Linker length in fluorophore-cholesterol conjugates directs phase selectivity and cellular localisation in GUVs and live cells

Abstract

Lipid membrane fluorescent probes that are both domain-selective and compatible with demanding microscopy methods are crucial to elucidate the presence and function of rafts and domains in cells and biophysical models. Whereas targeting fluorescent probes to liquid-disordered (L_d) domains is relatively facile, it is far more difficult to direct probes with high selectivity to liquid-ordered (L_o) domains. Here, a simple, one-pot approach to probe-cholesterol conjugation is described using Steglich esterification to synthesise two identical BODIPY derivatives that differ only in the length of the aliphatic chain between the dye and cholesterol. In the first, BODIPY-Ar-Chol, the probe and cholesterol were directly ester linked and in the second BODIPY-Ahx-Chol, a hexyl linker separated probe from cholesterol. Uptake and distribution of each probe was compared in ternary, phase separated giant unilamellar vesicles (GUVs) using a commercial L_d marker as a reference. BODIPY-Ar-Chol targets almost exclusively the L_d domains with selectivity of >90% whereas by contrast introducing the C₆ linker between the probe and cholesterol drove the probe to L_o with excellent selectivity (>80%). The profound impact of the linker length extended also to uptake and distribution in live mammalian cells. BODIPY-Ahx-Chol associates strongly with the plasma membrane where it partitioned preferably into opposing micron dimensioned do-mains to a commercial L_d marker and its concentration at the membrane was reduced by cyclodextrin treatment of the cells. By contrast the BODIPY-Ahx-Chol permeated the membrane and localised strongly to lipid droplets within the cell. The data demonstrates the profound influence of linker length in cholesterol bioconjugates in directing the probe.

Scheme 1. Synthesis of BODIPY–cholesterol conjugates, BODIPY-Ar-Chol (2) and BODIPY-Ahx-Chol (6).

Fig. 1. Confocal fluorescent imaging of phase separated GUVs DOPC/BSM/Chol (4 : 4 : 2) mol%. GUV stained with compound BODIPY-Ar-Chol (A), DiD (B) and overlay image (C). GUV stained with BODIPY-Ahx-Chol (D), DiD (E) and overlay image (F). Figures (A–F) are raw data images, without pre-processing. Fluorescence intensity plot of BODIPY-Ahx-Chol () vs. DiD () (G and H). Fluorescent lifetime image of BODIPY-Ahx-Chol(I) in a phase separated GUV of composition DOPC/BSM/Chol (4 : 4 : 2) with variation in lifetimes given by the colour bar. Fluorescent lifetime image of the difference in lifetime given by colour (L_d region = orange/yellow) and (L_o region = green). A 503 nm white light laser was used to excite the GUV samples and the emission collected between 511–570 nm.

Fig. 2. Confocal fluorescent image of a phase separated GUV of composition DOPC/BSM/Chol (4 : 4 : 2 mol%) co-stained with BODIPY-Ahx-Chol (green) and DiD (red) showing the different L_d (red) and L_o phases (green) (A). Fluorescent lifetime image (FLIM) of the same GUV showing BODIPY-Ahx-Chol in the L_o phase (B).

Fig. 3. Live HeLa (A and B) and CHO cells (C and D) stained with BODIPY-Ahx-Chol (5 μM, 3 h 37 °C). White arrows highlighting the dark regions where BODIPY-Ahx-Chol does not localise. (A) and (C) overlay of BODIPY-Ahx-Chol and background channels. (B) and (D) BODIPY-Ahx-Chol channel.

Fig. 4. Live HeLa (A–C) and CHO cells (D–F) stained with BODIPY-Ahx-Chol and co-stained with DiD, (800 nM). Overlay of channels (A and D), the BODIPY-Ahx-Chol channel in green (B and E) and the DiD channel in red (C and F).

Fig. 5. Live HeLa and CHO cells treated with MβCD. Cells were treated with 10 mM MβCD and after 2 h ((A and B) HeLa, (C and D) CHO) and 4 h, altering the cholesterol distribution in HeLa cells (E and F) and CHO cells (G and H). (A), (C), (E) and (G) showing the BODIPY-Ahx-Chol channel, and (B), (D), (F) and (H) showing the overlay of BODIPY-Ahx-Chol and background channels.

Fig. 6. Fluorescent lifetime images (FLIM) of a live HeLa cell (A) and CHO cell (B) stained with BODIPY-Ahx-Chol (5 μM, 2 h), and after treatment with MβCD (10 mM) in HeLa (C) and CHO (D) cells.