System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A0A011: : EEmergence of an artificial cellular modelmergence of an artificial cellular modelKenichi Yoshikawa, Hideki Seto, Hiroyuki, Kitahata (Kyoto Univ.)
1. Chromosome reconstitution in a test tube.2. Switching on transcription and translation by the transition in
the higher-order structure of DNA.3. System dynamics and function in an artificial cell model.
【Publications】
1) M. Hase and K. Yoshikawa, J. Chem. Phys., 124, 104903 (2006).2) A. A. Zinchenko, K. Yoshikawa, and D. Baigl, Phys. Rev. Lett., 95, 228101-1 - 228101-4 (2005).3) F. Luckel, K. Kubo, K. Tsumoto, and K. Yoshikawa, FEBS Lett., 579, 5119-5122 (2005).
5 mmFig. 1 Discrete on/off conformational change in reconstituted chromatin. Free energy of chromatin is deduced from the distribution nucleosomeparticles.
Fig. 2 Switching on the assembly of actin filament in a droplet.
K. Yoshikawa
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: A01: in vivoin vivo reconstitution and manipulation of higherreconstitution and manipulation of higher--order protein complexes using nanoorder protein complexes using nano--particlesparticles
Shige H. Yoshimura (Kyoto Univ.), Kohji Hizume (Kyoto Univ.)
S.H. Yoshimura K. Hizume
100 nm100 nm10 μm
[1] J. Kim, S.H. Yoshimura, K. Hizume, R.L. Ohniwa, A. Ishihama and K. Takeyasu, “Fundamental structural units of the Escherichia colinucleoid revealed by atomic force microscopy”, Nuc. Acids Res. 32(6), 1982-1992 (2004).[2] S.H. Yoshimura, H. Maruyama, R. Ohki, F. Ishikawa and K. Takeyasu, “Molecular mechanism of DNA end-loop formation by TRF2”, Genes Cells 9(3), 205-218 (2004).[3] K. Hizume, S.H. Yoshimura and K. Takeyasu “Linker histone H1 per se can induce three-dimensional folding of chromatin fiber”, Biochemistry, 44(39), 12978-12989 (2005).
1. Nano-scale observation of in vitro-reconstituted chromatin fiber
2. Structural and functional analysis of Nuclear Pore Complex
3. Single-molecule analysis of enzyme function using fast-scanning AFM
4. In-vivo manipulation of protein-coupled nano-particle
5. In vivo reconstitution of higher-order protein complex using nano-particles
HeLa cells carrying fluorescent nano-particles (~100 nm)
In vitro-reconstituted chromatin fiber
Nuclear envelope and nuclear pore complex of X. laevis oocyte
Nuclear envelope reconstituted around sepharose beads
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01:Development of a genomeA01:Development of a genome--scale scale in vitroin vitrotranscription and translation systemtranscription and translation system
Tadayuki Imanaka (Kyoto Univ.), Tamotsu Kanai (Kyoto Univ.)
【Publications】
1) T. Fukui, H. Atomi, T. Kanai, R. Matsumi, S. Fujiwara, and T. Imanaka, Genome Res., 15(3), pp. 352-363 (2005)
2) T. Endoh, T. Kanai, Y. T. Sato, D. V. Liu, K. Yoshikawa, H. Atomi, and T. Imanaka, J. Biotechnol., in press
T. Imanaka T. Kanai
1. To develop a system for in vitro transcription and translation using an extract from a hyperthermophile.
2. To explore the possibility of a genome-scale in vitrotranscription and translation reaction.
T. kodakaraensisDNA Microarray
Thermococcus kodakaraensis KOD1(hyperthermophilic
archaeon)
T. kodakaraensisGenome Project(2,088,737-base)
Gene Exchange System(first in the hyperthermophiles)
In vitro Translation(optimal temp. = 65 °C)
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Gene Transduction Using EpsteinA01: Gene Transduction Using Epstein--Barr VirusBarr Virus--based based Artificial ChromosomeArtificial Chromosome
Osam Mazda (Kyoto Prefectural University of Medicine)
Signal TransductionFunctional GenomicsGene Therapy, Molecular Therapyetc.
Multi-scale manipulationand measurement
EBV
非EBV
高発現
複製 核内移行
EBV非EBV
CD8発現量(対数) 01000200030004000500060007000
細胞数
DpnI MboI Sau3AI
高効率導入
Luc活
性
EBV
Conven-tional
High Intensity
Repli-cation
Nuclear Transfer
EBVConventional
01000200030004000500060007000
DpnI MboI Sau3AI
Highly Efficient Transfection/Expression
Luc A
ctivi
ty
High Frequency
Cell
Count
Log Expression Level
oriP sequence
EBNA1gene
EBNA1 protein
FRDSLinking region 1
GA repeat
Linking region 2Nuclear localization signalDimerization/DNA binding
EBV-Artificial Chromosome
NH2 COOH
Binding
Nuclear Transfer
Nuclear Retention
DNA duplication
Segregation
Transcriptional Enhancement
O. Mazda
T. Kishida
H. Asada
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A0A011: : Molecular basis ofMolecular basis of protein synthesis and its protein synthesis and its application application forfor reconstitution of cellular functionsreconstitution of cellular functions
Masatoshi Kataoka (Health Technology Research Center, AIST)
【Publications】[1] M. Kataoka, Y. Fukura, Y. Shinohara, Y. Baba, “Analysis of mitochondrial membrane potential in the cells by
microchip flow cytometry”, Electrophoresis 26, 3025-3031 (2005).[2] M. Kataoka, S. Inoue, K. Kajimoto, Y. Shinohara, Y. Baba, “Usefulness of microchip electrophoresis for reliable
analyses of non-standard DNA samples and subsequent on-chip enzymatic digestion”, Eur. J. Biochem. 271, 2241-2247 (2004).
[3] M. Hino, Y. Shinohara, K. Kajimoto, H. Terada, Y. Baba, “Requirement of continuous transcription for the synthesis of sufficient amounts of protein by a cell-free rapid translation system”, Protein Express. Purif. 24, 255-259 (2002).
Biochemical characterization of cell-size liposome
reaction chamber
semi-permeable membranesupply/wastecompartment
M. Kataoka
Studies on the interaction between model membrane protein and lipid bilayermembrane
MQSVITDVTGQLTAVQADITTIGGAIIVLAAVVLGIRWIKAQLF
membrane spanning region- -
[Pf3-WT]
LLL[Pf3-3L]
- -- -(N
)(C
)
LUV
(N)
(C)
++
--
--
++
LLL
[Pf3-WT] [Pf3-3L]
(N)
(C)
LUV
(N)
(C)
++
--
--
++
LLL
[Pf3-WT] [Pf3-3L]
(N)
(C)
LUV
(N)
(C)
++
--
--
++
LLL
[Pf3-WT] [Pf3-3L]
Development of the device seeking optimum condition of protein synthesis
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
H. Miyadera
Analysis on the protein interaction in the signal transduction pathway
1. Measurement of the activity of signal transduction.
2. Understanding the molecular basis of signal transduction by reconstitution and kinetic analysis.
A01: Reconstitution of Signal Transduction PathwayA01: Reconstitution of Signal Transduction Pathway
Hiroko Miyadera (Univ. Tokyo)
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Single Molecular Analyses of Genomic DNA and A01: Single Molecular Analyses of Genomic DNA and Biochemical Experiments in a CellBiochemical Experiments in a Cell--sized Vesicle sized Vesicle
Hidehiro Oana (The University of Tokyo)
H. Oana
burst cellsuspension intermediate
burst cell
higher saltsolution
cover glassTOPVIEW
SIDEVIEW
1. To achieve optical mapping of intact genomic DNA and recover DNA fragments of interest.
2. To achieve biochemical experiments in a cell-sized vesicle with the recovered DNA fragments.
transporting direction
10 μm
reaction in the vesicle
DNA fragment
cell-sized vesicle
【Publications】
1) K. Terao, H. Kabata, H. Oana and M. Washizu, Proc. 9th International Conference on Miniaturized Systems for Chemistry and Life Sciences (MicroTAS 2005), 1, 715-717 (2005).
2) H. Oana, K. Kubo, K. Yoshikawa, H. Atomi, and T. Imanaka, Appl. Phys. Lett., 85, 5090-5092 (2004).
Isolation of a genomic DNA Biochemical experimentsOptical mapping
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Design of artificial cell with membraneA01: Design of artificial cell with membraneproteinprotein--function by chaperone engineeringfunction by chaperone engineering
Kazunari Akiyoshi (Tokyo Medical and Dental Univ.)
cDNA
mRNA
Membrane protein
Chaperone
cDNA
mRNA
Membrane protein
Chaperone
1. To directly construct membrane protein-reconstituted liposomes by in vitro protein synthesis.
2. To develop artificial chaperone to assist membrane protein refolding.
【Publications】1. N. Morimoto, T. Endo, Y. Iwasaki, K. Akiyoshi, Biomacromolecule. 6, 1829-1834 (2005)2. Y. Nomura, M. Ikeda, N. Yamaguchi, Y. Aoyama, K. Akiyoshi, FEBS Lett. 553, 271-276 (2003)
Catching
Releasing
Nanogel
Folding protein
Nativeprotein
Inhibition
Aggregation
Catching
ReleasingReleasing
NanogelNanogel
Folding protein
Nativeprotein
InhibitionInhibition
AggregationAggregation
Artificial cellChaperonefunction
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: ArtificialA01: Artificial--cellular network model to cellular network model to work with chemicalwork with chemical--communicationscommunications
Shin-ichiro M. Nomura (Tokyo Medical & Dental Univ.)
S.-i. M. Nomura
【Publications】• S.-i. M. Nomura, Y. Mizutani, K. Kurita, A. Watanabe, K. Akiyoshi, BBA Biomembrane, 1669, 164 (2005).• S.-i. M. Nomura, K. Tsumoto, T. Hamada, K. Akiyoshi, Y. Nakatani, K. Yoshikawa, ChemBioChem, 4,
1172-1175 (2003).
We’d like to constract a model of artificial cellular network driven in chemical energy-dissipative system. However, biochemical reaction entrapped within liposomes MUST DIE because of substrates starvation. For living their LIFE, it should be refueled from their environmental system (filled with substrates).
The possible “refueling” routes are:-Gating channel on the membrane,-Fusion, connection with the other liposomes.
Expression of the membrane channel and connectable/demountable-liposome network are now under investigation.
Liposome network
REFUELING
Gene-expressing Liposome
AminoAcids,
ATP, etc.
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Response of Artificial Cells to Environment and A01: Response of Artificial Cells to Environment and Construction of Functional Artificial CellsConstruction of Functional Artificial Cells
Masahito Yamazaki (Shizuoka Univ.)
1. To construct artificial cells such as GUVs (Giant Unilamellar Vesicles) of lipid membranes containing cytoskelton, and to investigate their response to applied force and osmotic pressure by their environment.
2. To investigate membrane fusion and vesicle fission using the single GUV method, and to construct artificial cells with these functions.
M. Yamazaki
Membrane fusion and vesicle fission【Publications】
1) Y. Tamba, M. Yamazaki, Biochemistry, 44, 15823-15833 (2005)2) T. Tanaka, R. Sano, Y. Yamashita, M. Yamazaki, Langmuir, 20, 9526-9534 (2004)
Mechanical response of single filamin A molecules
Actin/filamin A gel
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Direct Observation of Transformation Process of Giant A01: Direct Observation of Transformation Process of Giant Liposome Induced by Reconstructed CytoskeletonsLiposome Induced by Reconstructed Cytoskeletons
TAKIGUCHI, Kingo (Nagoya Univ.)
1. To study membrane morphogenesis that is induced by cytoskeletons, behaviors of giant liposome under interaction with cytoskeletal proteins are directory observed with optical microscopes.
2. Construct artificial cells made of giant liposome and cytoskeletons, molecular motors, and membrane associating proteins.
【Publications】1) Inaba T., Ishijima A., Honda M., Nomura F., Takiguchi K., Hotani H. J. Mol. Biol. 348, 325-333 (2005)2) Tanaka-Takiguchi Y., Kakei T., Tanimura A., Takagi A., Honda M., Hotani H., Takiguchi K. J. Mol. Biol. 341, 467-476 (2004)3) Takiguchi K., Nomura F., Inaba T., Takeda S., Saitoh A., Hotani H. ChemPhysChem 3, 571-574 (2002)
K. Takiguchi
+ Actin&
Fascin
+ MT+ Actin
&Myosin-I
Bar = 5 μm
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01:Reconstitution of Functions of Membrane Protein A01:Reconstitution of Functions of Membrane Protein Complex Systems on Giant LiposomesComplex Systems on Giant Liposomes
Kanta Tsumoto (Mie Univ.)
1. To express transmembrane proteins related to response to extracellular environment using baculovirus system.
2. To reconstitute complex systems of the transmembrane proteins that can function on giant liposomes.
K. Tsumoto
Recombinant membrane protein expression
Giant liposome preparation
~10µm
Reconstitution
Giant proteoliposomeexpressing functions
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Synthesis and A01: Synthesis and In vitroIn vitro Evolution of RNAEvolution of RNA--Protein Protein (RNP)(RNP)--based gene regulatory networks based gene regulatory networks
Hirohide Saito (Kyoto Univ.)
1. To construct synthetic RNA-Protein (RNP) molecules that rewire cellular signaling.
2. To construct and evolve RNP-based translational regulators (synthetic RNP switch).
【Publications】
1) H. Saito et al. Chemistry & Biology, 11(6), 765-773 (2004)
2) H. Saito, Dimitrios Kourouklis, Hiroaki Suga EMBO J., 20(7), 1797-1806 (2001)
H. Saito
Synthetic RNP switch
5 mm
RNA-Protein(RNP)complex Design and In Vitro Evolution
RNA/Protein pool
Transcription
RT/PCR
Amplificationselection In Vitro Evolution
Synthetic RNP
QuickTimeý Dz�TIFFÅià èkǻǵÅj êLí£ÉvÉçÉOÉâÉÄ
�ǙDZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕïKóvÇ-Ç ÅB
RNA OFF switch RNA ON switch
Gene ON Gene OFF
Protein A Protein B
Gene OFF GeneON
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Artificial A01: Artificial NeuroendocrineNeuroendocrine Vesicles as Tools for Vesicles as Tools for System Cell EngineeringSystem Cell Engineering
Motonari Tsubaki (Kobe Univ.)
【Publications】
1) M. Tsubaki, F. Takeuchi, and N. Nakanishi, Biochim. Biophys. Acta, 1753, 174-190 (2005)
2) A. Asada, H. Orii, K. Watanabe, and M. Tsubaki, FEBS Journal, 272, 942-955 (2005)
1. Construction of proteoliposomes with multiple functions (transmembrane ET, substrate transfer, intravesicular enzymatic activities, membrane surface presentation).
2. Artificial neuroendocrine vesicles as tools for system cell engineering with various approaches (cell injection, pinocytosis).
Neuroendocrievesicles
Transmembraneproteins
Proteoliposomes with multiple functions
Artificial neuroendocrinevesicles
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Expansion of protein expression systemA01: Expansion of protein expression systemcontaining containing nonnaturalnonnatural amino acidsamino acids
Takashi Ohtsuki (Okayama Univ.)
【Publications】[1] H.Nakata, T.Ohtsuki, R.Abe, T.Hohsaka and M.Sisido, Binding efficiency of EF-Tu to tRNAs charged with
nonnatural fluorescent amino acids. Analytical Biochemistry, 348, 321-323 (2006)[2] T.Ohtsuki, T. Manabe and M. Sisido, Multiple incorporation of non-natural amino acids into a single protein using
tRNAs with non-standard structures. FEBS letters, 579, 6769-6774 (2005)
Incorporation of various amino acids
tRNA
aminoacyl-tRNA
growing polypeptide
mRNA 4-base codon/ anticodon pair
Protein
ribosome
nonnaturalamino acid
endogenousARS
EF-Tu
In vivosynthesis
Selection of efficient 4-base suppressor tRNA
Creation of EF-Tu mutantsfor large nonnatural amino acids
Expansion of protein biosynthesis system for protein engineering and drug discovery
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01:A01:In vitroIn vitro reconstruction of postreconstruction of post--transcriptional transcriptional modification machinery modification machinery
Hiroyuki Hori (Ehime Univ.)
1 construction of active multi-subunit RNA modification enzyme2 construction of RNA-enzyme complex
【Publications】1. K. Watanabe, O. Nureki, S. Fuakai, R. Ishii, H. Okamoto, S. Yokoyama, Y. Endo, and H. Hori, “Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.”J. Biol. Chem. 280, 10368-10377 (2005)2. H. Okamoto, K. Watanabe, Y. Ikeuchi, T. Suzuki, Y. Endo, and H. Hori, “Substarte tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus.”J. Biol. Chem. 279, 49151-49159 (2004)
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: Reconstitution of the replication cycle of A01: Reconstitution of the replication cycle of E. coliE. coliminimini--chromosome controlled by initiation switch chromosome controlled by initiation switch Masayuki Su’etsugu (Kyushu Univ.), Tsutomu Katayama (Kyushu Univ.)
【Publications】
1) Su'etsugu, M., Shimuta, T., Ishida, T., Kawakami, H. and Katayama, T. J. Biol. Chem. 280, 6528-6536 (2005)
2) Su'etsugu, M., Takata, M., Kubota, T., Matsuda, Y. and Katayama, T. Genes Cells 9, 509-522 (2004)
3) Fujimitsu, K. and Katayama, T. Biochem. Biophys. Res. Commun. 322, 411-419 (2004)
M. Su’etsugu T. Katayama
Purified proteins in the replication system DnaA-activity cycle
Reactivation system
DARS(DnaA Reactivating Sequense)
• Specific DNA segment
DnaA
DnaA
ATP
ADPATP hydrolysis
Pi
ADP
ATPActive
Inactive
DnaA protein initiates minichromosomal replication in an in vitro reconstituted system using purified proteins. We aim to develop a novel system that replicates minichromosome in a manner controled by inactivation / reactivation-cycle of DnaA.
GyraseGyrasedsDNA-bindingClamp loader & PolymeraseClampssDNA-bindingPrimaseHelicase loaderHelicaseInitiatorFunction
GyrBGyrAHU
pol III* β subunit
SSBDnaGDnaCDnaBDnaA
Protein Inactivation system
RIDA (Regulatory Inactivation of DnaA)
• DNA replicase(DNA-loaded clamp)
• Hda protein
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A01: The relationship between vesicle morphology and A01: The relationship between vesicle morphology and lateral phase separation on phospholipid membranelateral phase separation on phospholipid membrane
Masatoshi Ichikawa (Kyushu Univ.)
1. Laser manipulation and measurement on biological materials.
2. Membrane physics and polymer physics applied to biological systems.
【Publications】
1) M. Ichikawa, Y. Matsuzawa and K. Yoshikawa, J. Phys. Soc. Jpn., 74, 1958 (2005).
2) M. Ichikawa, Y. Matsuzawa, Y. Koyama and K. Yoshikawa, Langmuir, 19, 5444 (2003).
Biological systemsLife processes
Laser manipulation on a DNA molecule
Fabrication of DNA particles
Laser manipulation on a cell-sized liposome
5 μm
Micro-nano measurements
Masatoshi Ichikawa
System Cell Engineering Using Multi-scale Manipulation, Scientific Research in Priority Areas, MEXT, JAPAN
A0A011: Construction of a: Construction of artificialrtificial exocyexocytosistosis systemsystemNaohide Hirashima (Nagoya City Univ.)
1. To construct a artificial exocytosis system which secretes upon stimulation.
2. To construct an artificial cell which has an intracellular membrane systems.
【Publications】[1] N. Kato, M. Nakanishi, N. Hirashima, Journal of Immunology, 177, in press (2006)[2] S. Tadokoro, M. Nakanishi, N. Hirashima, Journal of Cell Science, 118, 2239-2246 (2005)[3] Y. Inoh, D. Kitamoto, N. Hirashima, M. Nakanishi, Journal of Controlled Release, 94, 423-431 (2004)
N. Hirashima
Exocytotic release is a important mechanism to release mediators, such as neurotransmitters and hormones.We have investigated such membrane fusion using small liposomes. stimulation
small liposome vesicles Secretion by exocytotic fusion
Large liposome
+ fuison
small liposome
Now we are developing a novel type of fusion system which exhibits exocytosis
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