Motohide Murate

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.—Makino, A., Abe, M., Murate, M., Inaba, T., Yilmaz, N., Hullin‐Matsuda, F., Kishimoto, T., Schieber, N. L., Taguchi, T., Arai, H., Anderluh, G., Parton, R. G., Kobayashi, T. Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis. FASEB J. 29, 477‐493 (2015). www.fasebj.org

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.

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.

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-β-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.

CARTS biogenesis requires VAP-lipid transfer protein complexes functioning at the endoplasmic reticulum-Golgi interface.

Biogenesis of the TGN-derived transport carriers CARTS requires the ER protein VAP and Golgi lipid transfer proteins, ceramide transfer protein and OSBP. Sac1 lipid phosphatase is recruited to a VAP–OSBP complex formed at an ER subdomain closely apposed to the trans-Golgi/TGN. Association–dissociation dynamics of ER–Golgi contacts are important for CARTS formation.

Selective incorporation of docosahexaenoic acid into lysobisphosphatidic acid in cultured THP-1 macrophages.

Lysobisphosphatidic acid (LBPA) is highly accumulated in specific domains of the late endosome and is involve in the biogenesis and function of this organelle. Little is known about the biosynthesis and metabolism of this lipid. We examined its FA composition and the incorporation of exogenous FA into LBPA in the human monocytic leukemia cell line THP-1. The LBPA FA composition in THP-1 cells exhibits an elevated amount of oleic acid (18∶1n−9) and enerichment of PUFA, especially DHA (22∶6n−3). DHA supplemented to the medium was efficiently incorporated into LBPA. In contrast, arachidonic acid (20∶4n−6) was hardly esterified to LBPA under the same experimental conditions. The turnover of DHA in LBPA was similar to that in other phospholipids. Specific incorporation of DHA into LBPA was also observed in baby hamster kidney fibroblasts, although LBPA in these cells contains very low endogenous levels of DHA in normal growth conditions. Our results, together with published observations, suggest that the specific incorporation of DHA into LBPA is a common phenomenon in mammalian cells. The physiological significance of DHA-enriched LBPA is discussed.

Revisiting transbilayer distribution of lipids in the plasma membrane.

Whereas asymmetric transbilayer lipid distribution in the plasma membrane is well recognized, methods to examine the precise localization of lipids are limited. In this review, we critically evaluate the methods that are applied to study transbilayer asymmetry of lipids, summarizing the factors that influence the measurement. Although none of the present methods is perfect, the current application of immunoelectron microscopy-based technique provides a new picture of lipid asymmetry. Next, we summarize the transbilayer distribution of individual lipid in both erythrocytes and nucleated cells. Finally we discuss the concept of the interbilayer communication of lipids.

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 family—pleurotolysin A2 (PlyA2; KD = 12nM), ostreolysin (Oly; KD = 1.3 nM), and erylysin A (EryA; KD = 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 (KD = 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.—Bhat, H. B., Ishitsuka, R., Inaba, T., Murate, M., Abe, M., Makino, A., Kohyama‐Koganeya, A., Nagao, K., Kurahashi, A., Kishimoto, T., Tahara, M., Yamano, A., Nagamune, K., Hirabayashi, Y., Juni, N., Umeda, M., Fujimori, F., Nishibori, K., Yamaji‐Hasegawa, A., Greimel, P., Kobayashi, T. Evaluation of aegerolysins as novel tools to detect and visualize ceramide phosphoethanolamine, a major sphingolipid in invertebrates. FASEB J. 29, 3920‐3934 (2015). www.fasebj.org

Evaluation of the influence of ionization states and spacers in the thermotropic phase behaviour of amino acid-based cationic lipids and the transfection efficiency of their assemblies

The influence of both the ionization states and the hydrocarbon chain spacer of a series of amino acid-based cationic lipids was evaluated in terms of gene delivery efficiency and cytotoxicity to the COS-7 cell line and compared with that of Lipofectamine 2000. We synthesized a series of amino acid-based cationic lipids with different ionization states (i.e., -NH(2), -NH(3)(+)Cl(-) or -NH(3)(+)TFA(-)) in the lysine head group and different hydrocarbon chain spacers (i.e., 0, 3, 5 or 7 carbon atoms) between the hydrophilic head group and hydrophobic moieties. In the 3-carbon series, the cationic assemblies formed a micellar structure in the presence of -NH(3)(+)Cl(-) and a vesicular structure both in the presence of -NH(2) and -NH(3)(+)TFA(-). Differential scanning calorimetry (DSC) data revealed a significantly lower (8.1°C) gel-to-liquid crystalline phase transition temperature for cationic assemblies bearing -NH(3)(+)TFA(-) when compared to their -NH(2) counterparts. Furthermore, the zeta potential of cationic assemblies having -NH(3)(+)TFA(-) in the hydrophilic head group was maximum followed by -NH(3)(+)Cl(-) and -NH(2) irrespective of their hydrocarbon chain spacer length. The gene delivery efficiency in relation to the ionization states of the hydrophilic head group was as follows: -NH(3)(+)TFA(-)>-NH(3)(+)Cl(-)>-NH(2).

Phospholipase Cβ1 induces membrane tubulation and is involved in caveolae formation

Significance Lipid membrane curvature plays important roles in various physiological phenomena. Using darkfield microscopy, we performed nonbiased screening of a protein that induces deformations of nonlabeled liposomes. We identified phospholipase Cβ1 (PLCβ1), which induces tubulation of the phosphatidylethanolamine and phosphatidylserine-containing membranes. The characteristic C-terminal sequence of PLCβ1, but not the conserved inositol phospholipid-binding pleckstrin homology (PH) domain or catalytic domains of PLCβ1, is involved in the tubulation of liposomes. The C-terminal sequence is predicted to have the Bin/amphiphysin/Rvs (BAR)-like conformation by computational modeling. Our results indicate that sensing and modulation of the curvature by the C-terminal BAR-like domains is involved in the activation of PLCβ1. The present results also reveal the role of PLCβ1 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 Cβ1 (PLCβ1) as a new candidate. PLCβ1 is well characterized as an enzyme catalyzing the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2). In addition to lipase activity, our results indicate that PLCβ1 possessed the ability of membrane tubulation. Lipase domains and inositol phospholipids binding the pleckstrin homology (PH) domain of PLCβ1 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 PLCβ1 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 PLCβ1: plasma membrane remodeling, and in particular, caveolae formation.