Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

A novel sterol-binding protein reveals heterogeneous cholesterol distribution in neurite outgrowth and in late endosomes/lysosomes.

We identified a mushroom-derived protein, maistero-2 that specifically binds 3-hydroxy sterol including cholesterol (Chol). Maistero-2 bound lipid mixture in Chol-dependent manner with a binding threshold of around 30%. Changing lipid composition did not significantly affect the threshold concentration. EGFP-maistero-2 labeled cell surface and intracellular organelle Chol with higher sensitivity than that of well-established Chol probe, D4 fragment of perfringolysin O. EGFP-maistero-2 revealed increase of cell surface Chol during neurite outgrowth and heterogeneous Chol distribution between CD63-positive and LAMP1-positive late endosomes/lysosomes. The absence of strictly conserved Thr-Leu pair present in Chol-dependent cytolysins suggests a distinct Chol-binding mechanism for maistero-2.

Cholesterol asymmetry at the tip of filopodia during cell adhesion.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

Phospholipase Cß1 induces membrane tubulation and is involved in caveolae formation.

Lipid membrane curvature plays important roles in various physiological phenomena. Curvature-regulated dynamic membrane remodeling is achieved by the interaction between lipids and proteins. So far, several membrane sensing/sculpting proteins, such as Bin/amphiphysin/Rvs (BAR) proteins, are reported, but there remains the possibility of the existence of unidentified membrane-deforming proteins that have not been uncovered by sequence homology. To identify new lipid membrane deformation proteins, we applied liposome-based microscopic screening, using unbiased-darkfield microscopy. Using this method, we identified phospholipase Cß1 (PLCß1) as a new candidate. PLCß1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCß1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCß1 were not involved, but the C-terminal sequence was responsible for this tubulation activity. Computational modeling revealed that the C terminus displays the structural homology to the BAR domains, which is well known as a membrane sensing/sculpting domain. Overexpression of PLCß1 caused plasma membrane tubulation, whereas knockdown of the protein reduced the number of caveolae and induced the evagination of caveolin-rich membrane domains. Taken together, our results suggest a new function of PLCß1: plasma membrane remodeling, and in particular, caveolae formation.

A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of sphingomyelin (SM), a major sphingolipid in mammalian cells, and cholesterol (Chol). The purified protein, termed nakanori, labeled cell surface domains in an SM- and Chol-dependent manner and decorated specific lipid domains that colocalized with inner leaflet small GTPase H-Ras, but not K-Ras. The use of nakanori as a lipid-domain-specific probe revealed altered distribution and dynamics of SM/Chol on the cell surface of Niemann-Pick type C fibroblasts, possibly explaining some of the disease phenotype. In addition, that nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrates the therapeutic value of targeting specific lipid domains for anti-viral treatment.-Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C.-G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., Kobayashi, T. A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C.

Transbilayer distribution of lipids at nano scale.

There is a limited number of methods to examine transbilayer lipid distribution in biomembranes. We employed freeze-fracture replica-labelling immunoelectron microscopy in combination with lipid-binding proteins and a peptide to examine both transbilayer distribution and lateral distribution of various phospholipids in mammalian cells. Our results indicate that phospholipids are exclusively distributed either in the outer or inner leaflet of human red blood cell (RBC) membranes. In contrast, in nucleated cells, such as human skin fibroblasts and neutrophils, sphingomyelin was distributed in both leaflets while exhibiting characteristic lipid domains in the inner leaflet. Similar to RBCs, lipid asymmetry was maintained both in resting and thrombin-activated platelets. However, the microparticles released from thrombin-activated platelets lost membrane asymmetry. Our results suggest that the microparticles were shed from platelet plasma membrane domains enriched with phosphatidylserine and/or phosphatidylinositol at the outer leaflet. These findings underscore the strict regulation and cell-type specificity of lipid asymmetry in the plasma membrane.

Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.

Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates.

Ceramide phosphoethanolamine (CPE), a sphingomyelin analog, is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. In this study, mushroom-derived proteins of the aegerolysin family­pleurotolysin A2 (PlyA2; K(D) = 12 nM), ostreolysin (Oly; K(D) = 1.3 nM), and erylysin A (EryA; K(D) = 1.3 nM)­strongly associated with CPE/cholesterol (Chol)-containing membranes, whereas their low affinity to sphingomyelin/Chol precluded establishment of the binding kinetics. Binding specificity was determined by multilamellar liposome binding assays, supported bilayer assays, and solid-phase studies against a series of neutral and negatively charged lipid classes mixed 1:1 with Chol or phosphatidylcholine. No cross-reactivity was detected with phosphatidylethanolamine. Only PlyA2 also associated with CPE, independent of Chol content (K(D) = 41 µM), rendering it a suitable tool for visualizing CPE in lipid-blotting experiments and biologic samples from sterol auxotrophic organisms. Visualization of CPE enrichment in the CNS of Drosophila larvae (by PlyA2) and in the bloodstream form of the parasite Trypanosoma brucei (by EryA) by fluorescence imaging demonstrated the versatility of aegerolysin family proteins as efficient tools for detecting and visualizing CPE.

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains.

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-ß-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.

Distribution of Glycolipids in the Plasma Membrane Monitored by Specific Probes in Combination with Sodium Dodecyl Sulfate-Digested Freeze-Fracture Replica Labeling (SDS-FRL).

Glycolipids are mainly distributed in the outer leaflet of the plasma membrane and are involved in cellular signaling by modulating the activity of cell surface receptor proteins. Glycolipids themselves also work as cell surface receptors of bacterial toxins. Anti-glycolipid antibodies are associated with various pathological conditions. The cellular distribution of glycolipids has been studied using specific toxins or antibodies. However, these proteins are multivalent and thus potentially induce the artificial aggregation of glycolipids. Since chemical fixative such as paraformaldehyde does not fix glycolipids, an alternative methodology is required to localize glycolipids with multivalent probes. Sodium dodecyl sulfate-digested freeze-fracture replica labeling (SDS-FRL) physically fixes glycolipids on the cast after quick freezing. Thus, SDS-FRL provides the opportunity to observe the natural distribution of glycolipids using multivalent probes. Here, we describe the application of SDS-FRL on the cell surface distribution of phosphatidylglucoside.

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains.

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly.

PMP2/FABP8 induces PI(4,5)P2-dependent transbilayer reorganization of sphingomyelin in the plasma membrane.

Sphingomyelin (SM) is a mammalian lipid mainly distributed in the outer leaflet of the plasma membrane (PM). We show that peripheral myelin protein 2 (PMP2), a member of the fatty-acid-binding protein (FABP) family, can localize at the PM and controls the transbilayer distribution of SM. Genetic screening with genome-wide small hairpin RNA libraries identifies PMP2 as a protein involved in the transbilayer movement of SM. A biochemical assay demonstrates that PMP2 is a phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-binding protein. PMP2 induces the tubulation of model membranes in a PI(4,5)P2-dependent manner, accompanied by the modification of the transbilayer membrane distribution of lipids. In the PM of PMP2-overexpressing cells, inner-leaflet SM is increased whereas outer-leaflet SM is reduced. PMP2 is a causative protein of Charcot-Marie-Tooth disease (CMT). A mutation in PMP2 associated with CMT increases its affinity for PI(4,5)P2, inducing membrane tubulation and the subsequent transbilayer movement of lipids.

The use of pore-forming toxins to image lipids and lipid domains.

Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.

Impact of Intrinsic and Extrinsic Factors on Cellular Sphingomyelin Imaging with Specific Reporter Proteins.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells. Although SM is enriched in the outer leaflet of the cell plasma membrane, lipids are also observed in the inner leaflet of the plasma membrane and intracellular organelles such as endolysosomes, the Golgi apparatus and nuclei. SM is postulated to form clusters with glycosphingolipids (GSLs), cholesterol (Chol), and other SM molecules through hydrophobic interactions and hydrogen bonding. Thus, different clusters composed of SM, SM/Chol, SM/GSL and SM/GSL/Chol with different stoichiometries may exist in biomembranes. In addition, SM monomers may be located in the glycerophospholipid-rich areas of membranes. Recently developed SM-binding proteins (SBPs) distinguish these different SM assemblies. Here, we summarize the effects of intrinsic factors regulating the lipid-binding specificity of SBPs and extrinsic factors, such as the lipid phase and lipid density, on SM recognition by SBPs. The combination of different SBPs revealed the heterogeneity of SM domains in biomembranes.

Extreme deformability of insect cell membranes is governed by phospholipid scrambling.

Organization of dynamic cellular structure is crucial for a variety of cellular functions. In this study, we report that Drosophila and Aedes have highly elastic cell membranes with extremely low membrane tension and high resistance to mechanical stress. In contrast to other eukaryotic cells, phospholipids are symmetrically distributed between the bilayer leaflets of the insect plasma membrane, where phospholipid scramblase (XKR) that disrupts the lipid asymmetry is constitutively active. We also demonstrate that XKR-facilitated phospholipid scrambling promotes the deformability of cell membranes by regulating both actin cortex dynamics and mechanical properties of the phospholipid bilayer. Moreover, XKR-mediated construction of elastic cell membranes is essential for hemocyte circulation in the Drosophila cardiovascular system. Deformation of mammalian cells is also enhanced by the expression of Aedes XKR, and thus phospholipid scrambling may contribute to formation of highly deformable cell membranes in a variety of living eukaryotic cells.

Phosphatidylglucoside forms specific lipid domains on the outer leaflet of the plasma membrane.

Phosphatidylglucoside (PtdGlc) is a recently discovered unique glycophospholipid involved in granulocytic differentiation of human promyelocytic leukemia cell line HL60 and in astrocytic differentiation in developing rodent brains. Using a PtdGlc-specific monoclonal antibody in immunofluorescence and immunoelectron microscopy, we showed that PtdGlc forms distinct lipid domains on the outer leaflet of the plasma membrane of HL60 cells and the human alveolar epithelial cell line, A549. Similar to glycosphingolipid, glucosylceramide (GlcCer), the natural form of PtdGlc exhibited a high main phase transition temperature in differential scanning calorimetry (DSC). However, unlike GlcCer, PtdGlc did not exhibit a large difference in the main phase transition temperature between the heating and cooling scans. DSC further indicated that GlcCer, but not PtdGlc, was miscible with sphingomyelin. In addition, DSC and small-angle X-ray scattering (SAXS) experiments revealed that PtdGlc was poorly miscible with phosphatidylcholine. Our results suggest that the lack of tight intermolecular interaction excludes PtdGlc from other lipid domains on the plasma membrane.

Cholesterol controls lipid endocytosis through Rab11.

Cellular cholesterol increases when cells reach confluency in Chinese hamster ovary (CHO) cells. We examined the endocytosis of several lipid probes in subconfluent and confluent CHO cells. In subconfluent cells, fluorescent lipid probes including poly(ethylene glycol)derivatized cholesterol, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol, and fluorescent sphingomyelin analogs were internalized to pericentriolar recycling endosomes. This accumulation was not observed in confluent cells. Internalization of fluorescent lactosylceramide was not affected by cell confluency, suggesting that the endocytosis of specific membrane components is affected by cell confluency. The crucial role of cellular cholesterol in cell confluency-dependent endocytosis was suggested by the observation that the fluorescent sphingomyelin was transported to recycling endosomes when cellular cholesterol was depleted in confluent cells. To understand the molecular mechanism(s) of cell confluency- and cholesterol-dependent endocytosis, we examined intracellular distribution of rab small GTPases. Our results indicate that rab11 but not rab4, altered intracellular localization in a cell confluency-associated manner, and this alteration was dependent on cell cholesterol. In addition, the expression of a constitutive active mutant of rab11 changed the endocytic route of lipid probes from early to recycling endosomes. These results thus suggest that cholesterol controls endocytic routes of a subset of membrane lipids through rab11.

Wrapping axons in mammals and Drosophila: Different lipids, same principle.

Plasma membranes of axon-wrapping glial cells develop specific cylindrical bilayer membranes that surround thin individual axons or axon bundles. Axons are wrapped with single layered glial cells in lower organisms whereas in the mammalian nervous system, axons are surrounded with a characteristic complex multilamellar myelin structure. The high content of lipids in myelin suggests that lipids play crucial roles in the structure and function of myelin. The most striking feature of myelin lipids is the high content of galactosylceramide (GalCer). Serological and genetic studies indicate that GalCer plays a key role in the formation and function of the myelin sheath in mammals. In contrast to mammals, Drosophila lacks GalCer. Instead of GalCer, ceramide phosphoethanolamine (CPE) has an important role to ensheath axons with glial cells in Drosophila. GalCer and CPE share similar physical properties: both lipids have a high phase transition temperature and high packing, are immiscible with cholesterol and form helical liposomes. These properties are caused by both the strong headgroup interactions and the tight packing resulting from the small size of the headgroup and the hydrogen bonds between lipid molecules. These results suggest that mammals and Drosophila wrap axons using different lipids but the same conserved principle.

Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes.

Ceramide phosphoethanolamine (CPE), a major sphingolipid in invertebrates, is crucial for axonal ensheathment in Drosophila. Darkfield microscopy revealed that an equimolar mixture of bovine buttermilk CPE (milk CPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (diC18:1 PC) tends to form tubules and helical ribbons, while pure milk CPE mainly exhibits amorphous aggregates and, at low frequency, straight needles. Negative staining electron microscopy indicated that helices and tubules were composed of multilayered 5-10 nm thick slab-like structures. Using different molecular species of PC and CPE, we demonstrated that the acyl chain length of CPE but not of PC is crucial for the formation of tubules and helices in equimolar mixtures. Incubation of the lipid suspensions at the respective phase transition temperature of CPE facilitated the formation of both tubules and helices, suggesting a dynamic lipid rearrangement during formation. Substituting diC18:1 PC with diC18:1 PE or diC18:1 PS failed to form tubules and helices. As hydrated galactosylceramide (GalCer), a major lipid in mammalian myelin, has been reported to spontaneously form tubules and helices, it is believed that the ensheathment of axons in mammals and Drosophila is based on similar physical processes with different lipids.

Protein probes to visualize sphingomyelin and ceramide phosphoethanolamine.

Sphingomyelin (SM) is a major sphingolipid in mammalian cells whereas its analog, ceramide phosphoethanolamine (CPE) is found in trace amounts in mammalian cells and in larger amounts in invertebrates such as insect cells like Drosophila melanogaster. To visualize endogenous SM or CPE, we need specific probes able to recognize the chemical structure of the lipid, rather than its physical property. A limited number of proteins is known to specifically and strongly bind SM or CPE. These proteins are either toxins produced by non-mammalian organisms, subunits or fragments of toxins or a protein that has similar structure to a toxin. These proteins labeled with small fluorophore (e.g. Alexa Fluor) or conjugated to fluorescent proteins (e.g. mCherry) or other types of markers (e.g. 125I, maltose-binding protein) are used to detect SM or CPE. Here we summarize the characteristics of specific SM-binding proteins, lysenin and equinatoxin II; CPE- and SM/cholesterol (Chol) binding aegerolysin proteins, pleurotolysin A2, ostreolysin and erylysin A and SM/Chol-binding protein, nakanori. Then we give examples of their applications including their limitations related not only to their lipid specificity and binding constants, but also to the lipid organization in the membrane.