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

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.lipid raft | Raman imaging | alkyne tag | supported monolayer | sphingomyelin

Electronic Preresonance Stimulated Raman Scattering Microscopy

“…44,45 Such bio-orthogonal chemical imaging offers a powerful platform for functional metabolic imaging in live cells and animals. 46−55 Its success underscores the importance of introducing vibrational probes to improve specificity and sensitivity of nonlinear Raman microscopy. However, even with extensive efforts of instrumentation improvement and small-tag optimization, the detection sensitivity of SRS is still in the range of 35 μM (i.e., diyne tags, double-conjugated alkynes) 51 to 200 μM (small alkyne tags).…”

Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior

“…The conjugated triple bonds in diyne-SM 2 show a significantly enhanced intensity and peak shifts to longer wavenumber at 2263 cm À1 , leading to an approximately five-fold greater peak height than that of the alkyne in 1; the Raman imaging of the distribution of 2 in phase-separated membranes has been published. 12 In the case of SM-d 9 3, the observed peaks can be assigned to symmetric CD 3 stretching at 2184 cm À1 , and Fermi resonance multiplets at 2132 cm À1 and 2081 cm À1 with overtone or combination tone of CD 3 deformation. 18 A peak due to asymmetric CD 3 stretching at $2276 cm À1 was unnoticeable owing to the overlap with rotation-vibrational Raman modes of nitrogen in air around 2332 cm À1 .…”

“…9,10 Initially, we attempted to introduce the conjugated diyne moiety using 3-bromoprop-2-yn-1-ol as the coupling partner under the classical CadiotChodkiewicz coupling conditions (copper(I) chloride, 5 mol %; hydroxylamine, 30 mol %; i-PrNH 2 in aqueous methanol), 11 as reported separately. 12 Unfortunately, it was difficult to drive the coupling to completion and only a modest conversion was achieved, as judged by 1 H NMR. Furthermore, the remaining alkyne 1 was hardly separable from compound 2 due to their similar polarities.…”

“…However, the clear phase separation of 5 is hardly observed in liposome preparation. 12 Thus, we adopted magnetically oriented membrane as a model to observe the minor signals due to 4 and 5 partitioned to the L d phase. Figure 4 shows the 2 H NMR spectra of A comparison of the spectra of 4 and 5 with that of SM-d 2 shows a high degree of conformity in phase redistribution.…”

Sphingolipids in mitochondria

“…Sphingolipids are a class of bioactive lipids found in cell membranes that modulate the biophysical properties of biological membranes (1) and exert a critical role in signal transduction (2,3). Multiple studies in the last decades revealed that sphingolipids control critical cellular functions such as cell cycle, senescence, apoptosis, cell migration and inflammation (4).…”