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