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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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Individual (A and B) and merged (C) single-plane CLSM images ofthe equator region of GUVs composed of SM/DOPC/CL 58:25:17 with 0.1mol % of 2 obtained by simultaneously recording emissionupon excitation at shorter (A, λex = 480 nm) andlonger wavelength (B, λex = 560 nm). (D) Immobilizedon a micropipette, complete GUVs were reconstructed from z-scans in0.8 μm-increments and color coded for emission from excitationat shorter (green) and longer wavelength (red). (E) CLSM images ofreconstructed GUVs composed of SM/DOPC/CL 56:24:20 with 0.1 mol %of 2 (red) and 0.01% of ATTO647N (cyan, λex = 630 nm). The diameters of all shown GUVs were around 5–10μm.
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fig3: Individual (A and B) and merged (C) single-plane CLSM images ofthe equator region of GUVs composed of SM/DOPC/CL 58:25:17 with 0.1mol % of 2 obtained by simultaneously recording emissionupon excitation at shorter (A, λex = 480 nm) andlonger wavelength (B, λex = 560 nm). (D) Immobilizedon a micropipette, complete GUVs were reconstructed from z-scans in0.8 μm-increments and color coded for emission from excitationat shorter (green) and longer wavelength (red). (E) CLSM images ofreconstructed GUVs composed of SM/DOPC/CL 56:24:20 with 0.1 mol %of 2 (red) and 0.01% of ATTO647N (cyan, λex = 630 nm). The diameters of all shown GUVs were around 5–10μm.

Mentions: Partition coefficients measured21 forflipper 2 indicated a weak preference for Ld membranes over So membranes at 25 °C (DPPC, Kx = 7.7 × 104; DOPC, Kx = 1.3 × 105, Figure S10). Confocal laser scanning microscopy (CLSM) ofGUVs composed of SM/DOPC/CL 58:25:17 (SM: sphingomyelin, CL: cholesterol)and labeled with flipper 2 showed two domains, whichlight up by exciting at different wavelengths (Figure 3A, λex = 480 nm, green; Figure 3B, λex = 560 nm, red; Figure 3C, merged). Co-labeling experiments with a commercial probefor the Ld phase (cyan, λex = 630 nm,Figure 3E) confirmed that the emission observedupon excitation at longer wavelength (red, λex =551 nm, Figure 3E) arises from flipper 2 in the Lo phase (red, Figure 3B).


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)

Individual (A and B) and merged (C) single-plane CLSM images ofthe equator region of GUVs composed of SM/DOPC/CL 58:25:17 with 0.1mol % of 2 obtained by simultaneously recording emissionupon excitation at shorter (A, λex = 480 nm) andlonger wavelength (B, λex = 560 nm). (D) Immobilizedon a micropipette, complete GUVs were reconstructed from z-scans in0.8 μm-increments and color coded for emission from excitationat shorter (green) and longer wavelength (red). (E) CLSM images ofreconstructed GUVs composed of SM/DOPC/CL 56:24:20 with 0.1 mol %of 2 (red) and 0.01% of ATTO647N (cyan, λex = 630 nm). The diameters of all shown GUVs were around 5–10μm.
© Copyright Policy
Related In: Results  -  Collection

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fig3: Individual (A and B) and merged (C) single-plane CLSM images ofthe equator region of GUVs composed of SM/DOPC/CL 58:25:17 with 0.1mol % of 2 obtained by simultaneously recording emissionupon excitation at shorter (A, λex = 480 nm) andlonger wavelength (B, λex = 560 nm). (D) Immobilizedon a micropipette, complete GUVs were reconstructed from z-scans in0.8 μm-increments and color coded for emission from excitationat shorter (green) and longer wavelength (red). (E) CLSM images ofreconstructed GUVs composed of SM/DOPC/CL 56:24:20 with 0.1 mol %of 2 (red) and 0.01% of ATTO647N (cyan, λex = 630 nm). The diameters of all shown GUVs were around 5–10μm.
Mentions: Partition coefficients measured21 forflipper 2 indicated a weak preference for Ld membranes over So membranes at 25 °C (DPPC, Kx = 7.7 × 104; DOPC, Kx = 1.3 × 105, Figure S10). Confocal laser scanning microscopy (CLSM) ofGUVs composed of SM/DOPC/CL 58:25:17 (SM: sphingomyelin, CL: cholesterol)and labeled with flipper 2 showed two domains, whichlight up by exciting at different wavelengths (Figure 3A, λex = 480 nm, green; Figure 3B, λex = 560 nm, red; Figure 3C, merged). Co-labeling experiments with a commercial probefor the Ld phase (cyan, λex = 630 nm,Figure 3E) confirmed that the emission observedupon excitation at longer wavelength (red, λex =551 nm, Figure 3E) arises from flipper 2 in the Lo phase (red, Figure 3B).

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