| 1. |
Ryo Ohtani, Yuka Anegawa, Hikaru Watanabe, Yutaro Tajima, Masanao Kinoshita, Nobuaki Matsumori, Kenichi Kawano, Saeko Yanaka, Koichi Kato, Masaaki Nakamura, Masaaki Ohba, Shinya Hayami, Metal complex lipids for fluid-fluid phase separation in co-assembled phospholipid membranes., Angewandte Chemie (International ed. in English), 10.1002/anie.202102774, 60, 24, 13603-13608, 2021.03, We demonstrate a fluid-fluid phase separation in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes using a metal complex lipid of type [Mn(L1)] (1; HL1 = 1-(2-hydroxybenzamide)-2-(2-hydroxy-3-formyl-5-hexadecyloxybenzylideneamino)ethane. Small amount of 1 produces two separated domains in DMPC, whose phase transition temperatures of lipids (Tc) are both lower than that of the pristine DMPC. Variable temperature fluorescent microscopy for giant-unilamellar vesicles of DMPC/1 hybrids demonstrates that visible phase separations remain in fluid phases up to 37 °C, which is clearly over the Tc of DMPC. This provides a new dimension for the application of metal complex lipids toward controlling lipid distributions in fluid membranes.. |
| 2. |
Manami Hieda, Akira Sorada, Masanao Kinoshita, Nobuaki Matsumori, Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation, Biochemistry and Biophysics Reports, 10.1016/j.bbrep.2021.100941, 26, 100941-100941, 2021.07, Amphidinol 3 (AM3), a polyhydroxy-polyene metabolite from the dinoflagellate Amphidinium klebsii, possesses potent antifungal activity. AM3 is known to interact directly with membrane sterols and permeabilize membranes by forming pores. Because AM3 binds to sterols such as cholesterol and ergosterol, it can be assumed that AM3 has some impact on lipid rafts, which are membrane domains rich in sphingolipids and cholesterol. Hence, we first examined the effect of AM3 on phase-separated liposomes, in which raft-like ordered and non-raft-like disordered domains are segregated. Consequently, AM3 disrupted the phase separation at 22 μM, as in the case of methyl-β-cyclodextrin, a well-known raft-disrupter that extracts sterol from membranes. The surface plasmon resonance measurements and dye leakage assays show that AM3 preferentially recognizes cholesterol in the disordered membrane, which may reflect a weaker lipid-cholesterol interaction in disordered membrane than in ordered membrane. Finally, to gain insight into the AM3-induced coalescence of membrane phases, we measured membrane fluidity using fluorescence correlation spectroscopy, demonstrating that AM3 significantly increases the order of disordered phase. Together, AM3 preferentially binds to the disordered phase rather than the ordered phase, and enhances the order of the disordered phase, consequently blending the separated phases.. |
| 3. |
Michio Murata, Nobuaki Matsumori, Masanao Kinoshita, Erwin London, Molecular substructure of the liquid-ordered phase formed by sphingomyelin and cholesterol: sphingomyelin clusters forming nano-subdomains are a characteristic feature, Biophysical Reviews, 10.1007/s12551-022-00967-1, 14, 3, 655-678, 2022.06. |
| 4. |
Masanao Kinoshita, Nobuaki Matsumori, Inimitable Impacts of Ceramides on Lipid Rafts Formed in Artificial and Natural Cell Membranes, Membranes, 10.3390/membranes12080727, 12, 8, 727-727, 2022.07, Ceramide is the simplest precursor of sphingolipids and is involved in a variety of biological functions ranging from apoptosis to the immune responses. Although ceramide is a minor constituent of plasma membranes, it drastically increases upon cellular stimulation. However, the mechanistic link between ceramide generation and signal transduction remains unknown. To address this issue, the effect of ceramide on phospholipid membranes has been examined in numerous studies. One of the most remarkable findings of these studies is that ceramide induces the coalescence of membrane domains termed lipid rafts. Thus, it has been hypothesised that ceramide exerts its biological activity through the structural alteration of lipid rafts. In the present article, we first discuss the characteristic hydrogen bond functionality of ceramides. Then, we showed the impact of ceramide on the structures of artificial and cell membranes, including the coalescence of the pre-existing lipid raft into a large patch called a signal platform. Moreover, we proposed a possible structure of the signal platform, in which sphingomyelin/cholesterol-rich and sphingomyelin/ceramide-rich domains coexist. This structure is considered to be beneficial because membrane proteins and their inhibitors are separately compartmentalised in those domains. Considering the fact that ceramide/cholesterol content regulates the miscibility of those two domains in model membranes, the association and dissociation of membrane proteins and their inhibitors might be controlled by the contents of ceramide and cholesterol in the signal platform.. |
| 5. |
Yasuda Hiroyuki, Torikai Kouhei, Kinoshita Masanao, Sazzad MA, Tsujimura Kouya, Slotte J. Peter, Matsumori Nobuaki., Preparation of nitrogen analogs of ceramide and studies of theiraggregation in sphingomyelin bilayers, 2021.10. |
| 6. |
Ohtani Ryo, Anegawa Yuka, Watanabe Hikaru, Tajima Yutaro, Kinoshita Masanao, Matsumori Nobuaki, Kawano Kenichi, Yanaka Saeko, Kato Kouichi, Nakamura, Ohba Masaaki, Hayami Sinya., Metal complex lipids for fluid–fluid phase separation in co-assembled phospholipid membranes, 2021.03. |
| 7. |
Manami Hieda, Akira Sorada, Masanao Kinoshita, Nobuaki Matsumori
, Amphidinol 3 preferentially binds to cholesterol in disordered domains and disrupts membrane phase separation., Biochim. Biophys. Rep., 2021.02. |
| 8. |
Masanao Kinoshita, Satoru Kato, Intermolecular interaction of phosphatidylinositol with the lipid raft molecules sphingomyelin and cholesterol, Biophysics, 10.2142/biophysics.4.1, 4, 1-9, 2008.06, Diacylphosphatidylinositol (PI) is the starting reactant in the process of phosphatidylinositide-related signal transduction mediated through the lipid raft domain. We investigated intermolecular interactions of PI with major raft components, sphingomyelin (SM) and cholesterol (Chol), using surface pressure-molecular area (π-A) isotherm measurements. The classical mean molecular area versus composition plot showed that the measured mean molecular areas are smaller in PI/Chol mixed monolayers and larger in PI/SM mixed monolayers than those calculated on the basis of the ideal additivity. These results indicate that PI interacts attractively with Chol and repulsively with SM. In addition, we energetically evaluated the interaction of PI with SM/Chol mixtures and found that the mixing energy of PI/SM/Chol ternary monolayers decreased as the molar ratio of Chol to SM increased. In order to quantitatively analyze the distribution of PI we calculated the chemical potentials of mixing of PI into the SM/Chol mixed monolayer and into the dioleoylphosphatidylcholine (DOPC) monolayer, which was used as a model for the fluid matrix, on the basis of partial molecular area analysis. Analysis using the chemical potential of mixing of PI suggested that partition of PI molecules between these two monolayers can be changed by a factor of about 1.7 in response to change in Chol molar fraction in the SM/Chol mixed monolayer from 0.3 to 0.6 when the concentration of PI in the DOPC monolayer is kept constant at 7 mol%. ©2008 The Biophysical Society of Japan.. |
| 9. |
Masanao Kinoshita, Satoru Kato, Hiroshi Takahashi, NaCl-dependent formation of the highly crystalline phase in sufficiently hydrated dimyristoylphosphatidylglycerol bilayers, CHEMISTRY AND PHYSICS OF LIPIDS, 10.1016/j.chemphyslip.2009.06.143, 161, 1, 1-10, 2009.09, We investigated the low-temperature phase behavior of dimyristoylphosphatidylglycerol (DMPG) bilayers in the presence of high concentration of NaCl (>= 100mM). Differential scanning calorimetry showed that the highly crystalline (HC) phase grew after an initial delay period when DMPG bilayers were sufficiently hydrated and incubated at 1 degrees C in the presence of more than 100 mM NaCl. The HC phase formation reached a plateau, the level of which depended on NaCl concentration; all the lipids were unable to be in the HC phase at the plateau stage without a quite high concentration of NaCl. Since electron microscopic observations suggested that the HC phase formed coexists with the precursor phases in a closed vesicle, elastic constrain and/or shortage of free sodium ions in the inside of the closed vesicle may prevent the complete transition into the HC phase. (C) 2009 Elsevier Ireland Ltd. All rights reserved.. |
| 10. |
Masanao Kinoshita, Keisuke Ito, Satoru Kato, Kinetics for the subgel phase formation in DPPC/DOPC mixed bilayers, CHEMISTRY AND PHYSICS OF LIPIDS, 10.1016/j.chemphyslip.2010.06.007, 163, 7, 712-719, 2010.09, We analyzed the kinetics for the subgel (SGI) phase formation in DPPC/DOPC binary bilayers paying attention to DOPC-induced modification of the bilayer physical properties. Differential scanning calorimetry and X-ray diffraction revealed that addition of DOPC reduced the apparent initial lag time to start the SGI phase formation, and that the SGI phase in the binary bilayers had basically the same structure as that in pure DPPC bilayers though addition of DOPC markedly increased the peak temperature and enthalpy of the subtransition in heating. Moreover, addition of DOPC abolished the prolongation of the initial lag time in pure DPPC bilayers induced by lowering the incubation temperature from 0 to -5 degrees C. Our results suggested that DOPC molecules work as a diffusion enhancer to promote the nucleation of the SCI phase, and relatively destabilize the gel phase so that the formed SGI phase transforms into the ripple phase in heating. (C) 2010 Elsevier Ireland Ltd. All rights reserved.. |
| 11. |
Sarah A. Goretta, Masanao Kinoshita, Shoko Mori, Hiroshi Tsuchikawa, Nobuaki Matsumori, Michio Murata, Effects of chemical modification of sphingomyelin ammonium group on formation of liquid-ordered phase, BIOORGANIC & MEDICINAL CHEMISTRY, 10.1016/j.bmc.2012.05.015, 20, 13, 4012-4019, 2012.07, Sphingomyelin (SM) and cholesterol form microdomains called lipid rafts in cellular membranes. To develop a versatile fluorescent lipid probe, chemical modifications to both the hydrophobic and hydrophilic portions of SM are essential. Few reports describing SM probes with a fluorophore at the polar head group have been published. This study examined the effect of substitution on an ammonium moiety of SM on the membrane properties of SM. Two SM analogs with small propargyl and allyl groups on the quaternary nitrogen atom were synthesized and subjected to analysis using differential scanning calorimetry, fluorescent anisotropy, detergent solubilization, surface pressure, and density measurements. Results demonstrated that the two SM analogs retained the membrane properties of SM, including formation of an ordered phase and the ability to interact with cholesterol. A dansyl-substituted SM was prepared for fluorescent measurements. Dansyl-SM showed less of a propensity to form microdomains. These findings imply the potential application of N-substituted SMs as a raft-specific molecular probe. (C) 2012 Elsevier Ltd. All rights reserved.. |
| 12. |
Tomokazu Yasuda, Masanao Kinoshita, Michio Murata, Nobuaki Matsumori, Detailed Comparison of Deuterium Quadrupole Profiles between Sphingomyelin and Phosphatidylcholine Bilayers, BIOPHYSICAL JOURNAL, 10.1016/j.bpj.2013.12.034, 106, 3, 631-638, 2014.02, Lipid rafts are microdomains rich in sphingomyelin (SM) and cholesterol (Chol). The essential question is why natural lipid rafts prefer SM rather than saturated diacyl glycerophosphocholine, although both form ordered membranes with Chol in model systems. Hence in this study, we synthesized site-specifically deuterated 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholines that match the acyl chain length of stearoyl-SM (SSM), and compared their deuterium quadrupole coupling profiles in detail. The results suggest a deeper distribution of Chol in the SSM membranes, a lower entropic penalty upon accommodation of Chol in SSM membranes, and a higher thermal stability of acyl-chain orders in the SSM-Chol bilayers than in the 1-palmitoyl2-stearoyl-sn-glycero-3-phosphocholine-Chol system at various Chol concentrations. The entropy effect and thermal stability should render SM a more preferred raft constituent than saturated diacyl glycerophosphocholine. Our data also demonstrate that the selective and comprehensive deuteration strategy is indispensable for accurate comparison of order profiles.. |
| 13. |
Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Coexistence of two liquid crystalline phases in dihydrosphingomyelin and dioleoylphosphatidylcholine binary mixtures, BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 10.1016/j.bbamem.2014.01.017, 1838, 5, 1372-1381, 2014.05, Recently, DHSM, a minor constituent in naturally occurring SMs, was indicated to form a raft-like ordered phase more effectively than a naturally occurring form of SM because DHSM has greater potential to induce the intermolecular hydrogen bond. In order to examine the influence of the DHSM-induced hydrogen bond on die phase segregation, the thermal phase behavior of stearoyl-DHSM/DOPC binary bilayers was examined using calorimetry and fluorescence observation and compared with that of SSM/DOPC binary bilayers. Results revealed that the DHSM/DOPC bilayers undergo phase segregation between two La phases within a limited compositional range. On the other hand, apparent phase separation was not observed above main transition temperature in SSM/DOPC mixtures. Our monolayer measurements showed that the lipid packing of DHSM is less perturbed than that of SSM by the addition of small amount of DOPC, indicating a stronger hydrogen bond between DHSM molecules. Therefore, in DHSM/DOPC binary bilayers, DHSM molecules may locally accumulate to form a DHSM-rich domain due to a DHSM-induced hydrogen bond. On the other hand, excess accumulation of DHSM should be prevented because the difference in the curvature between DHSM and DOPC assemblies causes elastic constraint at the domain boundary between the DHSM-rich and DOPC-rich domains. Competition between the energetic advantages provided by formation of the hydrogen bond and the energetic disadvantage conferred by elastic constraints likely results in L-alpha/L-alpha, phase separation within a limited compositional range. (C) 2014 Elsevier B.V. All rights reserved.. |
| 14. |
Jun Ando, Masanao Kinoshita, Jin Cui, Hiroyuki Yamakoshi, Kosuke Dodo, Katsumasa Fujita, Michio Murata, Mikiko Sodeoka, Sphingomyelin distribution in lipid rafts of artificial monolayer membranes visualized by Raman microscopy, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1418088112, 112, 15, 4558-4563, 2015.04, 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.. |
| 15. |
Jin Cui, Shigeru Matsuoka, Masanao Kinoshita, Nobuaki Matsumori, Fuminori Sato, Michio Murata, Jun Ando, Hiroyuki Yamakoshi, Kosuke Dodo, Mikiko Sodeoka, Novel Raman-tagged sphingomyelin that closely mimics original raft-forming behavior, BIOORGANIC & MEDICINAL CHEMISTRY, 10.1016/j.bmc.2015.05.014, 23, 13, 2989-2994, 2015.07, Three Raman probes of sphingomyelin (SM) were synthesized and evaluated for their applicability to imaging experiments. One probe containing a hydroxymethyl-1,3-butadiyne moiety in the polar head group showed strong scattering. The solid-state H-2 NMR spectra of this probe in oriented bilayer membrane revealed excellent compatibility with natural SM in phase behavior since the probe undergoes phase separation to form raft-like liquid ordered (L-o) domains in the raft-mimicking mixed bilayers. (C) 2015 Elsevier Ltd. All rights reserved.. |
| 16. |
Kimberly Cornelio, Rafael Atillo Espiritu, Yasuto Todokoro, Shinya Hanashima, Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Minoru Yoshida, Shigeki Matsunaga, Sterol-dependent membrane association of the marine sponge-derived bicyclic peptide Theonellamide A as examined by H-1 NMR, BIOORGANIC & MEDICINAL CHEMISTRY, 10.1016/j.bmc.2016.08.043, 24, 21, 5235-5242, 2016.11, Theonellamide A (TNM-A) is an antifungal bicyclic dodecapeptide isolated from a marine sponge Theonella sp. Previous studies have shown that TNM-A preferentially binds to 3 beta-hydroxysterol-containing membranes and disrupts membrane integrity. In this study, several H-1 NMR-based experiments were performed to investigate the interaction mode of TNM-A with model membranes. First, the aggregation propensities of TNM-A were examined using diffusion ordered spectroscopy; the results indicate that TNM-A tends to form oligomeric aggregates of 2-9 molecules (depending on peptide concentration) in an aqueous environment, and this aggregation potentially influences the membrane-disrupting activity of the peptide. Subsequently, we measured the H-1 NMR spectra of TNM-A with sodium dodecyl sulfate-d(25) (SDS-d(25)) micelles and small dimyristoylphosphatidylcholine (DMPC)-d(54)/dihexanoylphosphatidylcholine (DHPC)-d(22) bicelles in the presence of a paramagnetic quencher Mn2+. These spectra indicate that TNM-A poorly binds to these membrane mimics without sterol and mostly remains in the aqueous media. In contrast, broader H-1 signals of TNM-A were observed in 10 mol % cholesterol-containing bicelles, indicating that the peptide efficiently binds to sterol-containing bilayers. The addition of Mn2+ to these bicelles also led to a decrease in the relative intensity and further line-broadening of TNM-A signals, indicating that the peptide stays near the surface of the bilayers. A comparison of the relative signal intensities with those of phospholipids showed that TNM-A resides in the lipid-water interface (close to the C2' portion of the phospholipid acyl chain). This shallow penetration of TNM-A to lipid bilayers induces an uneven membrane curvature and eventually disrupts membrane integrity. These results shed light on the atomistic mechanism accounting for the membrane-disrupting activity of TNM-A and the important role of cholesterol in its mechanism of action. (C) 2016 Elsevier Ltd. All rights reserved.. |
| 17. |
Rafael Atillo Espiritu, Kimberly Cornelio, Masanao Kinoshita, Nobuaki Matsumori, Michio Murata, Shinichi Nishimura, Hideaki Kakeya, Minoru Yoshida, Shigeki Matsunaga, Marine sponge cyclic peptide theonellamide A disrupts lipid bilayer integrity without forming distinct membrane pores, BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 10.1016/j.bbamem.2016.03.019, 1858, 6, 1373-1379, 2016.06, Theonellamides (TNMs) are antifungal and cytotoxic bicyclic dodecapeptides derived from the marine sponge Theonella sp. These peptides specifically bind to 3 beta-hydroxysterols, resulting in 1,3-beta-D-glucan overproduction and membrane damage in yeasts. The inclusion of cholesterol or ergosterol in phosphatidylcholine membranes significantly enhanced the membrane affinity of theonellamide A (TNM-A) because of its direct interaction with 3 beta-hydroxyl groups of sterols. To better understand TNM-induced membrane alterations, we investigated the effects of TNM-A on liposome morphology. P-31 nuclear magnetic resonance (NMR) and dynamic light scattering (DLS) measurements revealed that the premixing of TNM-A with lipids induced smaller vesicle formation. When giant unilamellar vesicles were incubated with exogenously added TNM-A, confocal micrographs showed dynamic changes in membrane morphology, which were more frequently observed in cholesterol-containing than sterol-free liposomes. In conjunction with our previous data, these results suggest that the membrane action of TNM-A proceeds in two steps: 1) TNM-A binds to the membrane surface through direct interaction with sterols and 2) accumulated TNM-A modifies the local membrane curvature in a concentration-dependent manner, resulting in dramatic membrane morphological changes and membrane disruption. (C) 2016 Elsevier B.V. All rights reserved.. |
| 18. |
Masanao Kinoshita, Kenichi G. N. Suzuki, Nobuaki Matsumori, Misa Takada, Hikaru Ano, Kenichi Morigaki, Mitsuhiro Abe, Asami Makino, Toshihide Kobayashi, Koichiro M. Hirosawa, Takahiro K. Fujiwara, Akihiro Kusumi, Michio Murata, Raft-based sphingomyelin interactions revealed by new fluorescent sphingomyelin analogs, JOURNAL OF CELL BIOLOGY, 10.1083/jcb.201607086, 216, 4, 1183-1204, 2017.04, Sphingomyelin (SM) has been proposed to form cholesterol-dependent raft domains and sphingolipid domains in the plasma membrane (PM). How SM contributes to the formation and function of these domains remains unknown, primarily because of the scarcity of suitable fluorescent SM analogs. We developed new fluorescent SM analogs by conjugating a hydrophilic fluorophore to the SM choline headgroup without eliminating its positive charge, via a hydrophilic nonaethylene glycol linker. The new analogs behaved similarly to the native SM in terms of their partitioning behaviors in artificial liquid order-disorder phase-separated membranes and detergent-resistant PM preparations. Single fluorescent molecule tracking in the live-cell PM revealed that they indirectly interact with each other in cholesterol-and sphingosine backbone-dependent manners, and that, for similar to 10-50 ms, they undergo transient colocalization-codiffusion with a glycosylphosphatidylinositol (GPI)-anchored protein, CD59 (in monomers, transient-dimer rafts, and clusters), in CD59-oligomer size-, cholesterol-, and GPI anchoring-dependent manners. These results suggest that SM continually and rapidly exchanges between CD59-associated raft domains and the bulk PM.. |
| 19. |
Ryo Ohtani, Tsukasa Tokita, Tomohisa Takaya, Koichi Iwata, Masanao Kinoshita, Nobuaki Matsumori, Masaaki Nakamura, Leonard F. Lindoy, Shinya Hayami, The impact of metal complex lipids on viscosity and curvature of hybrid liposomes, CHEMICAL COMMUNICATIONS, 10.1039/c7cc07944c, 53, 99, 13249-13252, 2017.12, A morphology transformation of hybrid liposomes was shown to occur from spherical vesicles to tubular micelles when increasing the ratio of the metal complex lipid present. Phase transition temperatures increased while viscosities decreased, indicating that the hybrids exhibit stronger interaction between heads but weaker interaction between alkyl chains than occurs in pristine liposomes.. |
| 20. |
Masanao Kinoshita, Hikaru Ano, Michio Murata, Kenta Shigetomi, Junichi Ikenouchi, Nobuaki Matsumori, Emphatic visualization of sphingomyelin-rich domains by inter-lipid FRET imaging using fluorescent sphingomyelins, SCIENTIFIC REPORTS, 10.1038/s41598-017-16361-x, 7, 7, 16801, 2017.12, Imaging the distribution of sphingomyelin (SM) in membranes is an important issue in lipid-raft research. Recently we developed novel fluorescent SM analogs that exhibit partition and dynamic behaviors similar to native SM, and succeeded in visualizing lateral domain-segregation between SM-rich liquid-ordered (L-o) and SM-poor liquid-disordered (L-d) domains. However, because the fluorescent contrast between these two domains depends directly on their partition ratio for the fluorescent SMs, domain-separation becomes indeterminate when the distribution difference is not great enough. In this study, we propose the use of inter-lipid Forster resonance energy transfer (FRET) imaging between fluorescent SMs to enhance the contrast of the two domains in cases in which the inter-domain difference in SM distribution is inadequate for conventional monochromic imaging. Our results demonstrate that inter-lipid FRET intensity was significantly higher in the Lo domain than in the L-d domain, resulting in a clear and distinguishable contrast between the two domains even in poorly phase-separated giant unilamellar vesicles. In addition, we show that inter-lipid FRET imaging is useful for selective visualization of highly condensed assemblies and/or clusters of SM molecules in living cell membranes. Thus, the inter-lipid FRET imaging technique can selectively emphasize the SM-condensed domains in both artificial and biological membranes.. |
| 21. |
Masayuki Iwamoto, Ayumi Sumino, Eri Shimada, Masanao Kinoshita, Nobuaki Matsumori, Shigetoshi Oiki, Channel Formation and Membrane Deformation via Sterol-Aided Polymorphism of Amphidinol 3, SCIENTIFIC REPORTS, 10.1038/s41598-017-11135-x, 7, 7, 10782, 2017.09, Amphidinol 3 (AM3) is an anti-fungal polyene extracted from a marine dinoflagellate. Here, we examined the ion channel activity and membrane-embedded structure of AM3 using a lipid bilayer method and atomic force microscopy (AFM). AM3 exhibited large-conductance (similar to 1 nS) and nonselective single-channel activity only when sterols were present in the membrane leaflet of the AM3-added side. The variable conductance suggests the formation of a multimeric barrel-stave pore. At high AM3 concentrations, giant-conductance "jumbo" channels (similar to 40 nS) emerged. AFM revealed a thicker raft-like membrane phase with the appearance of a wrinkled surface, in which phase pores (diameter: similar to 10 nm) were observed. The flip-flop of ergosterol occurred only after the appearance of the jumbo channel, indicating that the jumbo channel induced a continuity between the outer and inner leaflets of the membrane: a feature characteristic of toroidal-like pores. Thus, AM3 forms different types of sterol-aided polymorphic channels in a concentration dependent manner.. |
| 22. |
Takaaki Matsufuji, Masanao Kinoshita, Anna Möuts, J. Peter Slotte, Nobuaki Matsumori, Preparation and Membrane Properties of Oxidized Ceramide Derivatives, Langmuir, 10.1021/acs.langmuir.7b02654, 34, 1, 465-471, 2018.01, Ceramide is a bioactive lipid with important roles in several biological processes including cell proliferation and apoptosis. Although 3-ketoceramides that contain a keto group in place of the 3-OH group of ceramide occur naturally, ceramide derivatives oxidized at the primary 1-OH group have not been identified to date. To evaluate how the oxidative state of the 1-OH group affects the physical properties of membranes, we prepared novel ceramide derivatives in which the 1-OH group was oxidized to a carboxylic acid (PCerCOOH) or methylester (PCerCOOMe) and examined the rigidity of their monolayers and the formation of gel domains in palmitoyloleoylphosphatidylcholine (POPC) or sphingomyelin (SM) bilayers. As a result, PCerCOOH and PCerCOOMe exhibited membrane properties similar to those of native ceramide, although the deprotonated form of PCerCOOH, PCerCOO-, exhibited markedly lower rigidity and higher miscibility with POPC and SM. This was attributed to the electrostatic repulsion of the negative charge, which hampered the formation of the ceramide-enriched gel domain. The similarities in the properties of PCerCOOMe and ceramide revealed the potential to introduce various functional groups onto PCerCOOH via ester or amide linkages
therefore, these derivatives will also provide a new strategy for developing molecular probes, such as fluorescent ceramides, and inhibitors of ceramide-related enzymes.. |
| 23. |
Masataka Inada, Masanao Kinoshita, Nobuaki Matsumori, Archaeal Glycolipid S-TGA-1 Is Crucial for Trimer Formation and Photocycle Activity of Bacteriorhodopsin., ACS chemical biology, 10.1021/acschembio.9b00756, 15, 1, 197-204, 2020.01, Although it has been demonstrated that membrane proteins (MPs) require lipids to ensure their structural and functional integrity, details on how lipid-MP interactions regulate MPs are still unclear. Recently, we developed a concise method for quantitatively evaluating lipid-MP interactions and applied it to bacteriorhodopsin (bR), a halobacterial MP that forms trimers and acts as a light-driven proton pump. Consequently, we found that the halobacterial glycolipid, S-TGA-1, has the highest affinity for bR, among other lipids. In this study, we examined the effects of S-TGA-1 on bR via visible circular dichroism spectroscopy, flash photolysis, and proton influx measurement. The results showed that S-TGA-1 efficiently promotes trimer formation, photocycle, and proton pumping in bR. Our data also suggested that the bR photocycle is restored as a consequence of the trimerization induced by the lipid. This study demonstrates clearly that lipids specifically interacting with MPs can have significant impacts on MP structure and/or function. The methodology adopted in our studies can be applied to other MPs and will help elucidate the physiological functions of lipids in terms of lipid-MP interactions, thus accelerating "lipid chemical biology" studies.. |
