SM-rich Domains Research Articles

Assembly formation of minor dihydrosphingomyelin in sphingomyelin-rich ordered membrane domains

The lipidome of mammalian cells not only contain sphingomyelin (SM) but also, as a minor component, dihydrosphongomyelin (DHSM), in which the double bond at C4–C5 in the sphingosine base is reduced to a single-bond linkage. It has been indicated that DHSM forms ordered domains more effectively than SM due to its greater potential to induce intermolecular hydrogen bonds. However, direct information on partition and dynamic behaviors of DHSM in raft-like liquid-ordered (Lo) and non-raft-like liquid-disordered (Ld) phase-segregated membranes has been lacking. In the present study, we prepared fluorescent derivatives of DHSM and compared their behaviors to those of fluorescent SM and phosphatidylcholine (PC) derivatives. Fluorescence microscopy showed that DHSM is more preferentially localized to the Lo domains in the Lo/Ld phase-segregated giant unilamellar vesicles than SM and PC. Most importantly, diffusion coefficient measurements indicated that DHSM molecules form DHSM-condensed assembly inside the SM-rich Lo domain of the SM/dioleoylphosphatidylcholine/cholesterol system even when DHSM accounts for 1–3.3 mol% of total lipids. Such heterogeneous distribution of DHSM in the SM-rich Lo domains was further confirmed by inter-lipid FRET experiments. This study provides new insights into the biological functions and significance of minor component DHSM in lipid rafts.

Young modulus of supported lipid membranes containing milk sphingomyelin in the gel, fluid or liquid-ordered phase, determined using AFM force spectroscopy

The biological membrane surrounding milk fat globules (MFGM) exhibits lateral phase separation of lipids, interpreted as gel or liquid-ordered phase sphingomyelin-rich (milk SM) domains dispersed in a fluid continuous lipid phase. The objective of this study was to investigate whether changes in the phase state of milk SM-rich domains induced by temperature (T < Tm or T > Tm) or cholesterol affected the Young modulus of the lipid membrane. Supported lipid bilayers composed of MFGM polar lipids, milk SM or milk SM/cholesterol (50:50 mol) were investigated at 20 °C and 50 °C using atomic force microscopy (AFM) and force spectroscopy. At 20 °C, gel-phase SM-rich domains and the surrounding fluid phase of the MFGM polar lipids exhibited Young modulus values of 10–20 MPa and 4–6 MPa, respectively. Upon heating at 50 °C, the milk SM-rich domains in MFGM bilayers as well as pure milk SM bilayers melted, leading to the formation of a homogeneous membrane with similar Young modulus values to that of a fluid phase (0–5 MPa). Upon addition of cholesterol to the milk SM to reach 50:50 mol%, membranes in the liquid-ordered phase exhibited Young modulus values of a few MPa, at either 20 or 50 °C. This indicated that the presence of cholesterol fluidized milk SM membranes and that the Young modulus was weakly affected by the temperature. These results open perspectives for the development of milk polar lipid based vesicles with modulated mechanical properties.

Non-senescent keratinocytes organize in plasma membrane submicrometric lipid domains enriched in sphingomyelin and involved in re-epithelialization

Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.

Orientational Properties of DOPC/SM/Cholesterol Mixtures: A PM-IRRAS Study

Sphingomyelins (SM) and phosphatidylcholines (PC) are major lipid classes in the external plasma membrane leaflet of mammalian cells. A preferential interaction between SM and cholesterol (Cho) in both cell and model membranes has been proposed as central for the formation of Cho- and SM-rich domains in membranes. In this context, the relevance of the SM hydrophobic moiety on its interaction with Cho for domain stabilization has been investigated by our group (1-2). We report here on the effects of sphingomyelin structure on the orientational and conformational properties of monolayers of pure lipids and of two ternary lipid mixtures (DOPC/16:0SM/Cho and DOPC/24:1SM/Cho), which are relevant as mammalian cell membrane models.

Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis.

Sphingomyelin (SM) and cholesterol (Chol) are the major lipids in the mammalian cells, which are mainly localized to the plasma membrane. Multiple lines of evidence suggest that these lipids form local lipid domains in the plasma membrane, playing functional roles in the cell. Several observations have suggested that these lipid domains are required for cytokinesis. In this chapter, we show the methods for visualizing SM-rich and/or Chol-rich membrane domains at cytokinesis by using specific lipid-binding proteins. Lysenin, equinatoxin II, perfringolysin O, and pleurotolysin A2 bind specifically to clustered SM-rich domain, dispersed SM-rich domain, Chol-rich domain, and SM/Chol mixtures, respectively. Nontoxic forms of these lipid-binding proteins fused to fluorescent proteins are used for imaging lipid domains in biological membranes at cytokinesis. The image analysis reveals the structures and functions of SM-rich and/or Chol-rich domains at the time of cytokinesis.

Visualization of Lipid Membrane Reorganization Induced by a Pore-Forming Toxin Using High-Speed Atomic Force Microscopy.

We examined the effect of a sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, on the dynamics of a phase-separated membrane of SM, where SM formed liquid-ordered (Lo) domains with cholesterol (Chol) within a phosphatidylcholine-rich liquid-disordered (Ld) phase. We visualized the lysenin-induced membrane reorganization using high-speed atomic force microscope (HS-AFM). Lysenin oligomerized on the SM-rich Lo domain and simultaneously its oligomers assembled into a hexagonal close-packed (hcp) structure. The phase boundary was stable during the assembling of lysenin on the SM-rich domain, indicating that lysenin did not affect the line tension between Lo and Ld phases. After the full coverage of the SM-rich domain by oligomers, their hcp assembly gradually expanded into the Ld phase and eventually covered the entire membrane. Our results suggest that pore formation, i.e., insertion of lysenin into the membrane in its oligomeric state, induced the exclusion of SM and Chol from the SM-rich domain, which was followed by further binding and oligomerization of lysenin.

Solid State 2H NMR Studies of the Disordering of Raft-Like Domains by n-3 PUFA

The health benefits of omega-3 polyunsaturated fatty acids (n-3 PUFA) contained in fish oils continue to be investigated intensively in pre-clinical and clinical studies. The major bioactive components are docosahexaenoic acid (DHA, 22:6) with 22 carbons and 6 double bonds and eicosapentaenoic acid (EPA, 20:5) with 20 carbons and 5 double bonds. An emerging hypothesis is that n-3 PUFA are incorporated into membrane phospholipids and modify the structure and organization of lipid rafts, thus affecting cell signaling. We used solid-state 2H NMR spectroscopy to compare molecular organization in mixtures of 1-palmitoyl-2-docosahexaenoylphosphatidylcholine (PDPC) and 1-palmitoyl-2-eicosapentaenoylphosphatidylcholine (PEPC) with the raft-stabilizing molecules sphingomyelin (SM) and cholesterol. Spectra for PDPC-d31 and PEPC-d31, analogs of PDPC and PEPC with a perdeuterated palmitoyl sn-1 chain, revealed that DHA and EPA incorporate into raft-like domains enriched in SM and cholesterol. Greater incorporation was seen for DHA than EPA. We used PSM-d31, an analog of SM with a perdeuterated N-palmitoyl chain, as a probe to directly investigate molecular order within raft-like domains. Our initial experiments looked at mixtures of (POPC) with SM and cholesterol as a control. In POPC/SM (1:1 mol) mixtures, the 2H NMR spectra revealed segregation into POPC-rich (less ordered) and SM-rich (more ordered) domains that are nano-scale in size and contain < 180 lipids. When cholesterol (1:1:1 mol) was added, both domains became more ordered and greater mixing of POPC and SM was observed. These results will be discussed together with the results from experiments on PSM-d31 in mixtures with DHA- and EPA-containing phospholipids and cholesterol.

Assembling of a Pore-Forming Toxin on a Model Membrane

The assembling of the sphingomyelin (SM)-binding pore-forming toxin (PFT), lysenin, to SM/cholesterol bilayer was examined by high-speed atomic force microscopy (HS-AFM) [1]. The HS-AFM images of SM/cholesterol bilayer preincubated with lysenin exhibited the hexagonal close packed (hcp) assembly of lysenin oligomers (Fig. 1A). The in-situ AFM images revealed that the formation of the hcp structure took place quickly (Fig. 1B). Before the full coverage of the membrane surface with a stable hcp assembly of lysenin oligomers, most of the oligomers underwent reorganization either by dissociating into monomers or by rapidly diffusing along the membrane in less than a second. The assembling of lysenin oligomers was also followed on SM/DOPC/cholesterol bilayer. Oligomers firstly formed at the edges of the SM-rich domains and covered these domains similarly to the SM/cholesterol bilayer. Our results revealed the dynamic nature of the oligomers of a lipid binding toxin during its assembling on SM-containing membranes.Reference[1] N. Yilmaz, T. Yamada, P. Greimel, T. Uchihashi, T. Ando, and T. Kobayashi, Biophys. J. 105, 1397-1405 (2013).View Large Image | View Hi-Res Image | Download PowerPoint Slide

A Role for Sphingomyelin-Rich Lipid Domains in the Accumulation of Phosphatidylinositol-4,5-Bisphosphate to the Cleavage Furrow during Cytokinesis

Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of phosphatidylinositol-4,5-bisphosphate (PIP(2)) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Aβ1−40 peptide in solid‐supported lipid bilayers

We utilized plasmon-waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid beta peptide(1-40) in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of approximately 2.5-fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high-resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM-rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC-enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mM zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.

Detergent-Resistant, Ceramide-Enriched Domains in Sphingomyelin/Ceramide Bilayers

When cell membranes are treated with Triton X-100 or other detergents at 4°C, a nonsolubilized fraction can often be recovered, the “detergent-resistant membranes”, that is not found when detergent treatment takes place at 37°C. Detergent-resistant membranes may be related in some cases to membrane “rafts”. However, several basic aspects of the formation of detergent-resistant membranes are poorly understood. To answer some of the relevant questions, a simple bilayer composition that would mimic detergent-resistant membranes was required. The screening of multiple lipid compositions has shown that the binary mixture egg sphingomyelin/egg ceramide (SM/Cer) exhibits the required detergent resistance. In detergent-free membranes composed of different mixtures of SM and Cer (5–30mol % of Cer) differential scanning calorimetry, fluorescence spectroscopy, and fluorescence microscopy experiments reveal the presence of discrete, Cer-enriched gel domains in a broad temperature range. In particular, at temperatures below SM phase transition (≈ 40°C) two gel (respectively Cer-rich and SM-rich) phases are directly observed using fluorescence microscopy. Although pure SM membranes are fully solubilized by Triton X-100 at room temperature, 5mol % Cer is also enough to induce detergent resistance, even with a large detergent excess and lengthy equilibration times. Short-chain Cers do not give rise to detergent resistance. SM/Cer mixtures containing up to 30mol % Cer become fully soluble at ∼50°C, i.e., well above the gel-fluid transition temperature of SM. The combined results of temperature-dependent solubilization and differential scanning calorimetry reveal that SM-rich domains are preferentially solubilized over the Cer-rich ones as soon as the former melt (i.e., at ∼40°C). As a consequence, at temperatures allowing only partial solubilization, the nonsolubilized residue is enriched in Cer with respect to the original bilayer composition. Fluorescence microscopy of giant unilamellar vesicles at room temperature clearly shows that SM-rich domains are preferentially solubilized over the Cer-rich ones and that the latter become more rigid and extensive as a consequence of the detergent effects. These observations may be relevant to the phenomena of sphingomyelinase-dependent signaling, generation of “raft platforms”, and detergent-resistant cell membranes.

Ligand Modulation of Lateral Segregation of a G-Protein-Coupled Receptor into Lipid Microdomains in Sphingomyelin/Phosphatidylcholine Solid-Supported Bilayers

A growing body of evidence supports the idea that the plasma membrane bilayer is characterized by a laterally inhomogeneous mixture of lipids, having an organized structure in which lipid molecules segregate into small domains or patches. Such microdomains are characterized by high contents of sphingolipids that form thicker liquid-ordered regions that are resistant to extraction with nonionic detergents. The existence of lipid lateral segregation has been demonstrated in both model and biological membranes, although its role in protein sorting and membrane function still remains unclear. In these studies, plasmon-waveguide resonance (PWR) spectroscopy was employed to investigate the properties of microdomains in a model system consisting of a solid-supported lipid bilayer composed of a 1:1 mixture of palmitoyloleoylphosphatidylcholine (POPC) and brain sphingomyelin (SM), and their influence on the partitioning and functioning of the human delta opioid receptor (hDOR), a G-protein coupled receptor (GPCR). Resonance signals corresponding to two microdomains (POPC-rich and SM-rich) were observed in such bilayers, and the sorting of the receptor into each domain was highly dependent on the type of ligand that was bound. When no ligand was bound, the receptor was incorporated preferentially into the POPC-rich domain; when an agonist or antagonist was bound, the receptor was incorporated preferentially into the SM-rich component, although with a 2-fold greater propensity for this microdomain in the case of the agonist. Binding of G-protein to the agonist-bound receptor in the SM-rich domain occurred with a 30-fold higher affinity than binding to the receptor in the PC-rich domain. The binding of the agonist to an unliganded receptor in the bilayer produced receptor trafficking from the PC-rich to the SM-rich component. Since the SM-rich domain is thicker than the PC-rich domain, and previous studies with the hDOR have shown that the receptor is elongated upon agonist activation, we propose that hydrophobic matching between the receptor and the lipid is a driving force for receptor trafficking to the SM-rich component.