. (E and F) Transcription of excitationshift to emission shift by FRET. (E) Excitation spectra of donor 3 (dashed, λem = 460 nm) and acceptor 2 (dotted, λem = 600 nm) in DPPC (blue) andDOPC (red). (F) Emission spectra of an equimolar mixture of donor 3 and acceptor 2 in DPPC (blue, solid) and DOPC(red, dashed, λex = 405 nm, blue arrow in E) withthe following controls: Emission spectra of donor 3 (cyan,dashed) and acceptor 2 (blue, dotted) in DPPC, excitationspectrum of acceptor 2 in DPPC (gray, dotted) and DOPC(black, dotted), all at 25 °C.
Mentions: The mechanosensitivity of flipper probe 2 was evaluatedin large unilamellar vesicles (LUVs). LUVs composed of DPPC (dipalmitoyl-sn-glycero-3-phosphocholine) have a So–Ld transition at 41 °C. The excitation spectrum of 1.0μM 2 (1.3 mol %) added to Ld DPPC LUVsat 55 °C showed two maxima at λex = 453 nm andλex = 329 nm of equal intensity (ΔFex1/Fex2 = 0.97, Figure 2A, red, solid). The Δλex = +18 nm from chloroform suggested that flipper probe 2 could already be partially planarized in Ld DPPC.Cooled down to 25 °C, an intense peak with a flat maximum λex = 498–533 nm emerged, accompanied by a sharper butweaker band at λex = 352 nm (ΔFex1/Fex2 = 1.48, Figure 2A, blue, solid). A red shiftof up to Δλex = +80 nm in response to Ld–So transition, obtained with the firstunoptimized flippers, clearly exceeded Δλex = +44 nm of the best flipper-free probe 1. This findingsupported that increasing surface area in twisted push–pullprobes increases mechanosensitivity, as expected from “fluorescentflippers.”

Bottom Line: Twisted push-pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first "fluorescent flippers" are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns.Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET).These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy.

View Article: PubMed Central - PubMed

Affiliation: School of Chemistry and Biochemistry, National Centre of Competence in Research (NCCR) Chemical Biology, University of Geneva , Geneva, Switzerland.

ABSTRACT
In this report, "fluorescent flippers" are introduced to create planarizable push-pull probes with the mechanosensitivity and fluorescence lifetime needed for practical use in biology. Twisted push-pull scaffolds with large and bright dithienothiophenes and their S,S-dioxides as the first "fluorescent flippers" are shown to report on the lateral organization of lipid bilayers with quantum yields above 80% and lifetimes above 4 ns. Their planarization in liquid-ordered (Lo) and solid-ordered (So) membranes results in red shifts in excitation of up to +80 nm that can be transcribed into red shifts in emission of up to +140 nm by Förster resonance energy transfer (FRET). These unique properties are compatible with multidomain imaging in giant unilamellar vesicles (GUVs) and cells by confocal laser scanning or fluorescence lifetime imaging microscopy. Controls indicate that strong push-pull macrodipoles are important, operational probes do not relocate in response to lateral membrane reorganization, and two flippers are indeed needed to "really swim," i.e., achieve high mechanosensitivity.

Show MeSH
Related in: MedlinePlus