Tensing Flipper: Photosensitized Manipulation of Membrane Tension, Lipid Phase Separation, and Raft Protein Sorting in Biological Membranes

Abstract

The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (¹O₂) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.

Figure 1

Mechanosensing and photosensitizing properties of Flipper. (a) FLIM images of DOPC and DPPC GUVs labeled with Flipper (left) and the corresponding τ (right). Data from ≥25 GUVs from at least 3 independent experiments. Scale bars are 10 μm. (b) Left: Changes in the absorption spectra of FOX incubated with solutions of 50 μg/mL DOPC (black to blue; first plot) or DPPC (black to orange; second plot) and 2.5 μM Flipper irradiated with blue light for different times. Solutions of Flipper and the corresponding lipid were incubated for 15 min in the dark prior to illumination. Insets show the difference spectra (ΔAbs) before and after 2, 4, and 6 min of irradiation. Right: Area under the FOX absorption curves (AUC) at different irradiation times (columns 2′, 4′ and 6′) integrated from 550 to 700 nm and normalized to nonilluminated solutions AUC₀ (column 0). Gray columns dubbed dark indicate solutions of the corresponding lipid incubated with Flipper for 45 additional minutes (total 60 min) at room temperature in the dark. Data represent the mean ± SD of 3 replicates, *P < 0.01; ns: not significant determined by unpaired one-way ANOVA. (c) Fluorescence photobleaching of 6 μM ADMA upon blue light irradiation of 2.5 μM Flipper in 50 μg/mL DOPC (left plot) or DPPC (right plot) D₂O solutions. Insets: ADMA bleaching rates for DOPC (blue) and DPPC (orange), and in the presence of the specific ¹O₂ quencher NaN₃ (10 mM, green). AUCs integrated from 390 to 500 nm at each irradiation time were normalized to AUC₀, corresponding to nonilluminated solutions. Data are shown as mean ± SD of at least 3 replicates.

Figure 2

Photoinduced effects of Flipper excitation on model lipid membranes. (a) Selected FLIM imaging frames over time of DPPC and DOPC GUVS labeled with 1 μM Flipper. Irradiation times are shown in min:sec on the top right of each frame. Scale bars are 5 μm. (b) Time-course evolution of Flipper τ in DPPC (orange) and DOPC (blue) GUVs measured from the whole GUV at every imaging frame. Data are presented as mean ± SD for 5 independent GUVs. (c) Phasor plot showing the shift in Flipper’s photon clouds in a DOPC GUV (from cyan to red) induced by prolonged FLIM imaging. (d) Intensity (left) and FLIM-phasor images (right) showing a DOPC GUV imaged over multiple scans (white arrow) surrounded by others that were only exposed to a single imaging frame to capture the image, highlighting the highly localized effect of Flipper photosensitization at the illuminated region. Scale bar is 10 μm.

Figure 3

Flipper excitation triggers phase separation in model ternary lipid vesicles. Selected frames over time of a GUV composed of POPC/DPPC/Chol (1:1:1) and doped with the Ld marker DPPE-Atto647N transitioning from a single phase to segregated domains upon Flipper excitation in consecutive FLIM scans. Dynamic phases enriched with DPPE-Atto647N correlate with lower Flipper intensity and shorter τ_ave (pointed by white arrows), indicative of lower membrane tension in the Ld regions. Irradiation times in min:sec are shown on the top left. Scale bar is 5 μm.

Figure 4

Photoinduced phase separation of biological membranes labeled with Flipper. (a) Selected frames over time of a GPMV stained with 1 μM Flipper under prolonged FLIM imaging at 488 nm. The dynamic formation of small domains with different membrane tension can be observed in both intensity (up) and τ_ave (down) images. Irradiation times in min:sec are shown on the top right panels. Scale bar is 10 μm. (b) Identification of Lo and Ld domains in photoinduced phase-separated GPMVs using the photon clouds in the Flipper phasor plot.

Figure 5

Photoinduced phase separation in biological membranes using Flipper directs raft and nonraft protein sorting into Lo and Ld domains, respectively. GPMVs expressing Halo-tagged variants of GPI-AP or TfR were labeled with 1 μM far-red Halo-JFX₆₅₀ (magenta) and 1 μM Flipper (white). GPMVs initially showed a single phase in both channels (before) that separated after 4–6 min of blue light illumination (after). Scale bars are 10 μm.