Direct chemical evidence for sphingolipid domains in the plasma membranes of fibroblasts

Supplementary key words toxin • His-mCherry-NT-lysenin • lateral membrane heterogeneity • vital confocal imaging • membrane tension • cholesterol • temperatureLipids at the outer leafl et of the mammalian plasma membrane are mainly composed of: i ) SM, the most abundant sphingolipid (SL), based on a ceramide backbone and bearing a phosphocholine polar head; ii ) glycosphingolipids (GSLs), another group of SLs bearing various sugars instead of phosphocholine, from simple glucosylceramide (GlcCer) to complex GSLs such as GM1 [for a review, see ( 1 )]; iii ) phosphatidylcholine (PC), the major glycerophospholipid, sharing the same phosphocholine polar head as SM; and iv ) nonpolar cholesterol. Lipid bilayers are no longer considered as a homogenous solvent for membrane proteins ( 2 ), but are now represented with lateral heterogeneity at two different scales of time and space: i ) transient nanometric "lipid rafts", defi ned as small clusters enriched in SLs, sterol, and GPI-anchored proteins ( 3, 4 ); versus ii ) submicrometric/mesoscale domains ( 5-15 ). These larger and more stable domains are well-characterized on artifi cial vesicles ( 16, 17 ), but their relevance for living cells has been questioned ( 18,19 ).The occurrence in living cells of submicrometric/mesoscale domains was fi rst inferred from unexpected behavior in fl uorescence recovery after photobleaching (FRAP)

of the protein called glycosylphosphatidylinositol (GPI)-anchored proteins. The basic structure of a GPI-anchored protein consists of phosphatidylinositol (PI) linked to an unusual non-N -acetyl glucosamine, which, in turn, is linked to three mannose residues followed by an ethanolamine covalently linked to the protein via an amide linkage (EtNP-6Man ␣ 1-2Man ␣ 1-6Man ␣ 1-4GlcN ␣ 1-6 myo inositolphospholipid, Fig. 1A ). Depending on the species and functional context, there may exist variations in the side chain associated with the glycan core. These have been summarized in ( 1 ). The lipid moiety is necessary for the incorporation of GPI-anchored proteins into so-called lipid rafts/microdomains ( 2, 3 ), which can serve as a sorting station for a number of cell signaling molecules, thereby functioning as a reaction center. GPI-anchored proteins can exist in different forms depending on the context and the tissue in which they are expressed. Alternate splicing can cause the same protein to exhibit TM, soluble, or GPI-anchored forms; for example, neural cell adhesion molecule (NCAM) can exist in its GPI-anchored and soluble form when expressed in muscles; whereas, it takes up a TM form instead of the soluble form in brain. GPI anchoring of proteins occurs at the luminal face of The plasma membrane of the cell is comprised of a diverse set of proteins, many of which are transmembrane (TM) proteins spanning the entire bilayer, but a significant proportion of proteins are also lipid tethered, containing a complex glycan core attached to the C terminus

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