Fluorescent flippers for mechanosensitive membrane probes

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.

Figure 1

(A) Planarizable push–pull probes are conjugated oligomers with electron donors (D) and acceptors (A) at their termini and bulky twist inducers along the scaffold (red circles); their planarization in lipid bilayers is expected to report on membrane order, potential and tension (horizontal gray arrows). (B) Fluorescent flippers, i.e., monomers in twisted push–pull probes with high surface area and fluorescence, are introduced to maximize mechanosensitivity and fluorescence lifetime. Double-flipper probe 2 is shown together with FRET donor 3 (B), push–pull control 4, single-flipper controls 5 and 6 (C) and original oligothiophene 1 (A).

Scheme 1

Figure 2

(A) Excitation spectra of 2 in DPPC LUVs (solid) and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λ_em = 600 nm). (B) Same for emission (λ_ex = 420 nm). (C) Excitation spectra of 4 in DPPC LUVs (solid) and DOPC LUVs (dotted) at 25 °C (blue) and 55 °C (red, λ_em = 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 excitation shift to emission shift by FRET. (E) Excitation spectra of donor 3 (dashed, λ_em = 460 nm) and acceptor 2 (dotted, λ_em = 600 nm) in DPPC (blue) and DOPC (red). (F) Emission spectra of an equimolar mixture of donor 3 and acceptor 2 in DPPC (blue, solid) and DOPC (red, dashed, λ_ex = 405 nm, blue arrow in E) with the following controls: Emission spectra of donor 3 (cyan, dashed) and acceptor 2 (blue, dotted) in DPPC, excitation spectrum of acceptor 2 in DPPC (gray, dotted) and DOPC (black, dotted), all at 25 °C.

Figure 3

Individual (A and B) and merged (C) single-plane CLSM images of the equator region of GUVs composed of SM/DOPC/CL 58:25:17 with 0.1 mol % of 2 obtained by simultaneously recording emission upon excitation at shorter (A, λ_ex = 480 nm) and longer wavelength (B, λ_ex = 560 nm). (D) Immobilized on a micropipette, complete GUVs were reconstructed from z-scans in 0.8 μm-increments and color coded for emission from excitation at shorter (green) and longer wavelength (red). (E) CLSM images of reconstructed GUVs composed of SM/DOPC/CL 56:24:20 with 0.1 mol % of 2 (red) and 0.01% of ATTO647N (cyan, λ_ex = 630 nm). The diameters of all shown GUVs were around 5–10 μm.