Masahiro Takagi

Name:

Organization: Japan Advanced Institute of Science and Technology , Japan

Department: School of Materials Science

Title: Professor(PhD)

Chemicals
References

Raft-dependent endocytic movement and intracellular cluster formation during T cell activation triggered by concanavalin A

Co-reporter:Satomi Yabuuchi, Satoshi Endo, KeangOk Baek, Kunihide Hoshino, ... Masahiro Takagi

Journal of Bioscience and Bioengineering 2017 Volume 124, Issue 6(Volume 124, Issue 6) pp:

Publication Date(Web):1 December 2017

DOI:10.1016/j.jbiosc.2017.06.009

Certain food ingredients can stimulate the human immune system. A lectin, concanavalin A (ConA), from Canavalia ensiformis (jack bean) is one of the most well-known food-derived immunostimulants and mediates activation of cell-mediated immunity through T cell proliferation. Generally, T cell activation is known to be triggered by the interaction between T cells and antigen-presenting cells (APCs) via a juxtacrine (contact-dependent) signaling pathway. The mechanism has been well characterized and is referred to as formation of the immunological synapse (IS). We were interested in the mechanism behind the T cell activation by food-derived ConA which might be different from that of T cell activation by APCs. The purpose of this study was to characterize T cell activation by ConA with regard to (i) movement of raft domain, (ii) endocytic vesicular transport, (iii) the cytoskeleton (actin and microtubules), and (iv) cholesterol composition. We found that raft-dependent endocytic movement was important for T cell activation by ConA and this movement was dependent on actin, microtubules, and cholesterol. The T cell signaling mechanism triggered by ConA can be defined as endocrine signaling which is distinct from the activation process triggered by interaction between T cells and APCs by juxtacrine signaling. Therefore, we hypothesized that T cell activation by ConA includes both two-dimensional superficial raft movement on the membrane surface along actin filaments and three-dimensional endocytic movement toward the inside of the cell along microtubules. These findings are important for developing new methods for immune stimulation and cancer therapy based on the function of ConA.

Formation of modulated phases and domain rigidification in fatty acid-containing lipid membranes

Co-reporter:Naofumi Shimokawa;Rieko Mukai;Mariko Nagata

Physical Chemistry Chemical Physics 2017 vol. 19(Issue 20) pp:13252-13263

Publication Date(Web):2017/05/24

DOI:10.1039/C7CP01201B

We investigated the phase behavior of lipid membranes containing fatty acids (FAs) by microscopy and differential scanning calorimetry. We used palmitic acid (saturated FA), oleic acid (cis-isomer of unsaturated FA), elaidic acid (trans-isomer of unsaturated FA), and phytanic acid (branched FA) and examined the effects of FAs on phase-separated structures in lipid bilayer membranes consisting of dioleolylphosphocholine (DOPC)/dipalmitoylphosphocholine (DPPC)/cholesterol (Chol). Palmitic acid and elaidic acid exclude Chol from the DPPC-rich phase. As a result, the liquid-ordered phase formed by DPPC and Chol transforms into a solid-ordered phase. Oleic acid and phytanic acid significantly reduce the line tension at the liquid domain boundary. This decrease in line tension leads to the formation of modulated phases, such as striped, hexagonal, and polygonal domains. We measured the line tension and the interdomain interaction in these specific domains by an image analysis. The result showed that oleic acid and phytanic acid-containing vesicles as well as palmitic acid-containing vesicles are not spherical, and this domain-induced deformation is explained theoretically.

Physical properties of the hybrid lipid POPC on micrometer-sized domains in mixed lipid membranes

Co-reporter:Naofumi Shimokawa, Mariko Nagata and Masahiro Takagi

Physical Chemistry Chemical Physics 2015 vol. 17(Issue 32) pp:20882-20888

Publication Date(Web):16 Jul 2015

DOI:10.1039/C5CP03377B

Macro-phase separation in mixed lipid membranes containing the hybrid lipid palmitoyloleoylphosphatidylcholine (POPC) was observed by fluorescent and confocal laser scanning microscopy. In a binary system consisting of the saturated lipid dipalmitoylphosphatidylcholine (DPPC) and the hybrid lipid POPC, the hybrid lipid forms a liquid-disordered (Ld) phase. In a ternary system consisting of this binary system and an unsaturated lipid dioleoylphosphatidylcholine (DOPC), three-phase coexistence is observed. The POPC-rich phase appears around DPPC-rich domains, and the hybrid lipid is expected to behave like a line-active agent (linactant). Finally, phase separation in a four-component system, composed of this ternary system and cholesterol, was examined. Domains with a size that is smaller than 1 μm are found, and domain-induced budding is also observed. To explain small domain formation and domain-induced budding, chain ordering was evaluated based on Laurdan generalized polarization measurements. Our observations revealed that the hybrid lipid acted like a linactant to solid domains and disturbed chain ordering in liquid-ordered (Lo) domains. In both cases, the hybrid lipid reduced line tension at the domain boundary.

Size-Dependent Partitioning of Nano/Microparticles Mediated by Membrane Lateral Heterogeneity

Co-reporter:Tsutomu Hamada ; Masamune Morita ; Makiyo Miyakawa ; Ryoko Sugimoto ; Ai Hatanaka ; Mun’delanji C. Vestergaard

Journal of the American Chemical Society 2012 Volume 134(Issue 34) pp:13990-13996

Publication Date(Web):August 8, 2012

DOI:10.1021/ja301264v

It is important that we understand the physical, chemical, and biological mechanisms that govern the interaction between nanoparticles (NPs) and heterogeneous cellular surfaces because of the possible cytotoxicity of engineered nanomaterials. In this study, we investigated the lateral localization of nano/microparticles within a biomimetic heterogeneous membrane interface using cell-sized two-phase liposomes. We found that lateral heterogeneity in the membrane mediates the partitioning of nano/microparticles in a size-dependent manner: small particles with a diameter of ≤200 nm were localized in an ordered phase, whereas large particles preferred a fluidic disordered phase. This partitioning behavior was verified by temperature-controlled membrane miscibility transition and laser-trapping of associated particles. In terms of the membrane elastic energy, we present a physical model that explains this localization preference of nano/microparticles. The calculated threshold diameter of particles that separates the particle-partitioning phase was 260 nm, which is in close agreement with our observation (200 nm). These findings may lead to a better understanding of the basic mechanisms that underlie the association of nanomaterials within a cell surface.

Physicochemical Profiling of Surfactant-Induced Membrane Dynamics in a Cell-Sized Liposome

Co-reporter:Tsutomu Hamada, Hideyuki Hagihara, Masamune Morita, Mun’delanji C. Vestergaard, Yoshio Tsujino, and Masahiro Takagi

The Journal of Physical Chemistry Letters 2012 Volume 3(Issue 3) pp:430-435

Publication Date(Web):January 17, 2012

DOI:10.1021/jz2016044

We used a cell-sized model system, giant liposomes, to investigate the interaction between lipid membranes and surfactants, and the membrane transformation during the solubilization process was captured in real time. We found that there are four distinct dynamics in surfactant-induced membrane deformation: an episodic increase in the membrane area prior to pore-forming associated shrinkage (Dynamics A), fission into many small liposomes (Dynamics B), the formation of multilamellar vesicles and peeling (Dynamics C), and bursting (Dynamics D). Classification of the diversity of membrane dynamics may contribute to a better understanding of the physicochemical mechanism of membrane solubilization induced by various surfactants.Keywords: cell-sized liposome; detergent; lipid bilayer; membrane pore; morphological change; solubilization; surfactant;

Photochemical control of membrane raft organization

Co-reporter:Tsutomu Hamada, Ryoko Sugimoto, Takeshi Nagasaki and Masahiro Takagi

Soft Matter 2011 vol. 7(Issue 1) pp:220-224

Publication Date(Web):09 Oct 2010

DOI:10.1039/C0SM00797H

Controllable membrane phase separation through the action of a synthetic photoresponsive amphiphile is reported. We studied multi-component giant vesicles formed from a ternary lipid mixture of saturated and unsaturated phospholipid and cholesterol together with the photoresponsive amphiphile. A change in the conformation of the photoresponsive amphiphile can switch membrane lateral segregation in a reversible manner. Cis-isomerization induces lateral phase separation in one-phase membranes or produces additional lateral domains in two-phase membranes. Membranes that are close to miscibility boundary show high photo-responsiveness. This is the first report on the reversible control of membrane lateral segregation triggered by a conformational change in a membrane-constituting molecule. These findings may lead to new methods for controlling membrane self-organization such as raft engineering.

Membrane Disk and Sphere: Controllable Mesoscopic Structures for the Capture and Release of a Targeted Object

Co-reporter:Tsutomu Hamada ; Ryoko Sugimoto ; Mun’delanji C. Vestergaard ; Takeshi Nagasaki

Journal of the American Chemical Society 2010 Volume 132(Issue 30) pp:10528-10532

Publication Date(Web):July 12, 2010

DOI:10.1021/ja103895b

Design of molecules for self-assembled mesoscopic structures with specific functions is an important and interesting challenge that spans across disciplines such as nanosciences. A closed lipid membrane is a good example of a self-assembled mesostructure. In this study, we developed controllable membrane formation by making a subtle change at the molecular level. We utilized a synthetic photosensitive amphiphile (KAON12) to achieve the photobased molecular manipulation of the opening and closing of membranes through reversible transitions between sphere and disk structures. We found that the mechanism is based on the photoswitching of the membrane line tension, as deduced from the fluctuation of the membrane edge, through the action of KAON12. Furthermore, we demonstrated the controllable capture and release of colloidal particles into and from a membrane sphere. The observation of Brownian motion of the particle confirmed colloidal encapsulation. This successful photomanipulation of mesoscopic membrane structures in a noncontact and reversible manner should lead to a better understanding of the mechanism of membrane self-organization and may see wider application, such as in microreactors and drug-delivery systems.

Biomimetic Microdroplet Membrane Interface: Detection of the Lateral Localization of Amyloid Beta Peptides

Co-reporter:Tsutomu Hamada, Masamune Morita, Yuko Kishimoto, Yuuki Komatsu, Mun’delanji Vestergaard and Masahiro Takagi

The Journal of Physical Chemistry Letters 2010 Volume 1(Issue 1) pp:170-173

Publication Date(Web):November 12, 2009

DOI:10.1021/jz900106z

Lateral membrane organization into domains, such as lipid rafts, plays an important role in the selective association of biological and nonbiological materials on heterogeneous membrane surfaces. The localization of such materials has profound influence on cellular responses. We constructed a biomimetic water-in-oil microdroplet membrane to study the lateral localization of these materials at heterogeneous biological interfaces. As a case study, we studied selective association of amyloid β peptide on the constructed membrane surface. Amyloid β peptide has attracted much attention as one of these membrane-associating proteins because of its “role” in Alzheimer’s disease pathology. Ternary lipid membranes covering microdroplets successfully produced lipid ordered structures, which mimicked biological lipid rafts. We revealed that amyloid β peptide selectively localizes within nonraft fluid membrane regions. The successful lateral organization in microdroplet membrane systems may lead to new opportunities for the study of molecular associations within heterogeneous membranes. Keywords (keywords): amyloid β peptide; lipid monolayer; lipid rafts; membrane interaction; water-in-oil microdroplets;

Role of C-terminal Cys-rich Region of Phytochelatin Synthase in Tolerance to Cadmium Ion Toxicity

Co-reporter:Sachiko Matsumoto;Mun’delanji Vestergaard

Journal of Plant Biochemistry and Biotechnology 2009 Volume 18( Issue 2) pp:175-180

Publication Date(Web):2009 July

DOI:10.1007/BF03263316

Phytochelatins (PCs) are Cys-rich peptides, synthesized by PC synthase in response to heavy metal ions. The C-terminal Cys-rich region of the PC synthase has homology with functional domains of metallochaperone, metallothionein and thioredoxin. To test the possibility that the C-terminal Cys-rich region of PC synthase has a role in regulating PC synthesis, we introduced point mutations into the PC synthase, replacing Cys358, Cys359 Cys363 and Cys366 residues with Ala. The mutant PC synthase had a lower PC synthesis ability than the wild-type enzyme. Further, oxidative conditions severely damaged mutant PC synthase whilst the wild-type enzyme suffered less damage, suggesting that the Cys-rich region of PC synthase may play an important role in anti-oxidation activity. Although the C-terminal of PC synthase is not conserved, our studies support the possibility that this region performs several important biological functions.

Construction of Asymmetric Cell-Sized Lipid Vesicles from Lipid-Coated Water-in-Oil Microdroplets

Co-reporter:Tsutomu Hamada, Yoko Miura, Yuuki Komatsu, Yuko Kishimoto, Mun’delanji Vestergaard and Masahiro Takagi

The Journal of Physical Chemistry B 2008 Volume 112(Issue 47) pp:14678-14681

Publication Date(Web):November 4, 2008

DOI:10.1021/jp807784j

We present a simple, rapid, and robust method for preparing asymmetric cell-sized lipid bilayer vesicles using water-in-oil (W/O) microdroplets transferred through an oil−water interface. The efficiency for producing cell-sized model membranes is elucidated in relation to the vesicular size and the weight of contained water-soluble molecules. We demonstrate the biological asymmetric nature and the formation of lipid raft microdomain structures using fluorescence microscopy.

Effect of phospholipids on conformational structure of bovine pancreatic trypsin inhibitor (BPTI) and its thermolabile mutants

Co-reporter:Naoshige Izumikawa;Shingo Nishikori;Mun'delanji Vestergaard;Tsutomu Hamada;Yoshihisa Hagihara;Noboru Yumoto;Kentaro Shiraki

Biopolymers 2008 Volume 89( Issue 10) pp:873-880

Publication Date(Web):

DOI:10.1002/bip.21029

Abstract

The effects of negatively charged phosphatidylserine-prepared membranes (PS) and neutral phosphatidylcholine-prepared membranes (PC) on the structure of wild-type and mutant bovine pancreatic trypsin inhibitor (BPTI) at neutral pH were investigated. The presence of PC did not have any effect on the protein structure while PS induced a non-native structure in three mutant BPTI proteins. However, the negatively charged membrane did not have any effect on wild-type BPTI. The findings revealed that (i) elimination of some disulphide bonds results in dramatic change in protein structure, and, (ii) that this biochemical interaction is surface-driven and electrostatic interactions may play a very strong role in influencing the fore-stated changes in protein structure. Of further interest were the results obtained from investigating the possible role of PS fluidity and concentration in altering mutant. When the value of Gibbs free-energy change of unfolding (ΔG_U) was positive, various non-native structures were formed in a concentration-dependent manner. However, when the value of ΔG_U was negative, only two types of non-native structures were formed: one with high β structure content at low PS fluidity state, and the other with a high α-helical content at high PS fluidity state. Our study reveals how particular combinations of phospholipid:protein interactions can induce a protein conformation transition from a native to a non-native one at neutral pH, especially when the native structure is predestabilized by amino acid substitutions. This revelation may open up opportunities to explore alternative ways in which phospholipids may play a role in protein mis-folding and the related pathologies. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 873–880, 2008.

This article was originally published online as an accepted preprint. The “Published Online” date corresponds to preprint version. You can request a copy of the preprint emailing the Biopolymers editorial office at biopolymers@wiley.com

Unfolding mechanism of a hyperthermophilic protein O6-methylguanine-DNA methyltransferase

Co-reporter:Shingo Nishikori, Kentaro Shiraki, Shinsuke Fujiwara, Tadayuki Imanaka, Masahiro Takagi

Biophysical Chemistry 2005 Volume 116(Issue 2) pp:97-104

Publication Date(Web):1 July 2005

DOI:10.1016/j.bpc.2005.03.003

Unfolding intermediates have been found only rarely in earlier studies, and how a protein unfolds is therefore poorly understood. In this paper, we show experimental evidence for multiple pathways and multiple intermediates during unfolding reaction of O6-methylguanine-DNA methyltransferase from hyperthermophile Thermococcus kodakaraensis (Tk-MGMT). The unfolding profiles monitored by far-UV CD and tryptophan fluorescence were both biphasic, and unfolding monitored by fluorescence was faster than that monitored by CD. GdnHCl-induced titration curves indicate that the intermediates with significant α-helical structure accumulate during unfolding. Dependence of kinetic phases on initial GdnHCl concentrations and cysteine reactivity of Tk-MGMT were investigated, suggesting that the heterogeneity of native conformations and parallel unfolding pathways.

Functional analysis of phytochelatin synthase from Arabidopsis thaliana and its expression in Escherichia coli and Saccharomyces cerevisiae

Co-reporter:Sachiko Matsumoto, Kentaro Shiraki, Naoki Tsuji, Kazumasa Hirata, Kazuhisa Miyamoto, Masahiro Takagi

Science and Technology of Advanced Materials 2004 Volume 5(Issue 3) pp:377-381

Publication Date(Web):May 2004

DOI:10.1016/j.stam.2004.01.005

Plants, like other organisms, have adaptive mechanisms whereby they are able to respond to both nutrient deficiencies and toxicities. Phytochelatins (PCs) play an essential role in heavy-metal detoxification in plants, fungi and worms. PCs chelate heavy metals and then PC-metal complexes are translocated across the tonoplast and sequestered in vacuoles. PCs are synthesized from glutathione by the enzyme PC synthase (PCS). Comparison of the deduced amino acid sequences of PCS suggests that the C-terminal domain may be important for activation of the enzyme. We established the method for purification of PCS from Arabidopsis thaliana to perform enzymatic characterization. Moreover, the PCS gene was expressed in E. coli and S. cerevisiae to enhance tolerance to toxicity of cadmium ion. The obtained results implied that the some regions of the PCS may serve as regulatory region through interaction with cadmium ion and/or oxygen related compounds. Moreover, PCS expression dramatically enhanced cadmium ion tolerance of different organisms. Based on our findings, functional mechanism for PCS activation was hypothesized.

The effect of oxycholesterols on thermo-induced membrane dynamics

Co-reporter:Mun'delanji C. Vestergaard, Tsuyoshi Yoda, Tsutomu Hamada, Yoko Akazawa (Ogawa), Yasukazu Yoshida, Masahiro Takagi

Biochimica et Biophysica Acta (BBA) - Biomembranes (September 2011) Volume 1808(Issue 9) pp:

Publication Date(Web):September 2011

DOI:10.1016/j.bbamem.2011.05.002

The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7β-hydroxycholesterol (7β) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7β; (iii) 7β and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.Research highlights► We demonstrate that oxy-cholesterol-containing lipid vesicles were more sensitive to temperature. ► We show that keto functional group rendered lipid vesicles more thermo-sensitive than OH group at carbon 7. ► We show that OH group at C 25 has the same effect as the keto group at C 7. ► Characterization of the membrane dynamics provide a new insight into oxidative-stressed membranes. ► We show that temperature change in conjunction with membrane oxidation induces membrane instability.

Damage in brain development by morpholino knockdown of zebrafish dax1

Co-reporter:Yoshifumi Yajima, Mun'delanji C. Vestergaard, Masahiro Takagi

Journal of Bioscience and Bioengineering (June 2012) Volume 113(Issue 6) pp:683-688

Publication Date(Web):1 June 2012

DOI:10.1016/j.jbiosc.2012.02.002

DAX1 is an orphan nuclear receptor and involved in development of steroidogenic organs, which activates transcription of genes involved in steroidogenesis. In this study, we analyzed the function of the zebrafish dax1 during early development of central nervous systems to appear unidentified aspects of DAX1 and decrease confusions concerned with functions of DAX1 in early development of vertebrates. By whole-mount in situ hybridization of embryo at the 32 h post fertilization (hpf), expression of zebrafish dax1 was detected around the forebrain, midbrain, hindbrain, and the extending tail tip. Embryos injected with zebrafish dax1 morpholino antisense nucleotide (MO) exhibited delayed development. When the developmental stage of wild type embryos was at Prim-15 (32 hpf), zebrafish dax1MO injected embryos were at Prim-5 (24 hpf). Concurrently with developmental delay, the MO injected embryos showed high mortality. At 48 hpf, the MO injected embryos exhibited abnormal development in the central nervous systems. The enlarged tectum and the protruded rhombomeres were observed. Moreover, development of central nervous systems, especially midbrain–hindbrain boundary, became narrower. At 5 day post fertilization, the MO injected embryos formed edemas around head, pericardial sac and abdomen. Collectively, our results indicated that the zebrafish dax1 is important for brain development.

Localization of amyloid beta (Aβ1-42) protofibrils in membrane lateral compartments: Effect of cholesterol and 7-Ketocholesterol

Co-reporter:Huong T.T. Phan, Mun’delanji C. Vestergaard, KeangOk Baek, Naofumi Shimokawa, Masahiro Takagi

FEBS Letters (17 September 2014) Volume 588(Issue 18) pp:3483-3490

Publication Date(Web):17 September 2014

DOI:10.1016/j.febslet.2014.08.007

•Cholesterol modulates the localization of Aβ in all membrane lateral compartments.•7-Ketocholesterol majorly increases Aβ association with liquid-disordered phase.•Cholesterol and 7-ketocholesterol influence the fluidity of membrane lateral compartments.Cholesterol plays an important role in the interaction of Alzheimer’s amyloid beta (Aβ) with cell membranes, an important event in Aβ-induced cytotoxicity. However, it is not fully understood how cholesterol influences the association of Aβ with membrane lateral compartments. We have shown that by modulating membrane fluidity, cholesterol decreased peptide localization in solid-ordered domains and increased that in liquid-ordered domains. It changed the amount of Aβ associating with liquid-disordered (Ld) phase with different tendencies depending on the composition of heterogeneous membrane systems. 7-Ketocholesterol, an oxidized derivative of cholesterol, majorly enhanced the fluidity of and Aβ interaction with Ld phase. These findings are useful for clarifying the impact of cholesterol and its oxidation in Aβ-induced toxicity.

Formation of modulated phases and domain rigidification in fatty acid-containing lipid membranes

Co-reporter:Naofumi Shimokawa, Rieko Mukai, Mariko Nagata and Masahiro Takagi

Physical Chemistry Chemical Physics 2017 - vol. 19(Issue 20) pp:NaN13263-13263

Publication Date(Web):2017/04/24

DOI:10.1039/C7CP01201B

Physical properties of the hybrid lipid POPC on micrometer-sized domains in mixed lipid membranes

Co-reporter:Naofumi Shimokawa, Mariko Nagata and Masahiro Takagi

Physical Chemistry Chemical Physics 2015 - vol. 17(Issue 32) pp:NaN20888-20888

Publication Date(Web):2015/07/16

DOI:10.1039/C5CP03377B