Structure, fonction et dynamique des lipides membranaires dans les infections T. Kobayashi

Biological membranes fulfill a range of functions that go far beyond shaping and maintenance of cells’ architectural features. Cell membranes are composed of numerous different lipid molecules that are not randomly distributed in the membrane but organized in a well-controlled and highly dynamic way. It is, for example, well established that lipid composition of outer and inner leaflets of the lipid bilayer are distinct and exhibit transbilayer asymmetry. Lipids are also asymmetrically distributed laterally and segregate to form specific lipid domains such as lipid rafts. Specific organization of lipids is proposed to provide platforms for protein assembly involved in signal transduction or to be responsible for membrane deformation critical for membrane trafficking. Pathogens such as bacteria and virus often hi-jack these different systems, but little is known about the changes in lipid distribution and organization in the host cell membranes during infection. This is mainly because of the lack of appropriate methods and tools to label lipids specifically and to examine their distribution dynamically at the sub-micron scale. This research axis develops tools and techniques to visualize lipids and apply them to elucidate structure, function and dynamics of lipid membrane in patho-physiological conditions (for recent review of the group see 1,2,3,4,5).

1. 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 or parasite Trypanosoma brucei. 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 (6).

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

CPE is a major sphingolipid in invertebrates and parasites, whereas only trace amounts are present in mammalian cells. Mushroom-derived proteins of the aegerolysin family—pleurotolysin A2 (PlyA2;KD = 12 nM), 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 SM/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 crossreactivity 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 (9).

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

We identified a novel, nontoxic mushroom protein that specifically binds to a complex of SM and 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, nakanori treatment of epithelial cells after influenza virus infection potently inhibited virus release demonstrating the therapeutic value of targeting specific lipid domains for anti-viral treatment (10).

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

CPE 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 (11,12).

3. 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 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 (13).


1. Murate, M.; Kobayashi, T., Revisiting transbilayer distribution of lipids in the plasma membrane. Chem Phys Lipids 2016, 194, 58-71.

2. Kishimoto, T.; Ishitsuka, R.; Kobayashi, T., Detectors for evaluating the cellular landscape of sphingomyelin- and cholesterol-rich membrane domains. Biochim Biophys Acta 2016, 1861 (8 Pt B), 812-29.

3. Yamaji-Hasegawa, A.; Hullin-Matsuda, F.; Greimel, P.; Kobayashi, T., Pore-forming toxins: Properties, diversity, and uses as tools to image sphingomyelin and ceramide phosphoethanolamine. Biochim Biophys Acta 2016, 1858 (3), 576-92.

4. Kobayashi, T.; Menon, A. K., Transbilayer lipid asymmetry. Curr Biol 2018, 28 (8), R386-R391.

5. Yilmaz, N.; Yamaji-Hasegawa, A.; Hullin-Matsuda, F.; Kobayashi, T., Molecular mechanisms of action of sphingomyelin-specific pore-forming toxin, lysenin. Sem Cell Dev Biol 2018, 73, 188-198.

6. Hullin-Matsuda, F.; Murate, M.; Kobayashi, T., Protein probes to visualize sphingomyelin and ceramide phosphoethanolamine. Chem Phys Lipids 2018, 216, 132-141.

7. Abe, M.; Kobayashi, T., Dynamics of sphingomyelin- and cholesterol-enriched lipid domains during cytokinesis. Methods Cell Biol 2017, 137, 15-24.

8. Tomishige, N.; Murate, M.; Didier, P.; Richert, L.; Mély, Y.; Kobayashi, T., The use of pore-formaing toxin to image lipids and lipid domains. Methods Enz 2021 in press.

9. 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 2015, 29(9), 3920-3934.

10. 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. FASEB J 2017, 31 (4), 1301-1322.

11. Inaba, T.; Murate, M.; Tomishige, N.; Lee, Y.-F.; Hullin-Matsuda, F.; Pollet, B.; Humbert, N.; Mély, Y.; Sako, Y.; Greimel, P.; Kobayashi, T., Formation of tubules and helical ribbons by ceramide phosphoethanolamine-containing membranes. Sci Rep 2019, 9 (1), 5812.

12. Murate, M.; Tomishige, N.; Kobayashi, T., Wrapping axons in mammals and Drosophila: Different lipids, same principle. Biochimie 2020, 178, 39-48.

13. Kishimoto, T.; Tomishige, N.; Murate, M.; Ishitsuka, R.; Schaller, H.; Mély, Y.; Ueda, K.; Kobayashi, T., Cholesterol asymmetry at the tip of filopodia during cell adhesion. FASEB J 2020, 34(5), 6185-6197.