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. 2015 Apr 14;112(15):4558-63.
doi: 10.1073/pnas.1418088112. Epub 2015 Mar 30.

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

Jun Ando  1 , Masanao Kinoshita  2 , Jin Cui  3 , Hiroyuki Yamakoshi  4 , Kosuke Dodo  5 , Katsumasa Fujita  6 , Michio Murata  7 , Mikiko Sodeoka  8
Affiliations

Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy

Jun Ando et al. Proc Natl Acad Sci U S A. .

Abstract

Sphingomyelin (SM) and cholesterol (chol)-rich domains in cell membranes, called lipid rafts, are thought to have important biological functions related to membrane signaling and protein trafficking. To visualize the distribution of SM in lipid rafts by means of Raman microscopy, we designed and synthesized an SM analog tagged with a Raman-active diyne moiety (diyne-SM). Diyne-SM showed a strong peak in a Raman silent region that is free of interference from intrinsic vibrational modes of lipids and did not appear to alter the properties of SM-containing monolayers. Therefore, we used Raman microscopy to directly visualize the distribution of diyne-SM in raft-mimicking domains formed in SM/dioleoylphosphatidylcholine/chol ternary monolayers. Raman images visualized a heterogeneous distribution of diyne-SM, which showed marked variation, even within a single ordered domain. Specifically, diyne-SM was enriched in the central area of raft domains compared with the peripheral area. These results seem incompatible with the generally accepted raft model, in which the raft and nonraft phases show a clear biphasic separation. One of the possible reasons is that gradual changes of SM concentration occur between SM-rich and -poor regions to minimize hydrophobic mismatch. We believe that our technique of hyperspectral Raman imaging of a single lipid monolayer opens the door to quantitative analysis of lipid membranes by providing both chemical information and spatial distribution with high (diffraction-limited) spatial resolution.

Keywords: Raman imaging; alkyne tag; lipid raft; sphingomyelin; supported monolayer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Chemical structures of SM, diyne-SM, DOPC, and chol.
Fig. 2.
Fig. 2.
Raman spectra of supported lipid monolayers of (A) SM, (B) diyne-SM, (C) DOPC, and (D) chol on a quartz substrate. The Raman peak of diyne at 2,263 cm−1 is marked by a red arrow. The supported sample was prepared at 12 mN/m and 25 °C using the LB technique. Raman measurement was performed 15 times at different positions in each membrane, with an exposure time of 6 s. Averaged Raman spectra are shown. Raman peaks of O2 at ∼1,555 cm−1 and N2 at ∼2,330 cm−1 have been deleted so that Raman peaks from lipid molecules can be clearly seen, which is explained in Fig. S1.
Fig. 3.
Fig. 3.
Raman images of a diyne-SM/DOPC/chol ternary monolayer (1:1:1 molar ratio) on a quartz substrate. The images were reconstructed based on (A) the intensity of the diyne peak at 2,264 cm−1 and (B) that of the peak bottom at 2,222 cm−1. The images consist of 54 × 28 pixels. (Scale bar: 10 μm.)
Fig. 4.
Fig. 4.
Raman images of diyne-SM/DOPC/chol ternary monolayers with composition ratios of (A) 1:1:1, (B) 3:7:3, and (C) 1:0:0 reconstructed using the intensity of the diyne peak at 2,263 cm−1. (D) Averaged Raman spectra from rectangular areas marked i–iv in A–C. Area i corresponds to an ordered domain, whereas area ii corresponds to a disordered region. Each rectangular area includes 9 (3 × 3) pixels. The Raman peak of N2 at 2,330 cm−1 was removed so that the diyne peak could be clearly seen. (E) Spatial distribution of Raman spectra of the diyne-SM/DOPC/chol ternary monolayer at a 1:1:1 ratio acquired along the yellow line marked in A. Wavenumber region includes the diyne peak and CH2/CH3 stretching vibrational mode. The images consist of 36 × 24 pixels. (Scale bar: 10 μm.)
Fig. 5.
Fig. 5.
(A) High-resolution Raman imaging of a 1:1:1 diyne-SM/DOPC/chol ternary monolayer taken with slit-scanning Raman microscopy. The image was reconstructed using the diyne peak intensity at 2,262 cm−1. Exposure time and laser power were 100 s per line and 10.5 mW/μm2. The image is shown in a 16-color display. The images consist of 412 × 400 pixels. (Scale bar: 10 μm.) (B) Raman and fluorescence images of a 1:1:1 diyne-SM/DOPC/chol ternary monolayer containing 0.2 mol% Bodipy-PC. Raman and fluorescence images were obtained in the same imaging area of the same sample. The Raman image was reconstructed using the diyne peak intensity at 2,264 cm−1. The fluorescence image was reconstructed using the average fluorescence intensity at 542–603 nm. Fluorescence background during Raman imaging was suppressed by photobleaching of Bodipy-PC under 532-nm laser exposure. Exposure time and laser power for Raman imaging were 60 s per line and 14.1 mW/μm2. Exposure time and laser power for fluorescence imaging were 0.5 s per line and 0.3 mW/μm2. Each image consists of 387 × 250 pixels. (Scale bar: 10 μm.) (C) Line profiles of lipid rafts calculated along the dotted lines of the Raman and fluorescence images in B (red and gray, respectively). The line profile from the Raman image was smoothed using the moving average.
Fig. 6.
Fig. 6.
Surface pressure vs. molecular area isotherms of (A) diyne-SM/chol and (B) SM/chol binary monolayers. Reported data for SM/chol mixtures in B, D, and G were redrawn for comparison (24). The molar fraction of chol xchol is directly shown. The plots show mean molecular area Amean vs. composition of (C) diyne-SM/chol and (D) SM/chol binary monolayers at 5 mN/m. Each result can be fitted to two lines as indicated by dashed lines. Theoretical mean molecular areas (additivity functions) are indicated by solid lines. (E) The PMAs APMA of chol in (blue) diyne-SM/chol and (red) SM/chol monolayers at 5 mN/m were estimated from C and D, respectively (27). The cross-sections between dashed lines and xchol = 1 in C and D show APMA values, which are sums of the lateral areas occupied by chol and the chol-induced lateral expansion of neighboring lipids. (F and G) The molecular compressional modulus Cmol−1 of the diyne-SM and SM in the ordered phase was estimated by fitting the data to the theoretical function in the region of xchol ≥ 0.5 (dashed lines in F and G), in which all monolayer domains form the ordered phase. The cross-sections between xchol = 0 and the dashed lines correspond to the values of Cmol−1 (24). The Cs−1 vs. composition plots are shown for (F) diyne-SM/chol and (G) SM/chol binary monolayers at 5 mN/m (27). The solid lines indicate the theoretical Cs−1 values of SM/chol mixtures, and the dashed lines were obtained by fitting the data to the theoretical equation in the region of xchol ≥ 0.5. Analysis is in SI Materials and Methods.
Fig. 7.
Fig. 7.
Chemical structures of (A) d2-SM and (B) d2-diyne-SM and the NMR spectra of (C) d2-SM/DOPC/chol and (D) d2-diyne-SM/DOPC/chol (molar ratio of 1:1:1) ternary bilayers at 30 °C. The d2-SM/DOPC/chol mixtures gave two Pake doublets at Δν = 51.9 kHz (Lo) and Δν = 36.3 kHz (Ld). The d2-diyne-SM/DOPC/chol mixtures gave a single Pake doublet at Δν = 48.1 kHz (Lo).
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