Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation

Abstract

Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.

Keywords: Amphidinol 3; Cholesterol; Fluorescence microscope; Lipid rafts; Membrane; Phase separation.

Graphical abstract

Fig. 1

Chemical structure of AM3. The stereochemistry was unambiguously elucidated by Oishi's synthetic studies [[19], [20], [21]].

Fig. 2

Fluorescence microscopy observation of phase-separated GUVs in the presence of AM3 and MβCD. GUVs were composed of SM/DOPC/Chol (1:1:1). The disordered and ordered phases were labeled with 0.2 mol% TexRed-DPPE (Ld marker, red) and 488neg-SM (Lo marker, green), respectively. The green fluorescence of 488neg-SM was hardly observed after the two phases became miscible at 22 and 44 μM AM3 or 4 mM MβCD, because the green fluorescence was quenched by the FRET with TexRed-DPPE. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

SPR sensorgrams for the binding of AM3 to the liposomes immobilized on a dodecylamine-modified CM5 sensor chip: DOPC liposomes in the absence or presence of 10 and 30 mol % Chol (A, B, and C), and SM liposomes in the absence or presence of 10 and 30 mol % Chol (D, E, and F). The concentrations of AM3 are 30 (red), 40 (blue), and 50 (green) μM. The association of AM3 was monitored from 0 to 300 s, and its dissociation from the surface was recorded from 300 to 600 s. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

AM3-induced calcein leakage from DOPC/Chol (7:3; pink, 9:1; red), SM/Chol (7:3; pale blue, 9:1; orange), DOPC (green), and SM (blue) liposomes. In all cases, the final lipid concentration was 27 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Diffusion coefficients of 594neg-SM in SM/Chol (9:1) GUV, and 594neg-DOPC in DOPC/Chol (9:1) GUV, determined by FCS. Error bars indicate standard errors (n = 23–28 GUVs). 6.5 μM of AM3 corresponds to twice the amount of Chol in GUVs.

Fig. 6

Diffusion coefficients of 594neg-SM (Lo marker) and 594neg-DOPC (Ld marker) in Lo-Ld phase-separated GUVs composed of SM/DOPC/Chol (1:1:1), determined by FCS. Error bars indicate standard errors (n = 20–39 GUVs). 13.5 μM of AM3 is equimolar to Chol in the GUVs.