Preface

Preface

The Graduate School of Biostudies, Kyoto University was founded in April,
1999. Heads of about twenty laboratories scattered in the campus decided withenthusiasm to unite and make a graduate school of biosciences. Although theirbackgrounds are diverse previously belonging to different schools (Agriculture,
Medicine, Pharmacology, Science), their researches share a common language of“genes”, “molecules” and “cells” thanks to very fast developments in the whole life
science research fields. For last three years, the school has experienced very
favorable interactions among people and gratifying outcome. It has emerged as acenter of excellence in research and education, not only in this country but also in
the world. In this fall the school was awarded a grant of the ‘21st Century COE
Program’ from the Monkasyo.
The mission of Graduate School for education is summarized below. (1)Training of graduate students to acquire the ability to pursue new biostudies at theworlds’ top level. (2) Training students to apply the new life sciences and technologies for human welfare and protection of the global environment. (3) Nurturing
individuals who can understand the various vital phenomena of the living organisms as a systemic function, and pursue these systemic functions.
The Graduate School of Biostudies has annual acceptance of about 85
students in the two-year master course and subsequently 40 to 50 graduate
students (the number dependent on the year) enter the three year doctor course.
Although the first graduate students will obtain the doctoral degree in the spring of
2004, the school has already 253 graduate students (157 in the master courseand 96 in the doctor course) and predicts to increase the number of graduate
student to about 300 in the spring of 2003. It is a most active and flourishing life
science graduate school in Japan. The ratio of students who want to continue fromthe master to the doctor course was the highest among the science and technology graduate schools in Kyoto University. The ratio of female students is currently
28%.
The Graduate School of Biostudies has two divisions, the Division of Integrated Life Science (ILS) and the Division of Systemic Life Science (SLS). Laboratories in ILS investigate fundamental life mechanisms at the levels of gene, chromosome, cell, muticellular organization and surrounding environment. Research
topics include gene mechanisms, chromosome segregation, cell cycle control, celland developmental biology, plant gene and totipotency, applied molecular biology,1Preface2

environmental signals and stresses. Education and research in SLS are focused on
the elucidation of the fundamentals of molecular and systemic biology, cell biology
and immunology, and immunobiology for the recognition of self-and non-self, in
response to various factors. Research areas include molecular and system biology, animal development and physiology, signal transduction, functional biology,
mammalian regulatory network.
ILS and SLS consist of 12 and 7 core laboratories, respectively. In addition,
ILS cooperate with two groups in the Institute of Virus Research and one in theCenter for Radiation Biology of Kyoto University. SLS cooperate with two laboratories in the Institute of Virus Research, one in the Experimental Unit of Genetic
information, one in the Osaka Bioscience Institute and one in the Center of Developmental Biology, RIKEN.
The governing body of the Graduate School of Biostudies is the monthlyheld meeting of laboratory heads (full professors) of the two divisions. Almost all
the issues are discussed and decided in this meeting chaired by the dean. The
division meetings are held only for discussions and primary decisions for appointing junior members or selecting graduate students.
It took us nearly ten years of preparations to make the Graduate School of
Biostudies. In most of these years, our effort was concentrated on mutual understandings among people in the core laboratories. For several years, regular joint
seminars attracted many graduate students by excellent talks and beer withsandwich. We understood the necessity of acquaintance at the level of sciences
but also of personality, before making any concrete plan for the new graduate
school. Enthusiasm was mounted year by year. Gladly, when we finally submitted
our plan of the new Graduate School to the University headquarter and also to the
Monbusyo (now Monkasyo, the Ministry of Education, Culture, Sports, Science andTechnology) in the early summer of 1998, it was immediately accepted. Each fivelabs from the Schools of Agriculture and Science, each two labs from the Schoolsof Medicine and Pharmacology, each one lab from Institute of Virus Research andIntegrated Human Studies and three new labs were united and opened the Graduate School of Biostudies as the first merging attempt in the country's national
universities.
Research activities of Graduate School of Biostudies may be judged by the
number of very large research grants obtained (10 are more than hundred million
yen) and highly-cited publications (seven are over 1,000 citations and 300 papers
more than 100 citation) and patents (over 100). Many laboratory heads playleading roles in their research areas, and work as the board members for anumber of academic journals. Visibility in the international scene might be ex-ampled by the foreign membership election of National Academy of Science, USA
and Royal Society of London for two professors. Only within three years after its
foundation, four junior faculty members were promoted to full professors in otheruniversities or other institute of Kyoto University, and one full professor to thedirector of a large research center of RIKEN. Even one full professor was ‘rented’
to be the dean of other graduate school in Kyoto University. Our graduate school
has ample interactions with industries as two full professors and nine junior faculty
members have had experiences to work in the company’s research institutes.
Every year average fifty grants are given by the private sectors. The ratio of female
faculty is not high (one full professor and two junior faculty members). The ratio of
foreign faculty is low (currently only one junior faculty). We will be obliged to
improve these two points.
For improving the research and educational environment for graduate
students and young investigators, we decided to use the grant of 21st Century COE
program for giving an opportunity to improve their speaking and writing abilities inEnglish. Our plan is to hire native foreign teachers who will come to the University
campus so that students can have personal or group lessons. We also decided touse the grant for hiring students as research and education assistants for easing
their financial difficulty. The grant will be also used for hiring post-doc fellows incertain research labs that really need active post-docs for promoting their own
researches.
To sum up, the Graduate School of Biostudies has made an excellent startsince 1999. For further development, we need to examine ourselves and seek foroutside opinions. For this end, we have annual two-day symposium where every
lab head presents their recent research activities. This symposium has been
extremely useful and stimulating for students and members of the graduate
school. In next December, we will have the first conference, which is intended forthe external examination by outside researchers. Eminent researchers (three
foreign and four Japanese) will attend the conference and give us valuable comments. In this year, approximately half of the lab heads will speak about theirstudy, and the remaining half will be scheduled for the next year conference. We
believe that this kind of activity together with publication of this booklet introducing
our graduate school in English will serve for the further development in researchand education and also for showing our aim and reality to the outside people.
Mitsuhiro Yanagida
Dean, Professor
October 30th, 2002

3

Laboratory of Chromosome Transmission
Mitsuhiro Yanagida (Professor, D.Sc.)
Yukinobu Nakaseko (Associate Professor, D.Sc.)
http://kozo.biophys.kyoto-u.ac.jp ･･･････････････････ 6Contents

Laboratory of Gene Biodynamics
Tan Inoue (Professor, D.Sc.)
Hideaki Shiraishi (Associate Professor, D.Sc.)
Yoshiya Ikawa (Instructor, D.Sc.)
http://kuchem.kyoto-u.ac.jp/seika/index.html ･･････････ 8Laboratory of Cell Cycle Regulation
Fuyuki Ishikawa (Professor, MD, Ph.D.)
Junko Kanoh (Instructor, D.Sc.)
http://www.lif.kyoto-u.ac.jp/labs/fish/index.html ･･･････ 10Laboratory of Signal Transduction
Eisuke Nishida (Professor, D.Sc.)
Yoshihiko Miyata (Instructor, D.Sc.)
Makoto Fukuda (Instructor, D.Sc.) ･･････････････････ 12
Laboratory of Plant Molecular Biology
Kanji Ohyama (Professor, D.Agr.)
Hideya Fukuzawa (Associate Professor, D.Agr.)
Katsuyuki Yamato (Instructor, D.Agr.) ･･･････････････ 14
Laboratory of Molecular
and Cellular Biology of Totipotency
Fumihiko Sato (Professor, D.Agr.)
Tsuyoshi Endo (Lecturer, D.Agr.)
Kentaro Ifuku (Instructor, D.Agr.)
http://callus.kais.kyoto-u.ac.jp/ ･･･････････････････ 16
Laboratory of Biosignals and Response
Masaya Nagao (Professor, D.Agr.)
Yuko Iwai (Associate Professor, D.Med.Sc.)
Taiho Kambe (Instructor, D.Agr.)
http://nucleus.lif.kyoto-u.ac.jp/labs/seitaijouho ････････ 18

Contents

Laboratory ofApplied Molecular Microbiology
Hidehiko Kumagai (Professor, D.Agr.)
Hideyuki Suzuki (Associate Professor, D.Agr.)
Hisanori Tamaki (Instructor, D.Agr.)
http://www.lif.kyoto-u.ac.jp/labs/bisei/ ･･････････････ 20

4

Laboratory of Molecular Biology ofBioresponse
Kenji Yamamoto (Professor, D.Agr.)
Seiji Masuda (Associate Professor, D.Agr.)
Takane Katayama (Instructor, D.Agr.)
http://www.lif.kyoto-u.ac.jp/labs/molecule/ ･･･････････ 22

Laboratory of Plant Physiology
Katsura Izui (Professor, D.Sc.)
Shingo Hata (Associate Professor, D.Sc.)
Tsuyoshi Furumoto (Instructor, D.Agr.)
http://www.plantp.kais.kyoto-u.ac.jp ････････････････ 24
Laboratory of Plasma Membrane
and Nuclear Signaling
Kunio Takeyasu (Professor, D.Sc., D.Med.Sc.)
Shigehiro Yoshimura (Assistant Professor, D. Hu.Env.Std.)
http://www.lif.kyoto-u.ac.jp/labs/chrom/ ･････････････ 26
Laboratory of Genome Stability
Tomohiro Matsumoto (Professor, D.Sc.)
Toshiyuki Habu (Instructor, D.Sc.)
http://www.rbc.kyoto-u.ac.jp/ ･････････････････････ 28
Laboratory of Cell Patterning
Tadashi Uemura (Professor, D.Sc.)
Tadao Usui (Instructor, D.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/MolGen/top.html ････ 30
Laboratory ofMammalian Molecular Biology
Yoichi Shinkai (Associate Professor, D.Med.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/SHINGO.HTM ･･･････ 32
Laboratory of Neuroscience
Shigetada Nakanishi (Professor, M.D., D.Med.Sc.)
Koki Moriyoshi (Associate professor, M.D., D.Med.Sc.)
Jun Kitano (Instructor, M.D., D.Med.Sc.)
http://www.lif.kyoto-u.ac.jp/nintijouhou.html ･･････････ 34
Laboratory of Immunology
and Cell Biology
Nagahiro Minato (Professor, M.D., D.Med.Sc.)
Masakazu Hattori (Associate Professor, D.Vet.Sc.)
Yoshimasa Tanaka (Instructor, D.Agr.) ･･･････････････ 36
Laboratory of Molecular
and Cellular Biology
Shin Yonehara (Professor, D.Sc.)
Kazuhiro Sakamaki (Associate Professor, D.Med.Sc.)
Kyung-Kwon Lee (Instructor, D.Sc.) ････････････････ 38
Laboratory of Immunobiology
Kayo Inaba (Professor, D.Sc.)
Kazuhiko Takahara (Lecturer, D.Eng.)Tomonori Iyoda (Instructor, D.Sc.)
http://zoo.zool.kyoto-u.ac.jp/imm/ ･････････････････ 40

Laboratory of Molecular Cell Biology
Hisataka Sabe (Professor, D.Med.Sc.)
http://www.obi.or.jp/ken1.html ････････････････････ 42
Laboratory of Molecular Cell Biology
and Development
Masatoshi Takeichi (Professor, D.Sc.)
http://www.cdb.riken.go.jp/ctp/
http://www.cdb.riken.go.jp/english/index.html ･････････ 44
Laboratory of Molecular Neurobiology
Manabu Negishi (Professor, D.Pharm.Sc.)
Hironori Katoh (Instructor, D.Pharm.Sc.) ･････････････ 46
Laboratory of Membrane Biochemistry
and Biophysics
Yasunori Kozutumi (Professor, D.Pharm.Sc.)
Katsumi Matsuzaki (Associate Professor, D.Pharm.Sc.)
Hiromu Takematsu (Instructor, D.Pharm.Sc.)
http://www.users.kudpc.kyoto-u.ac.jp/~o51267/index.html･･････････････････････････････････････････ 48
Laboratory of Functional Biology
Akira Kakizuka (Professor, M.D., D.Med.Sc.)
Seiji Hori (Lecuture, D.Med.Sc.)
Hiroshi Ohizumi (Instructor)
http://www.lif.kyoto-u.ac.jp/labs/funcbiol/ ････････････ 50
Laboratory of Viral Oncology
Kunitada Shimotohno (Professor, D.Pharm.Sc.)
Makoto Hijikata (Associate Professor, D.Med.Sc.)
Yasuo Ariumi (Instructor, D.Med.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/htv/jp/index.html ････ 52
Laboratory of Genetic Information
Akira Shimizu (Professor, D.Med.Sc.)
Kei Tashiro (Associate professor, D.Med.Sc.) ･･････････ 54
Laboratory of Cell Recognition
and Pattern Formation
Osamu Chisaka (Associate Professor, D.Med.Sc.) ･･････ 56
Laboratory of Molecular Neurobiology
Kazutoshi Mori (Associate Professor, D.Pharm.Sc.) ･････ 57
Laboratory of Cell Regulationand Molecular Network
Yota Murakami (Associate Professor, D.Sc.)
http://www.virus.kyoto-u.ac.jp/virus.html ････････････ 58

5

Laboratory of
Chromosome Transmission
Mitsuhiro Yanagida (Professor, D.Sc.)
Yukinobu Nakaseko (Associate Professor, D.Sc.)
http://kozo.biophys.kyoto-u.ac.jp

Laboratory of Chromosome Transmission

Mitsuhiro Yanagida

6

Chromosome dynamics in the cell cycle and mechanisms of eukaryotic chromosome segregation
Chromosomes are correctly segregated to daughter cells in each cell division. This laboratory’s long range
goal is full understanding of chromosome behaviour during mitosis, using an excellent model organism, thefission yeast Schizosaccharomyces pombe. We have tackled problems of chromosome condensation andsister chromatid separation, and try to understand the mechanisms at the molecular level. The genes
required for condensation and segregation are now known to be the same between fission yeast andhuman. Molecular functions of essential proteins discovered in this laboratory are investigated. Research
topics include kinetochore formation and its interaction with the spindle, the roles of condensin, cohesin,securin-separase and APC/cyclosome, and genome stability. As a post-genome project, the whole geneinteraction network is under construction.

Kinetochore structure and function
Kinetochore, DNA-protein complex crucial for equal
segregation, consists of a large number of proteins. Twofunctional domains, outer repetitive and central, exist andare bound to different proteins. We intend to identify
molecular activities of kinetochore proteins Mis6, Mis12,
Mis12-Mis22, identified in this lab. Our focused interests
are on how kintochore is formed and activated, and how itinteracts with spindle microtubules. Dis1, 2 and 3 proteins
are studied as they relate to dephosphorylation, acetylationand exoribonuclytic activity. We also employ HeLa with theRNAi method for making the eukaryotic model for the

Figure 1
Human hMis12-GFP (green) locates at kinetochore of mitotic
chromosomes. Checkpoint protein Mad2 (red) also situates at
kinetochore but slightly different positions. DNA, blue. HeLa
prometaphase cell (Tomomi Kiyomitsu)

kinetochore.
understanding chromosome dynamics that lead to condenCondensin, cohesin and securin-separase com-sation, cohesion and sister chromatid separation, respec-

plexes, and regulation of anaphase promoting

tively. In this laboratory, the genes encoding the essential

proteolysis

components of these complexes were identified throughCondensin, cohesin and securin-separase are crucial for

analyses of mutants that displayed characteristic pheno-

types. Our current research plans are to elucidate the roleof mitotic and interphase roles of condensin and cohesin,the cell cycle regulated interaction between cohesin andseparase, and the upstream regulators and the down
stream target of securin-separase. Specific mitotic roles ofindividual APC/cyclosome subunits are also investigated.

Genome stability and checkpoints
Crb2 is a major player in the fission yeast DNA checkpoint. Upon damage, Crb2 and Rad3/ATR activate Chk1

Figure 2
Three regulatory steps for securin-separase, and separasetargets other than Rad21 are the subjects of investigation (Koji
Nagao).

kinase. Crb2 is the target of Cdc2, and is regulated indamage-induced cell but also in normal cell cycle. Since
Crb2 is implicated in repair per se and replication, proteinsthat interact with Crb2 are investigated. Cnd2, a non-SMC
condensin subunit, is required for both repair and activation of checkpoint kinase Cds1/Chk2. A question is being
addressed how Cnd2 becomes essential for the genome
stability during interphase.

Figure 3
Mutant cnd2-1 defective in condensin subunit fails in mitotic
condensation at 36ºC and is hypersensitive to hydroxyurea, an
inhibitor for replication, at 26ºC (Nobuki Aono).

Post-genomic approach toward the whole gene
network
Over 1000 temperature-sensitive mutants individuallycharacterised are used for gene cloning. The mutant
genes as well as high-copy suppressers are obtained.
Combined with other post-genomic approaches, thefunctional gene network, which cover a significant portionof the whole genome, are being constructed.

Aono, N. et al (2002). Cnd2 has dual roles in mitotic condensation and interphase. Nature 417, 197-202.
Takahashi, K. et al (2000). Requirement of Mis6 centromere connector for localizing a CENP-A-like protein in fission yeast.

Science 288, 2215-9.
Goshima, G. et al (2000). Establishing biorientation occurs with precocious separation of the sister kinetochores, but not thearms, in the early spindle of budding yeast. Cell 100, 619-33.
Tomonaga, T. et al (2000). Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in
the S phase. Genes Dev 14, 2757-2770.
Esashi, F. et al (1999). Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage
checkpoint. Mol Cell 4, 167-74.

7

Laboratory of Gene Biodynamics
Tan Inoue (Professor, D.Sc.)
Hideaki Shiraishi (Associate Professor, D.Sc.)
Yoshiya Ikawa (Instructor, D.Sc.)
http://kuchem.kyoto-u.ac.jp/seika/index.html
Tan Inoue
Nanobio RNA/RNP architecture
The catalytic RNAs called ribozymes exist as single molecular RNAs, small RNA-protein complexes (RNPs),and central components of ribosomes and spliceosomes. We showed that a self-splicing ribozyme is composed of physically separable modular units which are commonly identified in other ribozymes. On thebasis of this finding, we have developed a new method for the molecular design and construction of RNAsand RNPs with difined 3-D structures (ca. 10 nm scale). Our current goal is to establish the method as a
new tool for the field of nanobio science and engineering, and to fully elucidate the architecture of natural
RNAs and RNPs by employing the method. Further research areas include the molecular mechanisms ofgene expression, development and senesence of one of the simplest multicellular organisms called Volvox.

Laboratory of Gene Biodynamics

Minimal catalytic domain of a self-splicing

8

intron RNA
The self-splicing intron ribozymes have been regarded asprimitive forms of splicing machinery for eukaryotic premRNAs. The splicing activity of group I self-splicing intron
is dependent on its absolutely conserved and exceptionallydense-packed core region composed of two characteristic
modular units. We found that only one unit is responsible
for the RNA catalysis. This establishes that two physicallyand functionally separable components compose the coreof the RNA, and presents a model for the architecture of a
prototype of this class of intron (Ikawa et al., 2000).

Figure 1
Secondary structure of minimal catalytic domain in the self-

Designed self-splicing RNPRibonucleoproteins consisting of a derivative of the
Tetrahymena group I self-splicing RNA, and an RNAbinding protein were designed and constructed based
upon high-resolution structures of the corresponding
prototype molecules: the protein contains two RNA-binding

splicing group I intron RNAs.

motifs from bacteriophage λ N and HIV Rev. The splicing
reaction of the RNP proceeds efficiently only when the
RNA associates with the protein. The fact that the conversion of a ribozyme to a ribonucleoprotein can be accomplished by a simple molecular design suggests that anatural catalytic RNP might have evolved readily from aribozyme (Atsumi et al., 2001).

Gene expression, development and evolution of

Figure 2

volvocaceans

Designed conversion of an RNA-RNA interaction with an RNA-

Volvox is known as one of the simplest multicellular

protein -RNA interaction (from left to right panel).

organism consisting of only two cell-types, that are,somatic cells and reproductive cells. Phylogenetic analysis
suggests that Volvox may have shared a common ancestor
with Chlamydomonas as recently as 35 million years ago.
To investigate the evolutional pathways and mechanisms
from single-cellular to multi-cellular organisms, we haveisolated stage-specific cDNAs from V. carteri that may be
acquired by this organism concomitant with the evolutionof multicellularity. Initial characterization of the cDNAs thatare specifically expressed during late somatic cell phasehas shown that extensin-family proteins fused to

Figure 3
Spheroids of Volvox carteri.

metalloproteinase domains or a PR (pathogenesis-related)domain are specifically expressed in this stage, suggestingthat the shuffling of functional modules of proteins has
occured during the evolution of this organism (Shimizu etal., 2002). (Figure 3)

Ikawa, Y. et al (2000). Minimal catalytic domain of a group I self-splicing intron. Nature Structural Biology 11, 1032-1035.
Atsumi, S. et al (2001). Design and development of a catalytic ribonucleoprotein. EMBO Journal 20, 5453-5460.
Ikawa, Y. et al (2002). Design, construction and analysis of a novel class of self-folding RNA. Structure 10, 527-534.
Shimizu, T. et al (2002). Cloning and characterization of novel extensin-like cDNAs that are expressed during late somatic phasein the green alga Volvox carteri. Gene 284, 179-187.

9

Laboratory of
Cell Cycle Regulation
Fuyuki Ishikawa (Professor, MD, Ph.D.)
Junko Kanoh (Instructor, D.Sc.)
http://www.lif.kyoto-u.ac.jp/labs/fish/index.html
Fuyuki Ishikawa

Laboratory of Cell Cycle Regulation

Chromosome behavior in cell cycle, differentiation, aging and cancer development
The continuity of life relies on the faithful transmission of genetic information from one cell to its daughter
cells and from one generation to the next. In most organisms, DNA encodes genetic information, but DNA
alone cannot accomplish the functions required for the continuity of life. Together with numerous protein
and RNA components, DNA forms a huge multi-functional complex called chromosomes. It is our goal tounderstand how chromosomes behave faithfully and adaptively, as if they embody the will of life to transmit
DNA. Because the basic mechanistics of chromosome biology are highly conserved among eukaryotes
ranging from yeast to human, we attempt to reach this goal by exploiting such model systems as fission
yeast, Xenopus, mouse and human cells.

10

Sex and Death, a link elucidated by the surpris-

eukaryotes have evolved to maintain linear chromosomes

ingly adaptive behaviors of chromosomes

due to the necessity to perform sexual reproduction. This

Eukaryotes are characterized by linear chromosomes,

hypothesis suggests that death (caused by telomere-based

whereas most prokaryotes, by circular chromosomes.

aging) and sex (meiosis) are two opposite faces of a single

Accordingly, eukaryotes possess specialized structurescoin.

called telomeres at both ends of a single DNA molecule.
Telomeres have been implicated in diverse pathologies,
such as aging and cancer. Then, why is it that eukaryotes
do not maintain chromosomes in circular forms asprokaryotes do? Fission yeast possesses only three linear
chromosomes. Upon introducing mutations that led totelomere dysfunction, most fission yeast cells died due tochromosome instability. However, we found that somefission yeast cells survived this crisis by self-circularizing
the three chromosomes. This indicates that circularchromosomes per se are not lethal to eukaryotes. However, we also found that fission yeast possessing circularchromosomes does not undergo meiosis, a processnecessary for sexual reproduction, thereby indicating that

Figure 1
Circular chromosomes in fission yeast. Three condensed circularchromosomes are visualized by arresting the telomere-defective
cell in metaphase.

A look into sister chromatid cohesion in higher

types not straightforwardly expected with a simple glue

eukaryotes

molecule: the kinetochores in the disjoined sister chroma-

Chromosomes are replicated during the S phase totids did not interact with the spindle microtubules. This

produce identical sets of chromosomes called sister

indicates that cohesin is not simply a molecular glue, but

chromatids. The two sister chromatids are associated withis responsible for multiple functions in chromosome

each other until the cells are ready to segregate each ofdynamics.

the chromatids into two daughter cells in the M phase.
The sister chromatid association, cohesion, is maintained

Current and Future Goals

by cohesin, a molecular glue. Cohesin has been exten-

In addition to chromosome dynamics during the cell cycle,

sively studied in yeast, but its functional role in higher

we are currently investigating how chromosomes change

eukaryotes is largely unknown. We have identified atheir functions either reversibly or irreversibly, duringdominant-negative allele of the human cohesin subunit

differentiation, aging and cancer development. These

Scc1. When we introduced a dominant-negative Scc1 into

efforts will lead us to understand how aging and cancer

cells, we found that cohesion does not take place, as

occur, and to the discovery of new therapeutic strategies

expected. However, we also observed additional pheno-

for these conditions.

Figure 2
The kinetochore-microtubule association in the M phase is not established in cells defective in cohesin. In the normal M phase (left), sister
chromatids (blue) are segregated in anaphase by the pulling force of the spindle microtubules (red) applied to the kinetochores (green). In
cohesin-defective cells (right), microtubules do not interact with kinetochores, leaving DNA masses separated in a direction perpendicular,
instead of parallel in wild-type cells, to the orientation of spindles.

Nakayama, J. et al (1998) Telomerase activation by the catalytic component gene TRT in human primary fibroblasts and hepatocellular carcinomas. Nature Genetics, 18: 65-68.
Naito, T. et al (1998) Circular chromosome formation in a fission yeast mutant defective in two ATM-homologues. Nature Genet-

ics, 20: 203-206.
Kanoh, J., and Ishikawa, F. (2001) SpRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function infission yeast. Current Biology, 11:1624-1630.
Hoque, Md. T. and Ishikawa, F. (2002) Cohesin defects lead to premature sister chromatid separation, kinetochore dysfunction
and spindle-assembly checkpoint activation. J. Biol. Chem. 277:42306-42314.

11

Laboratory of Signal Transduction
Eisuke Nishida (Professor, D.Sc.)
Yoshihiko Miyata (Instructor, D.Sc.)
Makoto Fukuda (Instructor, D.Sc.)
Eisuke Nishida
Molecular mechanisms of signal transduction
We are interested in identifying and elucidating molecular mechanisms that regulate cell proliferation, celldifferentiation, cell cycle and developmental processes. The current topics include 1) regulatory mechanisms and functions of the MAP kinase pathways, 2) control mechanisms of nucleocytoplasmic transport ofsignaling molecules, 3) roles of protein kinases in cell cycle progression and regulation, 4) growth factor
signaling mechanisms in developmental processes, 5) regulatory mechanisms for cell polarity, and 6)
molecular mechanisms of aging.

Regulatory mechanisms and functions of the MAP

Laboratory of Signal Transduction

kinase pathways

12

We have been analyzing function and regulatory mechanisms of the MAPK pathways since we described classical
MAP kinase (ERK MAPK) as serine/threonine kinase that iscommonly activated by various growth factors and tumor
promoters. We have identified vertebrate MAPK activator(MAPKK, MEK) and shown that the MEK/ERK pathway lies
downstream of Ras and plays a crucial role in various
biological processes. The receptor tyrosine kinase (RTK)/
Ras/ERK MAPK pathway has now been shown to be oneof the central signaling pathways that regulate cell

Figure 1
Activation and nuclear translocation of ERK MAPK

proliferation, cell differentiation and various developmental
processes. We have identified several upstream kinases
that activate other members of the MAPK family (JNK and(MAPK phosphatase) s, and their substrates includingp38), such as MKK6, MKK7, TAK1, ASK1 and MLTK. Our

MAPKAPK (MAPK-activated protein kinase) s. Most

recent studies revealed spatiotemporal control mecha-

recently, we have shown that Sprouty, a signaling inhibitor

nisms of ERK and its activator MEK, and established theoriginally found in Drosophila, is a novel type of a negative

importance of docking interactions in enzymatic reactions

feedback inhibitor in the RTK/Ras/ERK pathway and playsin the MAPK cascade pathways, such as interactions ofa key role in regulation of cell differentiation, cell prolifera-

MAPK with their activators MAPKKs, their inactivators MKPtion and various developmental processes.

Mechanisms of nucleocytoplasmic transport ofsignaling molecules
We identified a leucine-rich nuclear export signal (NES)
sequence in MEK that regulates cytoplasmic localization of
MEK. We have then identified NES in a number of key
molecules, including cyclin B1, Cdc25C, and Smad4. We
have shown that CRM1 is a receptor or transporter for
NES-containing cargoes. We are studying molecularmechanisms that regulate nucleocytoplasmic trafficking of

figure 2

key signaling molecules.

Sprouty regulates brain patterning

Identification and functional analyses of othersignaling pathways
We are interested in signaling pathways that regulate
longevity and aging. The best characterized pathway is an
insulin/IGF-1-like endocrine system that regulates the life
span of C.elegans. We are searching for crucial genes
whose expression extends the life span of this animal. We
are also analyzing signaling pathways that regulate
vertebrate developmental processes by using both

Xenopus and mouse embryos.

Fukuda, M. et al (1997). CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308311.
Tanoue, T. et al (2000). A conserved docking motif in MAP kinases common to substrates, activators and regulators. Nature Cell

Biol. 2, 110-116.
Harada, T. et al (2001). ERK induces p35, a neuron-specific activator of Cdk5, through induction of Egr1. Nature Cell Biol. 3,
453-459.
Toyoshima-Morimoto, F. et al (2001). Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.

Nature 410, 215-220.
Hanafusa, H. et al (2002). Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway. Nature Cell

Biol. 4, 850-858.

13

Laboratory of
Plant Molecular Biology
Kanji Ohyama (Professor, D.Agr.)
Hideya Fukuzawa (Associate Professor, D.Agr.)
Katsuyuki Yamato (Instructor, D.Agr.)
Kanji Ohyama

Laboratory of Plant Molecular Biology

Sexual development and carbon concentrating mechanism in plants
In contrast to most animals, most plant species are bisexual. Only a few plants are unisexual (“dioecious")and have sex chromosomes. Structure and molecular function of the sex chromosomes in plants are largely
unknown. We are investigating the structure of plant sex chromosomes and their function in sex determination and differentiation, using the dioecious liverwort, Marchantia polymorpha L. This plant can reproduce
asexually from a single cell as well. We are also interested in how plants acclimate to the environmental
stresses, especially shortage of CO2 and high light. In order to elucidate the “carbon concentrating mechanisms” or “molecular acclimation process” in plants, we are studying genes for the CO2-signal transduction
pathway using model photosynthetic organisms, Chlamydomonas and cyanobacteria.

14

Structure of sex chromosomes and sexual
development in liverwort
Since liverwort is haploid, a Y chromosome is present onlyin the male, and an X chromosome is found only in thefemale plants. This X-Y exclusiveness in liverwort makes it
possible to investigate the genetic content of each sex
chromosome separately. In addition, the sizes of the Y and
X chromosomes of liverwort are estimated to be approxi-

Figure 1

M. polymorpha is dioecious; male (left) and female (right) plants.
Structures which look like “a plate on a stick” and “a broken
umbrella” are male and female sex organs, respectively.

mately 10 Mb and 20 Mb, respectively, and thus liverwort

Chromosomes are shown in the insets with sex chromosomes

is an excellent model for molecular investigation of the sex

indicated by arrow heads.

chromosome system in plants. We have shown that uniquerepeat sequences are accumulated in a portion of the Y

Lipid metabolism in liverwort

chromosome (Okada et al., 2001) and that novel multicopy

Another interesting feature of liverwort is that it contains

genes are embedded among the repeat sequences (Okada

high levels of polyunsaturated fatty acids (PUFA), such as

et al., 2001; Ishizaki et al., 2002) We have isolated DNA

arachidonic and eicosapentaenoic acids. The accumulation

markers for the sex chromosomes (Fujisawa et al., 2001).

of these PUFAs indicates respective high activities of the

Sex chromosome-specific genes and their functions are

PUFA-synthesizing enzymes in liverwort. We have isolated

under investigation using transformation technique.

genes encoding fatty acid elongase and desaturase fromliverwort.

Molecular mechanisms of acclimation to CO2limiting conditions and high light
Aquatic photosynthetic organisms can adapt to CO2-limiting
conditions by inducing a set of genes for carbon concentrating mechanisms (CCM). High-CO2 requiring mutants ofa unicellular green alga, Chlamydomonas reinhardtii, wereisolated by gene-tagging. One of the mutants is aberrant

Figure 2

in most of the normal responses to CO2 limitation and

M. polymorpha can proliferate asexulally. Cross section of adoes not accumulate inorganic carbon into the cell. The
gene impaired in this mutant appeared to be a regulatory

gemma cup with immature gemmae at various stages (left), andan immature gemma still attatched to a basal cell at the bottomof the gemma cup (right).

gene, which responds to the decrease of environmental
CO2 level. We have isolated the regulatory gene, Ccm1,
and found that the Ccm1 gene codes for a protein with anovel zinc-finger motif (Fukuzawa et al., 2001). By
exploiting cDNA arrays, we have identified a number ofgenes involved in CCM, including those regulated by the

Ccm1 gene. Genome-wide analyses provide us excellent
opportunities to understand how cells acclimate to thechange of environmental CO2 concentration to attainefficient photosynthesis. Random transposon-knockout
library was generated and used to isolate a gene encoding
a new type of sodium-dependent bicarbonate transporterfrom a cyanobacterium (Shibata et al. 2002).

Okada, S. et al (2001). The Y chromosome in the liverwort Marchantia polymorpha has accumulated unique repeat sequences
harboring a male-specific gene. Proc. Natl. Acad. Sci. U. S. A. 98, 9454-9459.
Fujisawa, M. et al (2001). Isolation of X and Y chromosome-specific DNA markers from a liverwort, Marchantia polymorpha, byrepresentational difference analysis. Genetics 159, 981-985.
Fukuzawa, H. et al (2001). Ccm1, a regulatory gene controlling the induction of a carbon- concentrating mechanism in Chlamy-

domonas reinhardtii by sensing CO2 availability. Proc. Natl. Acad. Sci. U. S. A. 98, 5347-5352.
Shibata, M. et al (2002). Genes essential to sodium-dependent bicarbonate transport in cyanobacteria. J. Biol. Chem. 277,
18658-18664.
Ishizaki, K. et al (2002). Multicopy genes uniquely amplified in the Y chromosome-specific repeats of the liverwort, Marchantia

polymorpha. Nucl. Acids Res., in press.

15

Laboratory of Molecular and Cellular Biology of Totipotency

Laboratory of Molecular andCellular Biology of Totipotency

16

Fumihiko Sato (Professor, D.Agr.)
Tsuyoshi Endo (Lecturer, D.Agr.)
Kentaro Ifuku (Instructor, D.Agr.)
http://callus.kais.kyoto-u.ac.jp/

Fumihiko Sato

Functional differentiation and totipotency in plant cells
Plant cells have high potentials of totipotency, which enables the multiple functional differentiation from asingle cell. To understand the molecular and cellular basis of totipotency in plant cells, we have been
studying the molecular mechanism of functional differentiation in plant cells using in vitro cultured cells andtransgenic plants. Several unique cell lines with photoautotrophic potential or secondary metabolite productivity etc have been established and used for further molecular characterization. Using genes identified,molecular engineering of plant productivity and stress tolerance has been conducted to provide novel
genetic resources for the sustainable agriculture and green industry.

Chloroplast DNA-binding protease (CND41),
chloroplast development and plant growth
CND41, a 41-kD DNA-binding protease in chloroplasts,
has been isolated from cultured tobacco cells (Nakano etal., 1997). The physiological role of CND41 was investigated using transgenic tobacco with reduced CND41 (lowCND41 tobacco). Since low CND41 tobacco showedretarded senescence, we examined the function of CND41
as a protease during the senescence (Murakami et al.,2000). Further analyses indicated that CND41 plays animportant role in the in vivo degradation of Rubisco in

Figure 1
Different forms of DNA packaging in plastid nucleoids are foundin cultured tobacco cells. Undifferentiated plastids showed morecondensed blue fluorescence due to DAPI-staining of DNA,
whereas chloroplasts with red fluorescence have rather diffusedblue signals.

senescent leaves and the induction of senescence. On theother hand, low CND41 tobacco also showed a dwarf
phenotype, whereas plastid development in shoot apex
cells was more accelerated in low CND41 tobacco.
Analysis of gibberellin (GA) content confirmed the reduction of active GA in transformant. The characterization ofmolecular mechanism of multiple functions of CND41 inplant growth and chloroplast development is undergoing.

Figure 2
Control (L) and low CND41 tobacco (R) under nitrogen depletion
condition.

Secondary metabolism in plant cells

Molecular characterization of gene functions

Functional differentiation of secondary metabolism inusing mutants and transgenic plants

isoquinoline-alkaloid-producing plants has been investi-

Molecular mechanisms for the regulation of photosynthetic

gated. First, characterization of whole biosynthetic pathway

functions are investigated using mutants and transgenic

of berberine alkaloid in high berberine-producing Coptis

plants. The cyclic electron transport around PSI byjaponica cells has been conducted. Several biosynthetic

NAD(P)H dehydrogenase complex (NDH) in tobacco

enzymes have been purified and those cDNAs werechloroplast has been demonstrated using chloroplastic

isolated (e.g., Choi et al, 2002). Further characterization oftransformants in which ndh genes were insertionallygene expression using these cDNAs and ESTs suggested

inactivated. On the other hand, several mutants with highthat selected C. japonica cells are useful materials tolevel of the minimum yield of chlorophyll fluorescence,

understand the whole regulatory system of isoquinoline

possibly including mutation in oxidizing side of PSII,

alkaloid biosynthesis. Molecular engineering of secondary

photoihibition machinery or plastid gene expression were

metabolism also provided new insights of metabolic

also isolated and characterized (Murakami et al. 2002).

regulation of secondary metabolism and evolution of new
biosynthetic pathway (Sato et al., 2001).

Figure 3
Isoquinoline alkaloid biosynthetic pathway incultured Coptis japonica cells.
cDNAs of biosynthetic enzymes and a transporter in red have been isolated and characterized. 6-OMT, norcoclaurine 6-Omethyltransferase; NMT, coclaurine Nmethyltransferase; 4'-OMT, 3'-hydroxy Nmethylcoclaurine 4'-O-methyltransferase; SMT,
scoulerine 9-O-methyltransferase; CYP719,
canadine (tetrahydroberberine) synthase;
CoOMT, columbamine O-methyltransferase;
Cjmdr, berberine-import ABC transporter

Nakano, T. et al (1997). A novel protein with DNA binding activity from tobacco chloroplast nucleoids. Plant Cell 9, 1673-1682.
Murakami, S. et al (2000). Protease activity of CND41, a chloroplast nucleoid DNA-binding protein, isolated from cultured
tobacco cells. FEBS Lett. 468,15-18.
Sato, F. et al (2001). Metabolic engineering of plant alkaloid biosynthesis. Proc. Natl. Acad. Sci. USA 98, 367-372.
Choi, K.-B. et al (2002). Molecular cloning and characterization of coclaurine N-methyltransferase from cultured cells of Coptis

japonica. J. Biol. Chem. 277, 830-835.
Murakami, R. et al (2002). Characterization of an Arabidopsis thaliana mutant with impaired psbO, one of two genes encoding
extrinsic 33-kDa proteins in photosystem II. FEBS Lett. 523, 138-142.

17

Laboratory of Biosignals
and Response

Laboratory of Biosignals and Response

Masaya Nagao (Professor, D.Agr.)
Yuko Iwai (Associate Professor, D.Med.Sc.)
Taiho Kambe (Instructor, D.Agr.)
http://nucleus.lif.kyoto-u.ac.jp/labs/seitaijouho

18

Masaya Nagao

Erythropoietin and heavy metals
Research in this laboratory focuses on multiple physiological functions of erythropoietin (Epo). Epo is amajor physiological regulator of erythropoiesis. This laboratory has found that Epo is also produced in thebrain, uterus, oviduct and epididymis. Epo protects neurons from ischemia-induced cell death and is involved in the uterine angiogenesis. To understand the mechanisms that Epo exerts, the study of tripartite(erythropoietic, neurotrophic and angiogenic) functions is of great interest.
Another area of research is the studies on the mechanism of heavy metal metabolism. Trace heavy metals
such as iron and zinc are indispensable for life but toxic when they exist in excess. Uptake and excretion ofthem, therefore, must be tightly regulated. We employ a variety of biochemical, molecular biological andgenetic approaches to clarify the heavy metal metabolism.

Neurotrophic function of erythropoietin
We demonstrated that cerebroventricular infusion of Epo
prevents the ischemia-induced learning disability andrescues hippocampal CA1 neurons from lethal ischemic
damage. Molecular basis of therapies for ischemic damage
or neuronal degeneration is under investigation.

Figure 1
Erythropoiein protects hippocampal CA1 neurons from ischemic

Regulation of iron metabolism

damage.

Iron is an essential nutrient for virtually all organisms yet
toxic when exists in excess. The uptake of iron must
therefore be tightly regulated. In Saccharomyces

cerevisiae, the Aft1 transcriptional activator controls ironhomeostasis. Aft1 activates a battery of genes required foriron uptake under iron-starved conditions, whereas Aft1
function is inactivated under iron-replete conditions. Our

Figure 2

main focus is to understand the mechanism underling thisSubcellular loalization of Aft1 and Aft1-1up proteins in the cellschange in gene expression responding to iron status. We

grown in iron-starved (-Fe) and iron-replete (+Fe) medium.

have shown that the regulation is mainly governed by theiron-regulated nuclear localization of Aft1. (Yamaguchi-Iwai
et. al., 2002). We are currently studying the mechanism
how iron status is sensed and regulates subcellular
localization of Aft1 in the cells by using genetic, biochemical and chemical approaches. We also plan to examine theinvolvement of mitochondrial iron homeostasis on thisiron-dependent nuclear localization of Aft1.

Zinc transport into secretory granules in pancre-

Figure 3

atic β cells

ZnT-5 protein is specifically detected in insulin-containing β cells,

Zinc is an essential trace element required for the key

but not in other endocrine cell types or acinar cells. Scale bar =structural components of a large number of proteins such

50 mm.

as metalloenzymes and transcriptional factors. In addition,
zinc plays unique roles in some specialized cells. In
pancreatic β cells, zinc is required for insulin storage insecretory granules for formation of zinc-insulin crystals.
We have identified a candidate for transporting zinc into
secretory granules and named zinc transporter 5 (ZnT-5).
ZnT-5 is a vesicular zinc transporter abundantly expressedin pancreatic β cells and localized in association withinsulin crystals. We are studying the mechanism oftransporting zinc, regulation of gene expression andanother function of ZnT-5 by the use of biochemical
approaches.

Kobayashi T. et al (2002). Epididymis is a novel site of erythropoietin production in mouse reproductive organs. Biochemical

Biophysical Research Communications 296 145-151.
Kambe T. et al (2002). Cloning and characterization of a novel mammalian zinc transporter, Zinc transporter 5, abundantly
expressed in pancreatic β cells. Journal of Biological Chemistry 277, 19049-19055.
Yamaguchi-Iwai Y. et al (2002). Subcellular localization of Aft1 transcription factor responds to iron status in Saccharomyces
cerevisiae. Journal of Biological Chemistry 277, 18914-8.
Chikuma M. et al (2000). Tissue-specific regulation of erythrpoietin production in the murine kidney, brain, and uterus. American

Journal of Physiology, Endocrinology and Metabolism 279, E1242-E1248.
Sakanaka M. et al (1998). In vivo evidence that erythropoietin protects neurons from ischemic damage. Proceedings of National

Academy of Science USA 95, 4635-4640.

19

Laboratory of Applied
Molecular Microbiology

Laboratory of Applied Molecular Microbiology

Hidehiko Kumagai (Professor, D.Agr.)
Hideyuki Suzuki (Associate Professor, D.Agr.)
Hisanori Tamaki (Instructor, D.Agr.)
http://www.lif.kyoto-u.ac.jp/labs/bisei/

20

Hidehiko Kumagai

Basic and applied studies on microbial enzymes
The Laboratory of Applied Molecular Microbiology gives courses in (1) fundamental aspects of microorganisms and the application of microbiology to industrial fermentation, food production, and processing; (2)microbial technology including gene engineering and its application; (3) microbial physiology; (4) laboratory
work on applied molecular microbiology.
Research projects include (1) Studies on microbial enzymes and their genes concerned with metabolism ofamines, amino acids, and peptides; (2) Studies on microbial enzymes and their genes concerned withmetabolism of oligosaccharides and organic acids; (3) Studies on signal transduction systems in yeast.

Analysis and construction of L-DOPA production

Characteristics of γ-glutamyltranspeptidase as ansystem with bacterial cells

N-terminal nucleophile hydrolase: Elucidation of

L-DOPA is used in the treatment of Parkinson’s disease

the mechanisms of the enzyme reaction andand about 100 tons of L-DOPA is now produced by a

autocatalytic processing

method involving the enzyme tyrosine phenol-lyase. ThisThe reaction catalyzed by γ-glutamyltranspeptidase (GGT,

method was developed by Kumagai et al., employing

EC 2.3.2.2) has been speculated to proceed via a γ-

Erwinia herbicola cells exhibiting high activity of tyrosine

glutamyl enzyme intermediate. Using a novel mechanism-

phenol-lyase. The molecular mechanism of functional

based affinity labeling agent, we showed that the oxygen

proteins, transcription regulating factor and a tyrosine

atom of the side-chain of Thr-391, which is the N-terminaltransporter were investigated to analyze the cell system

residue of the small subunit, is a catalytic nucleophile andand to produce L-DOPA much more effectively (Katayama

that GGT is a new member of the N-terminal nucleophileet al. 2000 and 2002).

hydrolase superfamily. Enzymatically active GGT, which
consists of one large subunit and one small subunit, isCloning and expression of thermo-tolerant cellu-

generated from an inactive common precursor throughlases in a thermophilic yeast

post-translational proteolytic processing. The processing

Thermo-tolerant cellulases are useful in biomass utiliza-

mechanism for GGT has been analyzed by means of intion. The expression of the enzymes in a thermophilic

vitro studies using purified precursors. We showed thatyeast will be also useful to produce alcohol from biomass.

the processing of its precursor is an intramolecular
autocatalytic event and that the catalytic nucleophile forthe processing reaction is the same oxygen atom as thatmechanism of nutrient signal transduction.

for the enzymatic reaction (Suzuki and Kumagai, 2002).
Further study is being performed to elucidate the molecular mechanism of the processing reaction by crystallographical study.

Application of bacterial γ-glutamyltranspeptidase
to improve the taste of foodWe found that the taste of bitter amino acids was improved dramatically by γ-glutamylization and developed
an enzymatic method for the synthesis of γ-glutamyl
amino acids involving GGT (Suzuki et al., 2002). Theanine(γ-glutamylethylamide) is not only the major umami
component of tea, but also has favorable physiologicaleffects on mammals. An enzymatic method for synthesiz-

Figure 1
Localization of Gpr1-GFP

ing theanine involving GGT was also developed. A salttolerant GGT was found in Bacillus subtilis and we are
studying its application for food fermentation as glutaminase in industry.

Nutrient sensing and signal transduction in yeast
How cells monitor the availability of nutrition and transduce signals is a fundamental, unanswered question. We
have found that Gpr1p, a recently identified G-protein
coupled receptor in yeast Saccharomyces cerevisiae,
regulates the cellular cAMP level in response to glucose(Yun et al., 1998). We are studying the role and the

Katayama, T. et al (2000). Cloning and random mutagenesis of the Erwinia herbicola tyrR gene for high-level expression oftyrosine phenol-lyase. Applied and Environmental Microbiology 66, 4764-4771.
Katayama, T. et al (2002). Functional analysis of the Erwinia herbicola tutB gene and its product. Journal of Bacteriology 184,3135-3141.
Suzuki, H. et al (2002). Autocatalytic processing of γ-glutamyltranspeptidase. Journal of Biological Chemistry 277, in press.
Suzuki, H. et al (2002). Improvement of the bitter taste of amino acids through the transpeptidation reaction of bacterial γglutamyltranspeptidase. Journal of Agricultural and Food Chemistry 50, 313-318.
Yun, C.W. et al (1998). Gpr1p, a putative G-protein coupled receptor, regulates glucose dependent cellular cAMP level in yeast

Saccharomyces cerevisiae. Biochemical and Biophysical Research Communications 252, 29-33.

21

Laboratory of Molecular Biology
of Bioresponse

Laboratory of Molecular Biology of Bioresponse

Kenji Yamamoto (Professor, D.Agr.)
Seiji Masuda (Associate Professor, D.Agr.)
Takane Katayama (Instructor, D.Agr.)
http://www.lif.kyoto-u.ac.jp/labs/molecule/22

Kenji Yamamoto

Elucidation of the role of sugar chain
The sugar chains of some glycoconjugates have been shown to play important roles in biological phenomena such as cellular recognition, immune response, and lectin binding, among others. Recently,
glycoconjugates are gathering much attention, and the studies in this field of “glycobiology” or“glycotechnology”, are increasing rapidly. We study about microbial enzymes acting on sugar chains ofglycoconjugates and their applications. We apply such microbial glycosidases to synthesize bioactive compounds and to develop the enzymatic means for research of cell biology. We also study on interaction
between lactic acid bacteria and cell membrane of host intestine through sugar chains, and on investigation of factor that promoted adhesion of useful microorganisms to intestine.

Analysis of specific activity of microbial
endoglycosidase and its application
Endo-β-N-acetylglucosaminidase is a uniqueendoglycosidase that hydrolytically cleaves the diacetylchitobiose linkages in oligosaccharides bound to theasparaginyl residue of various glycoproteins. We found a
specific endo-β-N-acetylglucosaminidase in the culture
fluid of a fungus, Mucor hiemalis, isolated from soil. This
enzyme, Endo-M, acted on the complex type of sugar
chains as well as on high-mannose and hybrid types ofsugar chains, and had transglycosylation activity. We

Figure 1
Tertiary Structures of Glycosylated Calcitonin and Native Calcitonin
These are the tertiary structures of glycosylated calcitonin which
was chemo-enzymatically synthesized using thetransglycosylation activity of Endo-M, N-acetylglucosaminyl
calcitonin and native calcitonin, determined by NMR. All of themwere found to have an identical conformation in micelles
characterized by an amphipathic α-helix followed by an
unstructured region at the C-terminus.

analyzed this specific activity and applied it for adding thethe following three steps: the synthesis of an N-

oligosaccharide to various compounds.

acetylglucosaminyl asparagine derivative which is thebuilding block of N-acetylglucosaminyl peptide, the

1. Synthesis of bioactive glycopeptide

preparation of an N-acetylglucosaminyl peptide, and the

We chemo-enzymatically synthesized novel glycopeptides

transglycosylation of N-linked oligosacchareides of naturalusing the transglycosylation activity of Endo-M. The

origin to the N-acetylglucosaminyl moiety of the peptide,strategy of the synthesis of bioactive glycopeptide involves

which is catalyzed by Endo-M. Using this chemo-enzymaticmethod, we artifically added oligosaccharides to theAdhesion mechanism of lactic acid bacteria

asparagine or glutamine residue of various bioactive

bound to host intestine through sugar chain

peptides such as Peptide T, calcitonin, Substance P and

Many viruses, pathogenic bacteria, and bacterial toxins

yeast α-mating factor, originally having no sugar chain.

specifically bind to sugar chains of eukaryotic cell surface.

These glycosylated peptides were more stable against

For intestinal bacteria, adherence to cell surfaces forprotease digestion than the original molecule.

colonizing epithelial tissues is an important event. Lacto-

bacillus, a representative useful bacterium, in the intestinal
2. Remodeling of oligosaccharides of glycoproteinstract was found to bind to some specific

The oligosaccharide-tranferring reaction by Endo-M could

glycosphingolipids, like the pathogenic intestinal bacteria.

be used for the remodeling of oligosaccharides of glyco-

We found that Lactobacillus casei bound to specificproteins. Using the transglycosylation activity, we ex-glycosphingolipids. The bacteria generally bound to non-changed the high mannose type of oligosaccharides inacid glycosphingolipids having short sugar chains andglycoproteins that are produced by yeasts or molds to agalactosyl moiety in the non-reducing terminal, but not tocomplex type of oligosaccharides which are human

any acid-glycosphingolipids.

compatible oligosaccharide.

3. Effective preparation of monoclonal antibody
against sugar chain of glycoprotein
Various monoclonal antibodies directed to sugar chains ofglycolipids have been established using the glycolipids
itself as the immunogen. However, those directed to sugar
chains of glycoproteins have not been established because
the protein moiety has high antigenicity. We synthesized
novel glycolipids having a sugar chain of glycoproteinusing the transglycosylation activity of Endo-M, andprepared a monoclonal antibody against the N-linked
oligosaccharide using the novel glycolipid as immunogen.

Mizuno M. et al (1999). Synthesis of a glycopeptide containing oligosaccharides. J.Am. Chem.Soc. 121, 284-290.
Ashida H. et al (2000). Characterization of endo-α-N-acetylgalactosaminidase from Bacillus sp. and syntheses of neo-oligosaccharides using its transglycosylation activity. Arch.Biochem.Biophys. 373, 394-400
Haneda K. et al (2001). Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its
characterization. Biochim.Biophys.Acta 1526, 242-248.
Saskiawan I. et al (2002). Chemo-enzymatic synthesis of the glycosylated α-mating factor of Saccharomyces cerevisiae andanalysis of its biological activity. Arch.Biochem.Biophys. 406, 127-134.
Kato T. et al (2002). Identification of an endo-β-N-acetylglucosaminidase gene in C. elegans and its expression in E.coli.

Glycobiology 12, 581-587.

23

Laboratory of Plant Physiology
Katsura Izui (Professor, D.Sc.)
Shingo Hata (Associate Professor, D.Sc.)
Tsuyoshi Furumoto (Instructor, D.Agr.)
http://www.plantp.kais.kyoto-u.ac.jp
Katsura Izui
Regulatory mechanisms for carbon and nitrogen metabolism in plants
Our research is mainly focused on molecular mechanisms for the regulation of C4 photosynthesis and N2fixation. We are identifying and characterizing novel components of these metabolic processes using a
combined biochemical, molecular and transgenic approach. Our studies will hopefully provide basic knowledge for the improvement of crop productivity.

Structure and function of phosphoenolpyruvatecarboxylase (PEPC)PEPC plays a key role in the photosynthetic CO2 assimilation in C4 plants such as maize and sugarcane. We havelong been studying on PEPC, and cloned the gene in 1984

Laboratory of Plant Physiology

and recently elucidated the 3-D structure for the first time

24

(Kai et al., 1999). The reaction mechanism, and noncovalent (allosteric) and covalent (phosphorylation)
regulatory mechanisms are being studied, and geneticallyengineered enzymes are produced.
Figure 1

Signal transduction for the regulatory phosphory-

Tetrameric structure of PEPC from maize.

lation of PEPCPEPC for C4 photosynthesis is activated by phosphorylation under light. A cDNA for protein kinase (PK) specific to

Search of novel proteins indispensable for the C4

PEPC was cloned from a model C4 plant Flaveria trinervia

photosynthesis

and characterized (Tsuchida et al., 2001). The enzyme

There are still many unknown genes indispensable for C4

was purified to homogeneity from maize and possible

photosynthesis. By differential screening of cDNA libraries

redox regulation of its activity via thioredoxin was pro-

between mesophyll cells and bundle-sheath cells of maize

posed (Saze et al., 2001). On the other hand, sense and(Furumoto et al., 2000), and between the leaves of C3-

antisence expression of the PEPC-PK gene in F. trinervia is

and C4 Flaveria species, many novel candidate genes

also under progress to evaluate its physiological signifi-

have been obtained and now being characterized.

cance in C4 photosynthesis.

Nitrogen-fixing nodule formation of leguminous
plantsPromoter analysis of a nodule-enhanced PEPC gene ofsoybean was carried out and the process of molecular
evolution of the gene was investigated. Plant-microbe
interaction was also investigated using Lotus japonicus, amodel legume. We found that Rhisobium etli, which is a
microsymbiont of Phaseolus, forms early senescentnodules on L. japonicus roots (Figure 2). The mechanismof programmed cell death in the artificial nodules is under

Figure 2
Early-senescent nodules (left) and regular nodules (right).

investigation with the aid of a cDNA array of L. japonicus.
(This project is led by S.H.)

Kai, Y. et al (1999). Three-dimensional structure of phosphoenolpyruvate carboxylase: A proposed mechanism for an allostericinhibition, Proc. Natl. Acad. Sci. USA 96, 823-828.
Tsuchida, Y. et al (2001). Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervia: cDNA
cloning and characterization, FEBS Lett. 507, 318-322.
Saze, H. et al (2001). Thioredoxin-mediated reductive activation of a protein kinase for the regulatory phosphorylation of C4-form
phosphoenolpyruvate carboxylase from maize, Plant Cell Physiol. 42, 1295-1302.
Furumoto, T. et al (2000). Isolation and characterization of cDNAs for differentially accumulated transcripts between mesophyll
cells and bundle sheath strands of maize leaves, Plant Cell Physiol. 41, 1200-1209.
Banba, M. et al (2001). Lotus japonicum forms early senescent root nodules with Rhizobium etli. Molecular Plant-Microb Interac-tions 14, 173-180.

25

Laboratory of Plasma Membrane and Nuclear Signaling

Laboratory of Plasma Membrane
and Nuclear Signaling

26

Kunio Takeyasu (Professor, D.Sc., D.Med.Sc.)
Shigehiro Yoshimura (Assistant Professor, D. Hu.Env.Std.)
http://www.lif.kyoto-u.ac.jp/labs/chrom/
Kunio Takeyasu
Plasma membrane and nuclear signaling
We are interested in how the cell signaling is regulated by specific molecular elements. We have been
investigating the molecular structure and function relationships between the signaling elements (e.g.,
membrane receptors and transcription factors) and their targets (e.g., specific enzymes and DNA) by using
a variety of techniques in biochemistry, molecular and cell biology, and structural biology. It is particularly
exciting to elucidate the higher-order structures of cellular machinery (e.g., chromosomes in nucleus) by
scanning probe microscopy. Molecular evolutionary aspects of the P-type ATPase superfamily proteins arealso investigated. A comparative phylogenetic analysis demonstrates the relationship between the molecular
evolution of these subfamilies and the establishment of the kingdoms of living things.

Nuclear dynamics
A single molecule imaging technique (Atomic ForceMicroscopy: AFM) is applied to the nano-strucutural
biology of DNA, nucleosome, chromatin, chromosome andnucleus. We have reconstituted chromatin fibers andobserved them by AFM (Figure 1). With this technique, aloop formation of DNA with multiple enhancer proteins(Yoshimura et al., 2000a) and a relaxation of supercoiled
plasmid induced by a replication initiator protein(Yoshimura et al., 2000b) were observed. A “beads on astring” structure can now be obtained routinely by using
centromeric histone octomer in which CENP-A replaces
histone H3 (Yoda et al., 2000). When histone H1 wasadded the chromatin, the nucleosomes were compacted.
The structure and function of the condensin molecule havealso been elucidated (Yoshimura et al., 2002). We now
focus upon the nuclear structures of eukaryotes (Figure 2)
and prokaryotes (Figure 3). A series of basic structurescommon to human and Escherichia coli has been identi-

Figure 1
AFM images of the nucleosomes reconstituted by a salt dialysis
methodMono-nucleosomes were formed on 5S RNA gene (437 bp) thathas two nucleosome positioning signals (left-upper panel). Rightand left-bottom panels were reconstituted chromatin fiber on alinear (right) or a closed circular (left-bottom) 56 kb DNA.
Nucleosomes were formed much more efficiently on the closed
circular DNA than the linear DNA.

fied. Monoclonal antibodies against nuclear proteins havethe Na/K-ATPase, a multi-subunit membrane protein, is

now become powerful tools to study the 4-dimensional

regulated at the ER, Golgi, and plasma membrane levels.

dynamics of nucleus.

How they assemble, how they are transported to theplasma membrane, and how they function at the plasma

Molecular evolution and function of membrane

membrane, and how they are degraded, are the major

proteins

focuses of the questions. Phylogenetic analyses of the

Membrane transportors are essential for the cellular ion

primary sequences among the membrane transportors,

homeostasis. Some of them play an important role inspecifically the P-type superfamily, have postulated a newsensing and transducing the extracellular information into

evolutionary process of the 5 kingdoms (Takeyasu et al.,the nucleus. We study how the biogenesis and turnover of2001).

Figure 2
Fibrous structures in nuclear matrix revealed by AFM.
HeLa cells growing on a caver glass were treated with detergent
to remove membrane and cytoplasm and then with DNase toremove genome DNA. The remained structures were observed
under AFM.

Figure 3
Escherichia coli nucleoids revealed by AFM
E. coli cells in the late stationary phase were lysed mildly on acover glass. Left panel shows the incompletely broken cells(allow), which still retain the cell shape. The spread-out nucleoids(magnified in the right-bottom panel) and F-plasmids (right-top
panel) are clearly identifed. The nucleoids and plasmids show thesame coral leaf structures. A detail image (right-bottom panel)
depicts nodes on the coils that resemble the plectonemic
supercoiled structure.

Yoshimura, S. H., et al (2000a). Atomic force microscopy proposes a 'kiss and pull' mechanism for enhancer function. J Electron

Microsc (Tokyo) 49, 407-413.
Yoshimura, S. H., et al (2000b). DNA phase transition promoted by replication initiator. Biochemistry 39, 9139-9145.
Yoda, K., et al (2000). Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro. Proc.

Natl. Acad. Sci. USA 97, 7266-7271.
Takeyasu, K., et al (2001). P-type ATPase diversity and evolution: the origins of ouabain sensitivity and subunit assembly. Cell

Mol. Biol. (Noisy-le-grand) 47, 325-333.
Yoshimura, S. H., et al (2002). Condensin architecture and interaction with DNA: regulatory non-SMC subunits bind to the headof SMC heterodimer. Curr. Biol. 12, 508-513.

27

Laboratory of Genome Stability
Tomohiro Matsumoto (Professor, D.Sc.)
Toshiyuki Habu (Instructor, D.Sc.)
http://www.rbc.kyoto-u.ac.jp/
Tomohiro Matsumoto

Laboratory of Genome Stability

Checkpoints in the cell cycle
The cell cycle is a series of biochemical reactions required for faithful duplication of cells. Most of thesereactions are initiated in a step-wise manner and have to be completed once and only once in the cell
cycle. Radiation, chemical compounds and other stress prevent these reactions from their completion andthus bring the cell cycle to a crisis. Depending on the stage of the cell cycle, eukaryotic cells employ
various strategies to cope with the crisis. At the first step of the crisis control, eukaryotic cells make adecision that is “to survive or to die”. The signal cascades of checkpoints are activated if they chose tosurvive. The checkpoints delay progression of the cell cycle, and allow time for repair and removal oflesion. On the other hand, if they chose to die, the cascades for the programmed cell death (apoptosis) are
activated. Most of the anti-cancer drugs target a step-wise reaction in the cell cycle and activate a cascadefor checkpoint or apoptosis. Our research focus is on the molecular mechanisms of checkpoints.

28

Mad2 and its target in the spindle checkpoint
The spindle checkpoint delays the onset of anaphasewhen a kinetochore is not attached to the spindle. The
delay induced by the spindle checkpoint requires that
Mad2 binds to its target, fission yeast Slp1, which
normally promotes proteolysis required for sister chromatid
separation, an action occurs at anaphase. slp1-mr63, amutation on the slp1+ gene, abolishes the binding between
Slp1 and Mad2, however, restores activities essential forcell growth. In the slp1-mr63 mutant, a defect in thespindle attachment does not induce a delay in cell cycle
progression. The mutant exits from mitosis and initiates
the next round of DNA replication even when the spindle
attachment in incomplete (Kim et al., 1998). A similar
result has been reported for budding yeast Cdc20 that ishomologous to Slp1. In higher eukaryotes such as humanand mouse, p55CDC, a structural homolog of Slp1/Cdc20,

binds to Mad2 and is believed to be a target of the

p55CDC can promote the proteolysis. We have recently

spindle checkpoint.

identified a novel Mad2-binding protein, CMT2, throughyeast-two hybrid screen and shown that 1) Formation of

Silencing the spindle checkpoint

the CMT2-MAD2 complex coincides with or immediately

It has been well documented that sister chromatids

follows dissociation of the p55CDC-MAD2 complex in mid-

separate, with a lag-time of 20-30 minutes, after the last

mitosis, 2) Overexpression of CMT2 abolishes the function

kinetochore is attached to the spindle. The separation isof the checkpoint by interfering with stability of thehighly synchronous, suggesting that the attachment of the

p55CDC-MAD2 complex, 3) Inactivation of CMT2 results inspindle to the last kinetochore triggers a process thata transient metaphase arrest followed by cell death. These

promotes separation of all the sister chromatids at once.

results would suggest that CMT2 may play a central role

Perhaps, a diffusible element is responsible for triggeringin silencing the spindle checkpoint for transition fromsuch a process. Prior to sister chromatid separation, themetaphase to anaphase (Habu et al., in press).

spindle checkpoint must be silenced so that Cdc20/Slp1/

Kim, S. H. et al (1998). Fission yeast Slp1: An effector of the Mad2-dependent spindle checkpoint. Science 279:1045-1047.
Ikui, A. E. et al (2002). Control of localization of a spindle checkpoint protein, Mad2 in fission yeast. J. Cell Sci. 115: 1603-1610.
Matsumoto, S. et al (2002). Role of fission yeast Tsc1-Tsc2 complex in signaling and transport across cell membrane. Genetics
161: 1053-1063.
Liu, J. et al (2002). NEDD8 modification of CUL1 dissociates p120CAND1, an inhibitor of CUL1-SKP1 binding and SCF ligases. Mol.

Cell (in press)
Habu, T. et al (2002). A Mad2-binding protein, Cmt2, is required for silencing the spindle checkpoint. EMBO. J. (in press)

29

Laboratory of Cell Patterning
Tadashi Uemura (Professor, D.Sc.)
Tadao Usui (Instructor, D.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/MolGen/top.html
Tadashi Uemura
Neuronal and epithelial single-cell patterning
During development, individual cells decode multiple polarity cues, reorganize cytoskeleton, and eventuallyadopt a vast range of asymmetrical patterns. We have tackled mysteries of “single-cell patterning” of twocell types: neurons and epithelial cells. Epithelial cells construct actin-based extensions on their apicalsurfaces; and neurons develop processes such as dendrites that are specialized for signal reception. How
do the cells select specific intracellular sites for groundbreaking? How is the growth of the extensions
limited? How are branching complexities of dendritic arbors controlled? We are investigating those molecular mechanisms by using Drosophila and mammals as model systems. Our long-term goal is to shed lighton pathogenesis of human diseases that are caused by defects in the single-cell patterning.

Multiple roles of a seven-pass transmembranecadherin Flamingo

Laboratory of Cell Patterning

A seven-pass transmembrane cadherin, designated30

Flamingo, plays multiple roles in planar cell polarization(PCP) and limiting outgrowth of neuronal processes in
Drosophila. In epithelial cells, subcellular distribution of
Flamingo becomes highly asymmetric and this biased
redistribution appears to be prerequisite for normal cell
polarization. We are investigating how this asymmetricpattern generated by developing in vivo time-lapse
analysis of GFP-tagged molecules. In neurons, Flamingo

Figure 1

appears to function as a receptor for an as yet unidenti-

In the normal Drosophila wing, each epithelial cell produces afied ligand, and we are performing structure-function
analysis to isolate the ligand and downstream components.
In addition, we are challenging functional analysis ofseven-pass transmembrane cadherins in mouse development.

single hair that points distally (left). Mutations of polarity genes
including flamingo disrupt the oriented pattern (right).

Control of actin reorganization by Slingshot that
dephosphorylates actin depolymerizing factor(ADF)/Cofilin
ADF (actin depolymerizing factor)/cofilin is a stimulusresponsive mediator of actin dynamics. In contrast to
mechanisms of inactivation of ADF/cofilin by phosphorylation, much less was known about its reactivation throughdephosphorylation. We isolated slingshot (ssh) mutations
that dramatically increased levels of both F-actin andphospho-cofilin, and disorganized epidermal cell extensions. ssh encodes an evolutionary conserved phosphatase
and we showed that SSH family members dephosphorylate

Figure 2
Dendritic arborization neurons (da neurons) in the Drosophila

phospho-cofilin. We are studying whether SSH contributes

larva are classified into four categories, class I- IV, in order ofto stimuli-driven cell shape change including neurons, andincreasing arbor complexity. Two class I and a class IV neurons

if so, how the SSH activity is regulated.

are labeled green and purple, respectively.

Formation of dendritic arbors in vivo
Neurons extend many dendrites, specialized for signal
reception, as well as a single axon, specialized for signaltransmission. In contrast to a large body of knowledge
about axon guidance, a number of questions remain aboutmechanisms underlying dendritic outgrowth and branchingin vivo. We have developed GFP-markers for a subset of
Drosophila sensory cells, dendritic arborization neurons (daneurons), and taken both cell biological and genetic
approaches to full understanding of dendritic patterning.

Niwa, R. et al (2002). Control of actin reorganization by Slingshot, a novel family of phosphatases that dephosphorylate ADF/
cofilin. Cell 108, 233-246.
Shima, Y. et al (2002). Differential Expression of the Seven-pass Transmembrane Cadherin Genes Celsr1- 3 and Distribution of
the Celsr2 Protein during Mouse Development. Developmental Dynamics, 223, 321-332.
Shimada, Y. et al (2001). Asymmetric colocalization of Flamingo, a seven-pass transmembrane cadherin, and Dishevelled inplanar cell polarization. Current Biology 11, 859-63.
Usui, T. et al (1999). Flamingo, a seven-pass transmembrane cadherin, regulates planar cell polarity under the control of Frizzled.

Cell 98, 585-595.
Sugimura, et al. Distinct developmental modes and lesion-induced reactions of dendrites of two classes of Drosophila sensory
neurons. (submitted)

31

Laboratory ofMammalian Molecular Biology
Yoichi Shinkai (Associate Professor, D.Med.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/SHINGO.HTM

Laboratory of Mammalian Molecular Biology

Yoichi Shinkai

32

Chromatin higher order structure and its function
Our principal objective is to understand the molecular mechanisms which control chromatin function andgenome diversity & stability in mammals. Current our interest centers on two chromosomal-associatedmolecules, telomere binding proteins and SET (Suppressor of variegation, Enhancer of zeste and Trithorax)domain containing histone methyltransferases (HMTases). Both molecules were shown to be critical for
chromatin higher order structure and its functions. However, it is still poorly understood how they function
at a molecular level.

Function of mammalian telomere binding proteins
Telomeres are special structures at the end of eukaryotic
chromosomes and protect the chromosome end fromdegradation and fusion to other chromosomes (Niida et al.,1998, Niida et al., 2000). It has been described that
telomere binding proteins play important roles for thesetelomere functions. To elucidate functional telomere
structure, we are studying how mammalian telomere
binding proteins contribute to chromosomal stability andcellular proliferation.

Figure 1
Localization of telomere binding protein, TRF1

Histone methylation and chromosomal function
The covalent modification of histone tails has regulatory
roles in various nuclear processes, such as control oftranscription and mitotic chromosome condensation. The
different groups of enzymes are known to catalyze thecovalent modification, including acetylation, phosphorylation, ubiquitination, and methylation. Recent studies
revealed that lysine or arginine methylation is important fortranscriptional control, mitosis, and meiosis. Among
HMTases, G9a is a mammalian histone H3 lysine 9 (H3K9)-preferring HMTase (Tachibana et al., 2001). We haveestablished G9a KO mice and ES cells and shown that
G9a is a dominant euchromatic H3-K9 HMTase and plays
an essential role on murine early embryogenesis

Figure 2(Tachibana et al., 2002). G9a as a model system, we are

Euchromatic accumulation of histone methyltransferase, G9astudying the molecular basis of transcriptional control and

DAPI (Blue) and EGFP-G9a (Green)other chromosomal functions regulated by HMTases.

Niida H. et al. (1998) Severe growth defect in mouse cells lacking a telomerase RNA component. Nature Genetics. 19, 203-206.
Niida H. et al (2000) Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified
telomeric DNA. Mol. Cell. Biol. 20, 4115-4127.
Tachibana M. et al. (2001) SET-domain containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferasewith hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J. Biol. Chem. 276, 25309-25317.
Shinkai Y. et al. (2002) A testicular germ cell-associated serine-threonine kinase, MAK is dispensable for sperm formation. Mol.

Cell. Biol. 22, 3276-3280.
Tachibana M. et al. (2002) G9a histone methyltransferase plays a dominant role for euchromatic histone H3 lysine 9 methylationand is essential for early embryogenesis. Genes and Development 16, 1779-1791.

33

Laboratory of Neuroscience
Shigetada Nakanishi (Professor, M.D., D.Med.Sc.)
Koki Moriyoshi (Associate professor, M.D., D.Med.Sc.)
Jun Kitano (Instructor, M.D., D.Med.Sc.)
http://www.lif.kyoto-u.ac.jp/nintijouhou.html
Shigetada Nakanishi
Investigations of synaptic operation and regulation are important for a better understanding of mechanisms
responsible for brain function and dysfunction. The research projects in this laboratory are directed toward:
1) the structure, function and regulation of diverse members of glutamate receptors; 2) signal transduction
and functional organization of glutamate receptors; 3) synaptic integration of neurotransmission in theneural network and 4) neural disorders derived from synaptic dysfunction.

Molecular diversity and function of glutamate
receptors
Glutamate receptors are important in neural plasticity,neural development and neurodegeneration. Glutamategated ionotropic receptors are subdivided into NMDA
receptors and AMPA/kainate receptors, whereas
metabotropic glutamate receptors (mGluRs) are coupled toLaboratory of Neuroscience

intracellular signal transduction via G proteins. We cloned

34

NMDA receptors and mGluRs by developing a novel

Figure 1

cloning strategy that combined electrophysiology and a

A family of NMDA receptors (A) and mGluRs (B)Xenopus oocyte expression system. Our studies indicatedthat diverse members of receptors exist in both families(Fig. 1) and exhibit specialized functions in various brain

OFF bipolar cells. Two distinct types of glutamate recep-

regions.

tors thus effectively operate for segregating visual information in response to the common glutamate transmitter.

Segregation and integration of visual information

Direction-selective (DS) responses of retinal ganglion cells

in the retinal network

to a moving stimulus represent a primitive pattern

In the retina, light and dark signals generate ON and OFF

recognition. The role of starburst cells (a retinal amacrine

responses in distinct bipolar cells (Fig. 2A). Usingcell) in DS responses was investigated using themolecular cloning, immunohistochemistry and gene

immunotoxin-mediated cell targeting (IMCT) technology. In

targeting, we showed that mGluR6 in ON bipolar cells is a

this technology, we generated transgenic mice thatkey receptor responsible for ON responses. In contrast,

expressed human IL-2 receptor (hIL-2R)/GFP in starburst

ionotropic receptor is used as a postsynaptic receptor incells and ablated these cells by intravitreally injecting theimmunotoxin composed of the hIL-2R antibody fused to

Synaptic integration in motor balance and abusebacterial toxin. Selective elimination of starburst cellsof drugs

abolished not only direction selectivity of ganglion cell

The basal ganglia circuit is modulated by dopamine

responses but also an optokinetic eye reflex derived byneurotransmitter (Fig. 2B). To investigate the role ofstimulus movement. The synaptic integrity ofacetylcholine (ACh) in the basal ganglia circuit, weneurotransmittions at the level of starburst cells underlies

selectively ablated striatal cholinergic neurons by IMCT

as a mechanism for a pattern recognition of visualtechniques. Our studies indicated that the basal ganglia

information.

function is concertedly and adaptively regulated bysynaptic integration of cholinergic and dopaminergictransmissions. Futhermore, ACh in the nucleus
accumbens plays a pivotal role in the sensitivity to additionof cocaine and morphine, indicating the involvement of
ACh in synaptic plasticity in the basal ganglia circuit.

Figure 2(A) A retinal neural network for direction-selective responses ofganglion cells. BP, bipolar cell; SA, starburst amacrine cell; ONOFF DSG, ON-OFF direction-selective ganglion cell.
(B) A basal ganglia neural circuit. Arrow-headed open andclosed lines indicate excitatory and inhibitory pathways, respectively. DA, dopamine; Ach, acetylcoline; Enk, enkephalin; SP,substance P; D1 and D2, D1 and D2 receptors; GP, globus
pallidus; SNc, substantia nigra pars compacta; SNr, substantia
nigra pars reticulata; EPN, entopeduncular nucleus; STN,
subthalamic nucleus.

Kaneko S. et al (2000). Synaptic integration mediated by striatal cholinergic interneurons in basal ganglia function. Science 289:
633-637
Kunishima N. et al (2000). Structural basis of glutamate recognition by a dimeric metabotropic glutamate receptor. Nature 407:
971-977
Yoshida K. et al (2001). A key role of starburst amacrine cells in originating retinal directional selectivity and optokinetic eye
movement. Neuron 30: 771-780
Hikida T. et al (2001). Increased sensitivity to cocaine by cholinergic cell ablation in nucleus accumbens. Proc. Natl. Acad. Sci.

USA 98: 13351-13354
Kitano J. et al (2002). Tamalin, a PDZ domain-containing protein, links a protein complex formation of group 1 metabotropic
glutamate receptors and the guanine nucleotide exchange factor cytohesins. J. Neurosci. 22: 1280-1289

35

Laboratory of Immunology
and Cell Biology
Nagahiro Minato (Professor, M.D., D.Med.Sc.)
Masakazu Hattori (Associate Professor, D.Vet.Sc.)
Yoshimasa Tanaka (Instructor, D.Agr.)

Laboratory of Immunology and Cell Biology

Nagahiro Minato

36

Recognition and functions of immune system
T cells in the immune system play a central role for specifically recognizing invading pathogens or endogenous abnormal cells and eliciting diverse responses to protect the body. While recognition of antigens per
se is mediated by antigen receptors, it is a functional site formed by the cognate interaction between T
cells and antigen-presenting cells (APC) that determines whether the T cells respond to the antigen or not,and if they do how. One of our major research foci is directed to the understanding of molecular basis forsuch cognate cellular interactions to control the modes of immune responses. We are also studying possible modes for the immunological recognition of endogenously arising neoplasmic cells to explore a longstanding yet poorly corroborated concept of immune surveillance against cancer.

Integrin-mediated cognate cellular interactions inhematopoietic homeostasis, respectively.

immune and hematopoietic systems
Integrins are the major cell adhesion proteins involved in

Unique mode of antigen recognition by γδ T cells

the formation of immune synapse between T cells andWhile the mode of protein antigen recognition by αβT

APC for immune responses and hematopoietic niche forcells in the context of peptides -associated major histo-

generation of blood cells. We have reported that a Ras-

compatibility (MHC) molecules has been well established,family GTPase Rap1 plays a crucial role for integrin

we have shown that human γδ T cells exhibit a distinct

activation and thereby controls these cognate interactions.

mode of antigen recognition, namely recognition of a

Activation levels of Rap1 in T cells, for instance, regulate
the integrity of immune synapse and affect the fates ofantigen-recognized T cells, namely controlled activationand proliferation, forced unresponsiveness (anergy), or
exhaustive activation followed by death. Those in hematopoietic progenitors also regulate the cognate interaction
with stroma cells and control the extent of hematopoiesis.
We are currently investigating the molecular mechanisms
involved in the cell fate decision of T cells and hematopoi-

Figure 1

etic cells by the cognate cell-cell interactions, which

Intimate interaction of SPA-1-deficient leukemia cells with a boneultimately affect the modes of immune responses andmarrow stroma cell

series of non-peptide antigens independent of MHC.

Study on the immune surveillance against cancer

These antigens are presented to γδ T cells ubiquitously

Immune surveillance against cancer has been one of theby human tumor cells by yet undefined “presenting

most important concepts in immunology since 1960’s, butmolecules”. We are currently investigating the structural

still remains to be corroborated. The major hung-up has

basis of the interaction between γδ T cell-antigen

been the lack of feasible animal models. We havereceptors and antigenic ligands including crystallographic

developed SPA-1-gene targeted mice, which develop age-

analysis and intending to identify the antigen-presenting

dependent T cell-immunodeficiency and accelerated

molecules. Through understanding of the unique antigen

hematopoietic progenitor expansion by affecting therecognition modes, we expect to explore the immunologi-

cognate cellular interaction between T cells and APC, andcal roles of γδ T cells, a long enigmatic T cell subset.

hematopoietic progenitors and stroma cells, respectively.
By the collaborative effects, the mice ultimately develop
lethal myelogenous leukemia of late onset. We intend to
study the feasibility of immune surveillance against cancer
by using this animal model.

Tomita, K. et al (1999). The bHLH gene Hes1 is essential for proliferation of early T cell precursors. Genes and Development
13,1203-1210.
Nishimura, H et al (1999). Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM
motif-containing immunoreceptor: Implication for breakedown of peripheral tolerance. Immunity 11, 141-152.
Nishimura, H. et al (2001). Autoimmune dilated cardiomyopathy in PD-1 receptor deficient mice. Science 291,319-322.
Miyagawa, F. et al (2001). Essential contribution of germline-encoded residues in Jγ1.2 segment to the recognition of
nonpeptide antigens by human γδ T cells. J. Immunol. 167,6773-6779.
Katagiri, K. et al (2002). Rap1 functions as a key regulator of T cell and APC interactions and modulates T cell-responses. Mol.

Cell. Biol. 22, 1001-1015.
Iwai, Y. et al (2002). Involvement of PD-L1 on tumor cells in the escape from host immune system and tumor immunotherapy by
PD-L1 blockade. Proc.Natl.Acd.Sci.USA 99, 12293-12297.

37

Laboratory of Molecular
and Cellular Biology
Shin Yonehara (Professor, D.Sc.)
Kazuhiro Sakamaki (Associate Professor, D.Med.Sc.)
Kyung-Kwon Lee (Instructor, D.Sc.)

Laboratory of Molecular and Cellular Biology

Shin Yonehara

38

Fas-mediated apoptotic cell death
Apoptosis, or programmed cell death, plays an important role in many biological processes, including
embryogenesis, development of immune system, maintenance of tissue homeostasis, and elimination ofvirus-infected and tumor cells. We found cell surface Fas antigen (Fas) which can directly transduce
apoptosis-inducing signals into cells by stimulation with agonistic anti-Fas mAbs (Yonehara et al., 1989;
Itoh et al., 1991) or Fas ligand. Genetic and biochemical analyses of Fas and FasL have revealed that Fas
plays an important role in the elimination of autoreactive peripheral T and B cells, and tumor and virusinfected cells. Our main purpose is to understand intracellular signals via Fas, intracellular regulatory(inhibitory) signals for Fas-mediated apoptosis and physiological role of Fas and its related signaling molecules. In conjunction with these studies, we have been trying to identify other apoptosis-related molecules
which play a key role in immune system, embryogenesis or tumorigenesis.

Fas-mediated apoptosis-inducing signals
Fas-induced apoptosis (Figure 1) has been shown to be
mediated by adaptor molecule FADD and caspase-8 that
directly bind to Fas and FADD, respectively (Figure 2). We
cloned cDNA encoding a novel protein FLASH, which canbind caspase-8 and FADD, and FLASH was suggested to
be involved in Fas-mediated activation of caspase-8 (Imaiet al., 1999).

B. Anti-Fas mAb RK8-induced in vivo apoptosis on lymphadenopathy

Figure 1
Fas-mediated apoptosis.

Figure 2

A. Transmission electron micrograph.

Fas-mediated apoptosis-inducing signals

Inhibition of Fas-mediated apoptosis

and FADD which are derived from Xenopus levis and Zebra

To elucidate inhibitory mechanisms for Fas-mediatedfish, and found that they show similar function as those of

apoptosis in tumor cells and autoreactive immunocytes, we

mammals. Therefore, we suggest that the apoptotic

showed that Raf/MAPKK/MAPK (ERK)/CREB cascade,machinery using caspase-8 and FADD is conserved inwhich can be induced by bFGF, plays an important role invertebrates.

the inhibition of Fas-mediated apoptosis in fibroblasts(Kazama and Yonehara, 2000). Interestingly, viral

A novel scavenger receptor SR-PSOX with

oncoporotein p40Tax was also shown to inhibit Fas-chemokine activity

induced apoptosis in fibroblasts through activating CREB,

We cloned a novel scavenger receptor SR-PSOX. We

although p40Tax inhibits Fas-mediated apoptosis in T

demonstrate that membrane-bound SR-PSOX/CXCL16

lymphoblasts by a different molecular mechanism withefficiently mediates adhesion and phagocytosis of not only

activation of NF-κB. In addition, we found that cell surface

OxLDL but also Gram-negative and Gram-positive bacteria.

receptor Kit-induced signals negatively regulate Fas-

Strikingly, SR-PSOX was found to be identical to novel

mediated apoptosis by down-regulating Fas-expression

transmembrane-type chemokine CXCL16. SR-PSOX/

through activating Akt. Thus, various oncogene products

CXCL16 is likely to play a role in both innate and adaptive

and growth factors inhibit Fas-mediated apoptosis in

immunity by mediating phagocytosis of bacteria as well as

various cells by different and complicated molecular

recruitment of T cells by dendritic cells.

mechanisms, which must be clarified in the future.

Functions of caspase-8 and FADD in various
vertebrates
We demonstrated that caspase-8-deficient mice died with
deficiency in early embryonic development of heart, neuraltube and yalk sac, and caspase-8 is indispensable for
apoptosis induced through the death receptors such as
Fas and TNF receptor. We examined function of caspase-8

Yonehara, S. (2002). Death receptor Fas and autoimmune disease: from the original generation to therapeutic application ofagonistic anti-Fas monoclonal antibody. Cytokine Growth Factor Rev. 13, 393-402.
Kazama, H. et al (2000). Oncogenic K-Ras and basic FGF prevent Fas-mediated apoptosis in fibroblasts through activation of
MAP kinase. J. Cell Biol. 148, 557-566.
Imai, Y. et al (1999). The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis.

Nature 398, 777-785.
Itoh, N. et al (1991). The polypeptide encoded by the cDNA for human cell surface Fas can mediate apoptosis. Cell 66, 233-243.
Yonehara, S. et al (1989). A cell-killing monoclonal antibody (anti-Fas) to a cell surface antigen codownregulated with thereceptor of tumor necrosis factor. J. Exp. Med. 169, 1747-1756.

39

Laboratory of Immunobiology
Kayo Inaba (Professor, D.Sc.)
Kazuhiko Takahara (Lecturer, D.Eng.)Tomonori Iyoda (Instructor, D.Sc.)
http://zoo.zool.kyoto-u.ac.jp/imm/
Kayo Inaba
Immune regulation by the dendritic cell system
The physiologic function of the immune system is defense against infectious microbes. However, evennoninfectious foreign substances can elicit immune responses. Furthermore, mechanisms that normallyprotect individuals from infection and eliminate foreign substances are themselves capable of causing
tissue injury and disease in some situations. Dendritic cells (DCs) are the only antigen-presenting cells
capable of stimulating naïve T cells. Present in all tissues, they engulf, migrate to draining lymph nodes,
and present processed antigens to T cells. The DC system consists of heterogeneous populations in termsof differentiation stage and pathway. We focus on DC functions as a controller in innate and adaptive
immune responses at inflammatory as well as steady states. Our goal is to provide a new insight for
autoimmunity and chronic immune-based diseases.

Uptake of dying cells by DCs in situ

Laboratory of Immunobiology

Although both DCs and macrophages sample various

40

substances, only immature DCs continuously circulate
through tissues and into lymphoid organs, capturing selfantigens as well as innocuous environmental proteins. We
recently found that dying cells or its fragments were
engulfed by CD8+ CD11blow, but not CD8- CD11bhigh, DC
subset, though both DC subsets capture protein antigens
and microspheres (Fig 1). This ability was also confirmed

Figure 1

in the experiment of uptake in vitro. We are now looking

Dying cells phagocytosed by spleen DCs in situ.

for responsible molecule(s) on CD8+ DC subset to recognize dying cells.
state. C-type lectins recognize carbohydrate profiles on
Receptors involved in recognition and endocytosis

pathogens and also interact with self- glycoproteins toof microbes

mediate cellular processes, such as differentiation andDCs could be specialized to respond to specific microbes

migration. We cloned mouse Langerin (CD207) expressedby expressing distinct sets of TLRs and C-type lectins

primarily on epidermal Langerhans cells. Five mousedepending on their tissue localization and differentiation

homologues to human DC-SIGN (CD209) and DC-SIGNR/L-SIGN were also obtained (Fig 2). Studies are now carryingout to find ligands, as well as pathogen targets for theseC-type lectins. We are also interested in signals derived
from C-type lectins and from TLRs for DCs and macrophages to determine the possibility that pathogens escapeimmunity or are eradicated.

Antigen presentation and T cell activation
Formation of immunological synapse, a complex cluster ofmolecules organized at the contact area of cell conjugate,

Figure 2
Structure of C-type lectins currently investigated.

is the essential step to initiate immune responses.
However, it remains to be determined what are uniquecharacters restricted to DCs in addition to high expressionof MHC and adhesion/costimulatory molecules. We are
studying membrane microdomain formation using bonemarrow-derived DCs at different stage of maturation.

Dendritic cell subsets in vivo
Different subsets of DCs localize in all tissues andcontinuously move to draining lymphoid organs in steady

Figure 3

state (Fig 3). We have reported that DCs play an essential

Life cycle of DCs in the family.

role to induce tolerance in periphery in the absence of
inflammatory stimuli. However, functional differences of DC
subsets for inducing deletion or inactivation of T cells andfor maintaining regulatory/suppressor T cells are not fully
elucidated. Furthermore, compositions of DC subsets inlymphoid and nonlymphoid tissues and their lifespans arelargely unknown. These subjects are also investigating toclarify physiological role of DC subsets in situ.

Inaba, K. et al (2000). The formation of immunogenic MHC class II-peptide ligands in lysosomal compartments of dendritic cells
is regulated by inflammatory stimuli. J. Exp. Med. 191, 927-936
Turley, S. et al (2000). Transport of peptide-MHC class II complexes in developing dendritic cells. Science 288, 522-527, 2000.
Takahara, K. et al (2002). Identification and expression of mouse langerin (CD207) in dendritic cells. Int. Immunol. 14, 433-444
Iyoda, T. et al (2002). The CD8+ dendritic cell subset selectively endocytoses dying cells in culture and in vivo. J. Exp. Med.
195,1289-1302
Kang Liu, et al (2002). Immune tolerance following delivery of dying cells to dendritic cells in situ. J. Exp. Med. 196,1091-1097

41

Laboratory of
Molecular Cell Biology
Hisataka Sabe (Professor, D.Med.Sc.)
http://www.obi.or.jp/ken1.html
Hisataka Sabe

Laboratory of Molecular Cell Biology

Intracellular signaling for cell migration
Cell migration is a multifactorial process in which a number of distinct events occur simultaneously. The
fundamental question in modern cell biology is, what are the molecular mechanisms involved in the temporal and spacial coordination of distinct processes, as well as orchestration of distinct events occurring atdifferent parts within single cells, all of which result in unified, efficient, directional migration, and are also
necessary for the stop of migration. Our main research interests are the molecular and cellular mechanisms involved in cell adhesion, migration and contact inhibition. We are also interested in the roles ofmembranes and cytoarchitecture, and their remodeling in growth control.

42

Endosomal recycling and cell migration
Arf proteins belong to the Ras-superfamily of GTPases,
and are involved in membrane traffic and remodeling. We
have found that several members of ArfGAP proteins areassociated with paxillin, an integrin-assembly adaptor
protein, and shown that Arf6 activity is essential for cell
migration. We are analyzing the roles of the paxillinassociated ArfGAP proteins in controling cell motile

Figure 1

activity.

Subcellular localization of a paxillin-associated ArfGAP, AMAP2, inlive cells.

Integrin signaling of protein tyrosine phosphorylation and cell motility
We have found that tyrosine phosphorylation of p130Cas

stream signaling pathways.

and paxillin are the prominent events occurring upon
integrin activation, and demonstrated that these proteins

Membrane remodeling and cytoskeletal remodel-

have the potential to exert opposing effects on several

ing

integrin-mediated cellular events. Paxillin tyrosine phos-

Arfs are known to be involved in actin cytoskeletal

phorylation appears to be closely related to the mutuallyremodeling, besides their function in membrane remodel-

exclusive regulation of the activities of Cdc42, Rac1 anding. Through extending our studies on paxillin-associated

RhoA in cell migration, and we are analysing the down-

ArfGAPs, we aim to clarify the principal mechanisms

involved in the coordination between membranes and thecytoskeleton in motile cells.

LIM proteins in Drosophila development
Paxillin has multiple LIM domains at its COOH-terminus,
and its homologue is present in Drosophila. We have foundthat Drosophila uses a unique system to generate a LIMonly protein from the paxillin gene locus. Drosophila, on
the other hand, does not have other paxillin-related LIM

Figure 2proteins, such as Hic-5 and leupaxin, present in mammals.

Biochemical circuitry of paxillin-associated ArfGAPs in motile cells

Our findings provide clues regarding the origin as well as
the current functions of the LIM proteins and the LIM-onlyproteins, and we are currently investigating these issues
using Drosophila.

Cell migration in neuronal network formation
Normal cells have the ability to detect their collision withother cells or extracellular materials and to stop migrating,that is, contact inhibition. Contact inhibition of cells has
been believed to be essential for efficient and proper
formation of neuronal networks as well as for the development of correct tissue morphology. We have started toaddress these issues using paxillin and paxillin-associatedArfGAP proteins, coupled with their gene manipulations inmice.

Sabe, H. Paxillin-associated ArfGAPs -their isoform specificities and roles in coordination- In ‘The ARF book’ (Editor, R. Kahn;
Kluwer Academic Publishers). (in press).
Yagi, R. et al (2001). A novel muscle-specific LIM-only protein is generated from paxillin gene locus in Drosophila. EMBO Report
2, 814-820.
Uchida, H. et al (2001). PAG3/Papα/KIAA0400, a GTPase-activating protein for ADP-Ribosylation factor, regulates ARF6 in FcγRmediated phagocytosis. J. Exp. Med. 193, 955-966.
Yano, H. et al (2000). Paxillinα and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of
growth through tyrosine phosphorylation. Proc. Natl. Acad.Sci. USA 97, 9076-9081.
Kondo, A. et al (2000). A new paxillin-binding protein, PAG3/Papα/KIAA0400, bearing an ARF GTPase-activating protein activity
is involved in paxillin recruitment to focal adhesions and cell migration. Mol. Biol. Cell 11, 1315-1327.

43

Laboratory of Molecular Cell Biology
and Development
Masatoshi Takeichi (Professor, D.Sc.)
http://www.cdb.riken.go.jp/ctp/
http://www.cdb.riken.go.jp/english/index.html

Laboratory of Molecular Cell Biology and Development

Masatoshi Takeichi

44

Cell adhesion, recognition, and morphogenesis
During animal development, cells undergo dynamic morphogenetic movement and rearrangement, leading
to the formation of complex tissue structures. We are studying the molecular basis of such cell behavior,focusing on the roles of a family of cell-cell adhesion molecules, the cadherins, and associated molecules.
We are also seeking to unravel the mechanisms of synaptic junction formation; this project includes testing
the idea that cadherin family members may play a role in interneuronal recognition during neural network
formation. Furthermore, we hope to identify the molecules that regulate the lamination of cortical layers in
the central nervous system, as a model of tissue patterning. Our studies are expected to benefit not only
basic developmental biology but also a number of medical fields, such as those seeking to identify themechanisms underlying neural circuit defects and cancer metastasis.

Regulatory mechanisms of cell-cell adhesion, andtheir roles in morphogenesis
Cadherin/catenin complexes are crucial for the establishment of stable cell-cell association. We are interested in
identifying signaling mechanisms that modulate cadherinmediated adhesion, which we consider to be essential formorphogenetic processes such as cell relocation and cell
shape changes, and even in cancer metastasis. The
cadherin cytoplasmic tail has regulatory functions, and ourcurrent studies include identifying novel moleculesinteracting with the cadherins and catenins, and analyzing
their functions.

Synapse formation and interneuronal recognition

Figure 1

The neural network is generated through specific connec-

Double-staining for cadherin (red) and actin (green) in epithelial

tions between neurons, but their mechanisms are poorly

cells.

understood. Our group discovered that cadherin/catenincomplexes are localized in synaptic junctions, playing

important roles in synapse formation. We are currently

retinal cell differentiation and laminated layer formation.We

investigating the following problems: How the cadherin

are now working to uncover the molecular and cellular

system regulates synapse morphology and function, how

events downstream of this Wnt signaling. In addition, we

cadherin-dependent specific cell adhesion can contributeare studying the role of αN-catenin signaling in corticalto interneuronal recognition, and how axons and dendrites

morphogenesis, as its loss causes cell migration defects incan be distinguished from each other.

the brain.

Seeking novel functions of cadherin superfamily
genes
The cadherin gene superfamily comprises more than 120
members, as estimated from the human genome database, and this number seems to have dramaticallyincreased after vertebrate evolution. The biological
functions of these diversified molecules are, however,mostly undetermined. We have started attempts to uncover
the roles of some of the cadherin superfamily members,
including protocadherins and other newly identified
molecules, expecting to shed light on their evolutional

Figure 2
Blocking cadherin activity in the neural retina causes disruptionof its laminated structures. Left, normal retina; right, cadherinblocked retina, stained for Pax-6 to visualize the ganglion,
amacrine, and horizontal cell layers.

roles.

Identification of mechanisms controlling neuronalpositioning and patterning
In the nervous system, during cortical development, eachdifferentiating neuron is placed in a specific layer, resulting
in the formation of laminated structures. We are investigating the mechanisms of neuronal positioning, using theneural retina as a model. Our recent studies revealed thata subtype of Wnt genes play a central role in controlling

Takeichi, M. (1991) Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455.
Suzuki, S.C. et al (1997). Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal
mouse brains. Mol. Cell. Neuroscience 9, 433-447.
Nakagawa, S. et al (1998). Neural crest emigration from the neural tube depends on regulated cadherin expression. Development
125, 2963-2971.
Aono, S. et al (1999). p120ctn acts as an inhibitory regulator of cadherin function in colon carcinoma cells. J. Cell Biol. 145,
551-562.
Togashi, H. et al (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, 77-89.

45

Laboratory of
Molecular Neurobiology
Manabu Negishi (Professor, D.Pharm.Sc.)
Hironori Katoh (Instructor, D.Pharm.Sc.)

Laboratory of Molecular Neurobiology

Manabu Negishi

46

Neuronal functions of Rho family GTPases
The mission of the lab is to understand the way in which neurons elaborate and guide their neurites andneural circuits are formed. In the developing nervous system, neurite outgrowth is an essential process
underlying the formation of the highly specific pattern of connections between neurons.
One of our major focuses has been on the intracellular signal transduction systems involving the Rho familyof small GTPases that allow extracellular guidance signals to instruct the neurite formation, elongation andguidance of the neurites. Rho family GTPases and their signaling partners play important roles in the
reorganization of the actin cytoskeleton for neuronal morphological changes. Among them, Rho has been
known to induce neurite retraction, while Rac and Cdc42 have been shown to be involved in neurite outgrowth.

Neuronal function of RhoG
We have examined a signal transduction pathway for Rhoinduced neurite retraction, and we revealed that G12family of heterotrimeric G proteins activates Rho andactivation of Rho triggers neurite retraction through Rhoassociated kinase. On the other hand, concerning the Rac
and Cdc42-mediated neurite outgrowth, we revealed that
RhoG, another Rho family GTPase, is a key regulator in
NGF-induced neurite outgrowth in PC12 cells, acting
downstream of Ras and upstream of Rac1 and Cdc42.
We have started two research projects in the neuronal
functions of Rho family GTPases. The first is to understandthe molecular basis of regulation of neuronal morphology
by Rho, Rac and Cdc42: how G12 family transduces thesignals via Rho activation and how RhoG extends the

Figure 1

neurites through activation of Rac and Cdc42.

Primary cultured hippocampal neurons expressing GFP.

Neuronal function of Rnd subfamily
The second is to investigate neuronal functions of a novel
type of Rho family GTPases, Rnd subfamily, two of them,Rnd1 and Rnd2, being expressed mainly in brain. We haverecently revealed several downstream novel effectors of
Rnd GTPases. We are now examining the molecular routes
for the Rnd signaling in neuronal network formation.

Figure 2
Structure and function of Rapostlin, an effector of Rnd2.

Katoh H. et al (2000). Small GTPase RhoG is a key regulator for neurite outgrowth in PC12 cells. Molecular and Cellular Biology
20, 7378-7387.
Katoh H. et al (2002). Socius is a novel Rnd GTPase-interacting protein involved in disassembly of actin stress fibers. Molecular

Cellular Biology 22, 2952-2964.
Nakamura K. et al (2002). The rostral Raphe Pallidus nucleus mediates pyrogenic transmission from the preoptic area. The

Journal of Neuroscience 22, 4600-4610.
Yamaguchi Y. et al (2002). Gα12 and Gα13 interact with Ser/Thr protein phosphatase type 5 and stimulate its phosphataseactivity. Current Biology 12, 1353-1358.
Fujita H. et al (2002). Rapostlin is a novel effector of Rnd2 GTPase inducing neurite branching. The Journal of Biological Chemis-

try in press.

47

Laboratory of Membrane Biochemistry
and Biophysics

Laboratory of Membrane Biochemistry and Biophysics

Yasunori Kozutumi (Professor, D.Pharm.Sc.)
Katsumi Matsuzaki (Associate Professor, D.Pharm.Sc.)
Hiromu Takematsu (Instructor, D.Pharm.Sc.)
http://www.users.kudpc.kyoto-u.ac.jp/~o51267/index.html

48

Yasunori Kozutumi

Research on the biological function of membrane lipids and glycoconjugates
Outline of Research Activities are as follows:
1. Sphingolipid-mediated signal transduction in mammals and yeasts
2. Molecular biological studies on the sialic acid converting enzyme and its function using knockout mice
3. Elucidation of action mechanisms of antimicrobial self-defense peptides and the development of potent
antibiotic peptides
4. Application of membrane-permeable peptides to intracellular drug delivery
5. Molecular recognition between membrane-spanning helices
6. Interactions of amyloid peptides with glycolipids

Biological function of sphingolipids
We are focusing on sphingolipids as potential regulators of
the induction of multinuclear cell formation through the
inhibition of cytokinesis. The first approach is to study
pathologically occurring multinuclear cells. A sphingolipid,
psychosine, was demonstrated to be a trigger lipid for the
induction of multinuclear giant cells associated with asphingolipid metabolic disease (Kanazawa et al., 2000)(Fig.1). The second approach is to investigate the action
mechanism of a new immunosuppressant that inhibits thebiosynthesis of sphingolipids. The inhibition was shown to
induce multinuclear cells and apoptotic cells. A serine/
threonine kinase potentially involved was identified using
yeast (Sun et al., 2000).

Figure 1
A typical multinuclear cell induced by psychosine. The

Onset mechanism of Alzheimer's disease
The critical step in the development of Alzheimer's disease
is considered to be the conversion of soluble, nontoxic

psychosine-treated cells were stained with FITC-phalloidin (green)
for actin filament and with PI (red) for nuclear staining. The barcorresponds to 10 μm.

amyloid β-protein (Aβ) to aggregated, toxic Aβrich in
β-sheet structures. We have proposed that the conformational transition occurs in lipid rafts rich in sphingolipids
and cholesterol. We discovered that soluble Aβselectively
binds to a ganglioside cluster, the formation of which isfacilitated by cholesterol (Kakio et al., 2001), and thatamyloid fibril formation is facilitated in the presence ofGM1 ganglioside-bound Aβin a β-sheet-rich structure,

Figure 2that works as a seed (Kakio et al., 2002) (Fig.2).

Our model for Alzheimer amyloid formation in lipid rafts. SolubleAβ with unordered structures does not bind to rafts without

Study on the biology of sialic acid mediatedganglioside clusters. Once ganglioside clusters are formed bymolecular recognitionchanges in local lipid composition, such as increases inSialic acids comprise a family of sugars found at thecholesterol content, which is a risk factor for the disease, Aβtermini of the cell surface glycoconjugates. N-

recognizes the cluster, forming a helix-rich structure. An

Glycolylneuraminic acid (NeuGc) is one of the major sialic

increase in membrane-bound Aβ density induces a helix-to-

acids, and is a structural derivative of the most abundant

sheet conformational transition of Aβ. Under conditions whereN-acetylneuraminic acid (NeuAc). In contrast to NeuAc, theβ-sheet-rich Aβ is observed, especially in the presence of

expression of NeuGc varies between animal species.

GM1, amyloid fibril formation is markedly accelerated.

NeuAc hydroxylase is the rate-limiting enzyme for thebiosynthesis of NeuGc in mammalian cells. Recently, thehuman gene for CMP-NeuAc hydroxylase was reported tohave suffered an exon-deletion mutation whereas themouse and other mammals including our closest relative,
the chimpanzee retain the active gene. In our laboratry, weare persueing the biological consequences of the targeted
disruption of NeuGc biosynthetic pathway in mice.

Kanazawa, T. et al (2000). Inhibition of cytokinesis by a lipid metabolite, psychosine. J. Cell Biol. 149, 943-50.
Sun, Y et al (2000). Sli2 (Ypk1), a homologue of mammalian protein kinase SGK, is a downstream kinase in the sphingolipidmediated signaling pathway of yeast. Mol. Cell Biol. 20, 4411-9.
Kakio A. et al (2001). Cholesterol-dependent formation of GM1 ganglioside-bound amyloid β-protein, an endogenous seed for
Alzheimer amyloid. J. Biol. Chem. 276, 24985-24990.
Kakio A. et al (2002). Interactions of amyloid β-protein with various gangliosides in raft-like membranes: importance of GM1
ganglioside-bound form as an endogenous seed for Alzheimer amyloid. Biochemistry, 41, 7385-7390.

49

Laboratory of
Functional Biology
Akira Kakizuka (Professor, M.D., D.Med.Sc.)
Seiji Hori (Lecuture, D.Med.Sc.)
Hiroshi Ohizumi (Instructor)
http://www.lif.kyoto-u.ac.jp/labs/funcbiol/

Akira Kakizuka

The molecular and biochemical basis of neurodegeneration
Many neuodegenerative disorders manifest disease-specific phenotypes, and thus it was thought impossible to deduce a common
molecular mechanism underlying many, if not all, neurodegenerative
disorders. However, protein aggregates and vacuoles are almost
universally found in degenerating neurons, suggesting the existence ofsimilar molecular processes in neuronal cell death. In 1994, we
identified the gene responsible for Machado-Joseph disease (MJD),
the most common inherited spinocerebellar ataxia. Since then we
have elucidated common molecular mechanisms underlying
neurodegeneration via molecular analysis of MJD. Not only MJD but

Laboratory of Functional Biology

also another 8 inherited neurodegenerative diseases, including

50

Figure 1
VCP is a potential key mediator of neurodegeneration
caused by abnormal protein precipitation

Huntington’s disease are caused by the expansion of CAG repeats
encoding polyglutamines. Indeed, we have showed that polyglutamines have the ability to self-aggregate and induce
neurodegeneration and neuronal cell death, leading to a proposal of ‘polyglutamine disease’. Furthermore, we have identified
ter94/VCP, a member of the AAA+ ATPase, as a key molecule in neurodegenration. Interestingly, VCP co-localizes not only withpolyglutamine aggregates but also other protein aggregates and Lewy bodies. Moreover, profound deficits in its ATPase activity
are found to severely affect ER quality control, leading to abnormal ER expansion and cell death. These lines of evidence
indicate that VCP functions not only as a common sensor for abnormal protein accumulations but also as a mediator ofneurodegenerative phenotypes; excessive accumulation of abnormal proteins may inactivate VCP’s ATPase in several
neurodegenerative disorders, eventually leading to the neurodegenerations. A proper regulation of VCP function is thus proposed
to lead to novel treatments that are effective in a broad spectrum of neurodegenerative diseases.

The molecular basis of gene expression networks

effect is still unclear. We are analyzing ERRL1 (for ERR

determining energy expenditure and obesity

Ligand 1), a protein related to PPARγ coactivator-1, which

A balanced body energy budget controlled by proper

can bind and activate orphan ERRs (estrogen receptor-

physical exercise is most effective against obesity andrelated receptors) in vitro. Consistently, ERRL1 transgenic

diabetes mellitus, but the precise molecular basis of thismice exhibit increased expression of the medium chainacyl coenzyme A dehydrogenase (MCAD), a known ERR
target and a pivotal enzyme of mitochondrial β-oxidationin skeletal muscle. As a result, the ERRL1 mice show astate similar to an athlete; namely the mice are hyperphagic and of elevated energy expenditure, and are resistantto obesity induced by a high-fat diet or by a genetic
abnormality. Indeed, expression of both MCAD and ERRL1

KKAy mice (Yellow)

is induced in the skeletal muscle after exercise in mice.
These results demonstrate that ERRL1 can function as a‘protein ligand’ of ERR and that its level contributes to the

KKAy (Yellow)+ ERRL1

control mice (black)

Figure 2
ERRL1 expression can antagonize the obesity induced by agenetic mutation

control of energy balance in vivo, and provide a strategy
for developing novel anti-obesity drugs, or ‘exercise
mimics’.

Kakizuka, A. (1998) Protein Precipitation: A common etiology in neurodegenerative disorders? Trend. Genet., 14: 396-402.
Yamamoto, Y., et al. (2001) Isolation of neuronal cells with high processing activity for the Machado-Joseph disease protein. Cell

Death Differ. 8: 871-873.
Hirabayashi, M., et al. (2001) VCP/p97 in abnormal protein aggregates, cytoplasmic vacuoles, and cell death, phenotypes
relevant to neurodegeneration. Cell Death Differ. 8: 977-984.
Higashiyama, H., et al. (2002) Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced
neurodegenerations in Drosophila. Cell Death Differ. 9: 264-273.
Kobayashi, T., et al. (2002) Functional ATPase activity of p97/VCP is required for the quality control of endoplasmic reticulum inneuronally differentiated mammalian PC12 cells. J. Biol. Chem. (in press)

51

Laboratory of Viral Oncology
Kunitada Shimotohno (Professor, D.Pharm.Sc.)
Makoto Hijikata (Associate Professor, D.Med.Sc.)
Yasuo Ariumi (Instructor, D.Med.Sc.)
http://www.virus.kyoto-u.ac.jp/Lab/htv/jp/index.html
Kunitada Shimotohno
Molecular mechanisms of virus induced carcinogenesis
We focus on the carcinogenesis caused by infection with human oncogenic viruses, in particular, human Tcell leukemia virus type 1 (HTLV-1) and hepatitis C virus (HCV). Clarification of the molecular mechanismsof viral carcinogenesis may not only facilitate development of methods for disease prevention but may also
contribute to our understanding of how cell proliferation is regulated by internal and external signals. Ourcurrent research projects are concerned with clarification of the role of virus proteins in virus replicationand modulation of cell proliferation. We are investigating the processes by an HCV replicon system that can
mimic HCV replication in cultured cells and by screening for oncogenic gene alteration(s) in hepatocellular
carcinoma (HCC) and in adult T-cell leukemia (ATL).
Multiple functions of Tax-1 of HTLV-I
Tax-1, an oncoprotein encoded by the HTLV-1 genome,
has potential to immortalize normal human T-lymphocytes.
We have suggested that several physiological phenomena

Laboratory of Viral Oncologyof HTLV-1 infected cells are associated with the modula-

52

tions of NFκB, CREB, or p53 activation pathways by Tax-1.
During analysis of the trans-acting function of Tax-1, we
observed that Tax-1 was associated with transcriptional
cofactors CBP/p300 and suppressed transcriptional
activities of p53 and its family member p73 throughcompetition with these transcription factors for theinteraction with transcriptional coactivators CBP/p300. We
are further extending our work to clarify molecularmechanisms of Tax-1 functions on the modulation of

Figure 1
Co-localization of Tax-1 with the RNA splicing bodies
Tax-1 ectopically produced in COS7 cells was detected in theparticular nuclear structures as well as SC-35 (panels a-c) but
was not colocalized with coproduced PML (panels d-f), indicatingthat Tax-1 is located in the RNA splicing bodies in the nucleus.

expression of various cellular genes.
tion which seems to be related with these events. The
HCV proteins that modulate cell proliferationcore, an HCV structural protein, has potential to modulate

Persistent infection of HCV and resultant chronic hepatitis

growth, transformation, and apoptosis by affecting thefor a few decades is believed to be important for thecellular signaling pathways, including Ras/Raf and NFκB

pathogenesis of HCC. We have shown that some HCV

pathways. NS5A, an HCV non-structural protein, hasproteins have functions in the modulation of cell prolifera-

potential to interfere with the function of PKR, a double-

stranded RNA dependent protein kinase induced byinterferon. We are studying the molecular mechanisms ofthese phenomena exhibited by the HCV proteins throughthe identification of cellular molecules targeted by theseviral proteins.

Replication of HCV genome reconstituted in celllines
The most effective way to prevent the development ofvirus-caused cancer is to block virus replication in carriers.
Thus, development of anti-HCV agents is quite important
for the cancer prevention. There was, however, no experimental system supporting efficient HCV proliferation which
was likely to be essential for development of anti-viral
agents. We have shown that some human-derived celllines, including hepatocytes and mononuclear cells,
support HCV replication, although not very efficiently. Using
the subgenomic sequences from these culture cell-derived

Figure 2
Establishment of the cell lines in which the HCV subgenome RNA
is efficiently replicated
The HCV genome bearing the neomycin resistance gene (neor)
instead of the region encoding the structural proteins(subgenomic RNA, upper panel) was synthesized in vitro andtransfected into the hepatocellular carcinoma derived HuH-7 cells(middle panel). After selection by geneticin, the subgenomic RNA
was found to be maintained in the total RNA from the selected
cell lines (lower panel), suggested the subgenomic RNA wasautonomously replicated in the cells and was supplying theneomycin resistance gene product.

HCVs, we have recently established a highly efficient HCV
replication system that mimics the replication of HCV using
cell lines, and have been studying the molecular mechanism of HCV genomic replication. Based on this system weare now trying to establish an in vitro system reconstituting the whole HCV life cycle.

Marusawa, H. et al (1999). Hepatitis C virus core protein inhibits Fas- and tumor necrosis factor alpha mediated apoptosis via
NF-κB activation. J. Virol. 73, 4713-4720.
Ariumi, Y. et al (2000). HTLV-1 tax oncoprotein represses the p53-mediated trans-activation function through coactivator CBPsequestration. Oncogene 19, 1491-1499.
Kaida, A. et al (2000). Functional impairment of p73 and p51, the p53-related proteins, by the human T- cell leukemia virus type1 Tax oncoprotein. Oncogene 19, 827-830.
Fukuda, K. et al (2001). Hepatitis C virus core protein enhances the activation of the transcription factor Elk1 in response tomitogenic stimuli. Hepatology 33, 159-165.
Watashi, K. et al (2001). Cytoplasmic localization is important for transcription factor nuclear factor-κB activation by hepatitis C
virus core protein through its amino terminal region. Virology 286, 391- 402.

53

Laboratory of Genetic Information
Akira Shimizu (Professor, D. Med. Sc.)
Kei Tashiro (Associate professor, D. Med. Sc.)
Akira Shimizu
Molecular mechanisms underlying highly systemic functions
Vertebrate animals have highly organized systems, such as neural and immune systems, which are characteristic to higher animals. To understand the molecular basis of such highly systemic functions, we focusedon the analyses of structure and regulation of genetic information responsible for such systemic functions
using model animals, for example, transgenic or gene disrupted mice. In the immune system, genetic
information of the antigen receptors is reorganized during the lymphocyte development in a cell stage
specific manner. Cell to cell contact and signaling, cell movement, fine organization of multi cellular system are also known to be very important for immune and neural systems. Our recent subjects are genes
and molecules involved in the regulation of such functions.

Accessibility of TCR Vγ genes are developmentally

Laboratory of Genetic Information

regulated by histone acetylation

54

Histone acetylation correlates with accessibility, since
histone acetylation at the fetal-type Vγ3 gene in accord
with germline transcription is relatively high in fetal
thymocytes, but specifically becomes low in adult thymocytes within the entirely hyperacetylated locus. Furthermore, inhibition of histone deacetylation during thedevelopment of adult bone marrow-derived thymocytes by
a specific histone deacetylase inhibitor, trichostatin A,
leads to elevated histone acetylation, germline transcrip-

Figure 1
Accessibility regulation of TCRg genes.

tion, cleavage, and rearrangement of the Vγ3 gene. These
data demonstrate that histone acetylation functionallyenhanced in B cells lacking Id2, through activation of εdetermines the chromatin accessibility for V(D)J recombi-

germline transcription. This augmentation is because ofnation in vivo and that an epigenetic modification of

the highly augmented activity of E2A. In contrast, Id2 ischromatin plays a direct role in executing a developmental

induced by TGF-β1 and suppresses IgE CSR in wild-type

switch in cell fate determination. (Figure 1)

B cells, but this suppression did not occur in Id2 deficient
B cells. Our results provide evidence for the inhibitory andId2 is an essential negative regulator of classselective role of Id2 in IgE CSR, especially in response toswitch recombination to IgE

TGF-β1. Id2 might act as molecular safeguard toClass switch recombination (CSR) to IgE was specificallysuppress the production of IgE to prevent serious compli-cations such as allergic hypersensitivity during the normal
course of immune response.

Th1 cells have higher trans-endothelial migration
activity than Th2
The TEM abilities of Th1 cells from mice bearing autoimmune diseases and antigen specific Th1 cell lines wereseveral folds higher than those of Th2 cells and lines ofthe same origin. These preferences were observed without
exogenous chemoattractant. Antibodies against LFA-1 andICAM-1 as well as CD44 markedly blocked TEM of Th1
cells. TEM ability was also blocked by pharmacologicalinhibitors for Src-family protein tyrosine kinases andphosphatidylinositol-specific phospholipase C. Cross-

Figure 2
Two signaling pathways for trans-endothelial migration.

linking of CD44 strongly induced highly elongated morphology in Th1 lines but weakly in Th2 lines. These data
indicate that there are signaling pathways for TEM
independent of chemokine attraction but through adhesion
molecules including CD44 and that preferences in TEM
ability of Th1 over Th2 is formed, at least in part, by
intrinsic differences in these pathways.

Other than above, Dr. Tashiro’s research group is working
on identification of novel regulatory proteins using highefficiency cDNA cloning systems for those encoding cellsurface and secretary proteins.

Figure 3
Members of Prof. Shimizu’s research group.

Sugai, M. et al. (2003) Essential role of Id2 in negative regulation of IgE class switching. Nature Immunol. in press.
Katakai, T. et al. (2003) Chemokine-independent preference of T-helper-1 cells in trans-endothelial migration. J. Biol. Chem. inpress.
Agata, Y. et al (2001). Histone acetylation determines the developmentally regulated accessibility for T cell receptor-γ generecombination. J. Exp. Med. 193, 873-879.

55

Laboratory of Cell Recognition
and Pattern Formation

Laboratory of Cell Recognition and Pattern Formation

Osamu Chisaka (Associate Professor, D.Med.Sc.)

56

Cranial Neural Crests
In vertebrates, cranial neural crest cells contribute to organs/tissues such as thymus, parathyroid, great
vessels, etc., that are indispensable for survival of an animal. We are interested in how these cranial neuralcrest cells find their way to the destined place and how they can differentiate into such a broad range oforgans/tissues. To address these questions, we have been analyzing mutant mice which show cranial
neural crest defects.

Hox code and Neural Crest Cells

tions, i.e., neural crest derived and epibranchial placode

Cranial neural crest cells arise from dorsal hindbrain

derived. We are currently investigating the cell autonomy

where regional identity is governed by the “Hox-code”. Weof the nerve defect focusing on the interaction between

found that in Hoxa3 mutant mouse embryos, a specificHoxa3 expressing neural crest cells and placode cells at

population of neural crest cells show abnormal migration

the level of presumptive glossopharyngeal nerve forming

pattern and results in the truncation of the glossopharyn-area. Recently we found another type of neural crest

geal nerve (Watari et al., 2001). Sensory neurons of thedefect (growth/differentiation) in Hoxa3 mutant mice

glossopharyngeal nerve consist of two different popula-(Kameda et al., 2002).

Watari, N. et al (2001). Hoxa3 regulates integration of glossopharyngeal nerve precursor cells. Dev Biol. 240, 15-31.
Kameda, Y. et al (2002). Homeobox gene hoxa3 is essential for the formation of the carotid body in the mouse embryos. Dev

Biol.,247, 197-209.

Laboratory of
Molecular Neurobiology

Unfolding or misfolding of proteins constitutes a fundamental threat to all living cells. In eukaryotes, proteins can be unfolded or misfolded in a variety of subcellular compartments such as cytoplasm, mitochondria, and peroxisomes, but the risk of protein misfolding is particularly acute in the endoplasmic reticulum(ER), in which newly synthesized secretory and transmembrane proteins attain their proper tertiary structure. Efficient quality control systems have evolved to prevent incompletely folded molecules from moving
along the secretory pathway. Thus, accumulation of misfolded proteins in the ER would detrimentally affectthe function and/or localization of the approximately one-third of all cellular proteins that translocate into
the ER after synthesis on membrane-bound ribosomes. Furthermore, misfolded proteins can exert
proteotoxicity through hydrophobic and non-productive interactions with other cellular proteins. Eukaryotic
cells have developed three different mechanisms for dealing with an accumulation of unfolded proteins in
the ER: transcriptional induction, translational attenuation, and degradation (see the figure below). Our goal
is to achieve comprehensive understanding of how eukaryotic cells control the quality of proteins in the ER
by pursuing the molecular mechanisms of these homeostatic responses.
Current Projects
We have identified two intracellular signaling pathways
from the ER to the nucleus important for the transcriptional induction in mammalian cells, which ultimately
utilize the transcription factors ATF6 and XBP1 activatedby regulated intramembrane proteolysis and frame switch
splicing, respectively. We are currently investigating theroles of these transcriptional induction systems in the ER
quality control.

Figure 1Mori, K. (2000). Tripartite Management of Unfolded Proteins in the Endoplasmic Reticulum. Cell 101, 451-454.
Yoshida, H. et al (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active
transcription factor. Cell 107, 881-891.

Laboratory of Molecular Neurobiology

Kazutoshi Mori (Associate Professor, D.Pharm.Sc.)

57

Laboratory of Viral OncoloLaboratory of Cell Regulation and Molecular Networkgy

Laboratory of Cell Regulationand Molecular Network

58

Yota Murakami (Associate Professor, D.Sc.)
http://www.virus.kyoto-u.ac.jp/virus.html
Nuclear/Chromatin structure and chromosome functions
Nuclear and chromatin structure is involved in the regulation of chromosome functions, such as transcription, DNA replication and recombination, but its molecular mechanism is unclear. To understand how suchhigher order structure regulates the chromosome functions, we mainly use fission yeast as a model systemand focus on establishment and maintenance of heterochromatin, which is a structurally and functionally
important constituent of the chromosome and nuclear structure.
Establishment and maintenance of heterochromatin in fission yeast
It has been unclear how heterochromatin is established at
specific regions. Human CENP-B is a centromere specific
DNA binding protein, and fission yeast has three CENP-B
homologs. We indicate that the CENP-B homologs act assite-specific nucleation factors for the formation ofcentromeric heterochromatin by heterochromatin-specificmodifications of histone tails. (Nakagawa et al. 2002,
Figure). We are investigating the process of heterochromatin establishment by detail analysis of the nucleation ofheterochromatin induced by CENP-B homologs. Little isFigure 1

also known about the heterochromaitn maintenance.

Establishment and Maintenance of heterochromatin

Several proteins, which are involved in DNA replication,
checkpoint and chromatin assembly, seem to be involvedin this process. We start to tackle this subject by molecular analysis of these proteins.

Nakagawa et al (2002). Fission yeast CENP-B homologs nucleate centromeric heterochromatin by promoting heterochromatin
specific histone tail modifications. Genes & Dev. 16, 1766-1778

The Graduate School of Biostudies, Kyoto University
Bioscience: a key solution to creating a brighter future– Aiming toward the Welfare and Happinessof Humanity in the 21st Century –Preface
At the start of the 21st century, we are witnessing the beginning of a new
era in life sciences: biological phenomena are finally being elucidated at anorganismal level, and vast amounts of genetic, molecular, and cellular information
is being generated. Anticipating such advancements, we founded the Graduate
School of Biostudies at Kyoto University in 1999 by combining research groups
from the Faculties of Science, Agriculture, Pharmaceutical Science, and the Schoolof Medicine. As we were the first academic institution in Japan to combine theseresearch fields to study bioscience, we have received much attention throughout
Japan and abroad. Bioscience is a new concept and breakthrough beyond the
traditional educational and research systems, and could not have been achieved
within the conventional concept and fields of the life sciences.
As a research institution, day-to-day production of valuable research results
and the future development of this research are the most important issues; however, considering our role as an educational institution, we seek to establish along-term objective and vision to achieve our commitments. That is, we need toraise a specific awareness of future education and research activities. We will
make every endeavor to realize our vision – “Aiming toward the Welfare andHappiness of Humanity in the 21st Century” –, which we set at the institution of
the Graduate School of Biostudies.

Kayo Inaba
Dean, Professor
October 30th, 2004

Preface1About the Graduate School of Biostudies– Institution and History –It has taken approximately 10 years for our frontier scientists

independent research projects and educational activities.
To enrich education at the Graduate School, we added inschool cooperative laboratories (four labs from the Institute ofto realize their vision. The researchers who filled the full-time

Virus Research, one lab from the Developmental Biology

positions at the Graduate School spent these seeding years bothResearch Center at the Graduate School of Science, and one lab

to carry out academic planning and to establish mutual under-from the Genetic Experiment Facility), and off-campus coopera-

standing and personal communications among the founding

tive labs for joint reseasrch (two labs from the Osaka Bioscience

members. During this period, to attract researchers and graduate

Institute). After an organizational restructuring, we currently havestudents, we invited internationally renowned researchers from

6 in-school cooperative labs (4 labs from the Institute of Virus

Japan and abroad and held joint seminars on a regular basis.

Research, one lab from the Radiation Biology Center, and one lab

Our enthusiasm and strong desire to establish an institution withfrom the Center for Genomic Medicine), and three off-campus

a novel vision of bioscience have been confirmed through thesecooperative labs (one lab from the Osaka Bioscience Institute andactivities and efforts. In the beginning of 1998, we finally

two labs from RIKEN). In addition, a new lab that studies

submitted the final proposal to institute the Graduate School of

Bioscience communication has joined the graduate school in

Biostudies at Kyoto University to the Monbusho (current Ministry

2004. In this way, we have made every endeavor to take "Life"of Education, Sports, Science, Culture and Technology); thisscience to new heights.

proposal was readily welcomed and accepted. A total of 19

The primary aim of the Graduate School of Biostudies was tolaboratories, including 16 existing labs (five labs each from theintegrate laboratories that were scattered throughout the campus

Faculty of Agriculture and the Faculty of Science, two labs eachof Kyoto University. A building for advanced comprehensivefrom the School of Medicine and the Faculty of Pharmaceuticalresearch that will house the Graduate School and the School of

Science, and one lab from the Institute for Virus Research andMedicine is currently under construction on the South campus,

the Faculty of Integrated Human Studies) and three new labs,

and the construction of a new building for joint use with themake up the Graduate School of Biostudies.

Faculty of Agriculture is also underway on the North campus;

To achieve our mission, the Graduate School of Biostudies

both buildings are to be completed in the spring of 2005. As

consists of two divisions, Integrated Life Science and Systemic

described above, the Graduate School of Biostudies has a brightLife Science. Laboratories in Integrated Life Science investigate

future.

fundamental life mechanisms at the levels of gene, chromosome,cell, muticellular organization and surrounding environment.

PrefaceResearch topics include gene mechanisms, chromosome

2

Division of Intergarted Life Science
Department of Gene Mechanisms

segregation, cell cycle control, cell and developmental biology,

Laboratory of Chromosome Transmission

plant gene and totipotency, applied molecular biology, environ-Laboratory of Gene Dynamics

mental signals and stresses. Education and research in Systemic

Laboratory of Cell Cycle Regulation

Life Science are focused on the elucidation of the fundamentals

Department of Cell and Developmental Biology

of molecular and systemic biology, cell biology and immunology,

Laboratory of Cell Recognition and Pattern Formationand neurology for the recognition of self-and non-self, inLaboratory of Signal Transduction

response to various factors. Research areas include molecular

Department of Plant Gene and Totipotency

and system biology, animal development and physiology, signal

Laboratory of Plant Molecular biology

transduction, functional biology, mammalian regulatory network.

Laboratory of Molecular and Cellular Biology

The two divisions work closely together, while each division
maintains its own specific viewpoint and engages in uniqueDepartment of Applied Molecular Biology
Laboratory of Biosignals and Response

Laboratory of Applied Molecular Microbiology

technologies. Thus, it is our commitment to develop frontier

Laboratory of Molecular Biology and Bioresponse

researchers, who are not bound only to their own specific field,

Department of Responses to Environmental Signals andbut have profound understanding of the multi-dimensionalStressesadvancement of the life sciences, the ability to recognize theLaboratory of Plant Physiology

importance of the environment surrounding human society, andLaboratory of Plasma Membrane and Nuclear

the ability to study the relationships between the environment

Signalingand society from a broader point of view. Our mission is to

Department of Molecular and Cellular Biology*Laboratory of Cellular and Developmental Genetics*Laboratory of Mammalian Molecular Biology
Department of Genome Stability

develop human resource who are capable of the following tasks:(1) To advance novel biostudies to the highest level in theworld.
In order to meet the demands of industry, universities,*Laboratory of Genome Stability

and research institutions, our rigorous program educates
students so that they will be equipped with advanced

Division of Systemic Life Scienceknowledge, master the technologies of novel bio-

Department of Molecular and System Biology
Laboratory of Neurosciencesciences, and establish a sense of “self” in society.
(2) To apply novel biosciences to protect the global

Laboratory of Immunology and Cell Biology

environment for human welfare and happiness.

Department of Animal Development and Physiology

By integrating knowledge and technologies of conven-Laboratory of Developmental Gene Regulation

tional sciences, agriculture, medicine, and pharmaceuti-

Laboratory of Immunobiology

cal science, we aim to develop researchers who can*Laboratory of Molecular Cell Biology

understand the complex biosphere and become leaders*Laboratory of Molecular Cell Biology and Developmentin 21st century human society.

Department of Signal Transductions(3) To observe and elucidate various biological phenomena

Laboratory of Molecular Neurobiology

of living organisms as systemic functions.

Laboratory of Membrane Biochemistry and Biophysics

In a 21st century society seeking human welfare andhappiness, we promise to develop human resource who

Department of Functional Biology
Laboratory of Functional Biology

will become leaders in the global community and who

Department of Biostudies and Society

will promote harmony between the human race andLaboratory of Science Communication and Bioethics

other living organisms.

Department of Mammalian Regulatory Network*Laboratory of Cell Regulation and Molecular Network*Laboratory of Viral Oncology*Laboratory of Genetic Information(*cooperate laboratories)

Research Activities
The number of grants-in-aid for Scientific Research andother competitive funds the Graduate School has received truly
reflects our research achievements. For the academic years of
2002, 2003, and 2004, the Graduate School of Biostudies

Our Mission
The outcomes of new life sciences are increasingly permeat-

received grants-in-aid that have surpassed a billion yen.
Furthermore, the number of grants per faculty member has been

ing into our daily lives; therefore, it is very important to achieve aat the top in Kyoto University. In addition, among our publica-

proper prospective of living in harmony with nature and thetions, eight papers have been cited as references more thanenvironment, in addition to pursuing the development of

1,000 times, and 300 papers have been cited more than 100

3

times. Many of the lab heads play leading roles in their research

entrance examination and the comprehensive examination andfields. Also, many of the lab heads have served as editors ofthesis defense for the Master's and doctoral degrees. In addition

academic journals. Two professors have been nominated as

to these routine meetings, lab meetings, which are called andmembers of the National Academy of Science, USA and the Royal

presided over by the head of each lab, are held to discuss theSociety of London, suggesting that they are indeed world-daily operation and management of each lab, as well as personal

renowned leaders in their research fields. Moreover, since thematters regarding young researchers working in the lab, prior to

institution of the Graduate School in 1999, five professors havethe Faculty Council and department meetings. Furthermore, allreceived prestigious prizes, such as the Japan Academy Prize,

topics and reports that are to be discussed at the Faculty Council

the Imperial Award, the Asahi Award, the Osaka Science Prize,

and Department meetings are to be discussed a priori by the

the Toray Science and Technology Prize, and the Inoue Prize forsteering committee.

Science.
We also have strong ties to industries: two of our professors

To date, six young researchers from our department havebecome professors at other universities or research institutions,and nine other faculty members have experience working atand one was promoted to associate professor. Two professors

institutes attached to private corporations. We have up to 100

have retired since our institution began. As a result, (number)contract research agreements and academic grants-in-aid, which

professors have been newly recruited from inside and outside thetruly indicate that our research direction meets social needs.

University. Furthermore, the number of female faculty members

In order to seek further development, and disclose thehas doubled since the institution began (two at the time ofoutcomes of our research projects to the public, we maintaininstitution: Professor (1), Assistant Professor (1); four at presenthigh standards, and are ready to accept evaluation from outside(Professor (1), Associate Professor (1), and Assistant Professor

sources. As one component of sharing our research results, all of(2)). However, the employment of foreign researchers has not

the labs at the Graduate School have participated in an annual

increased (currently we have only one researcher working as an

two-day open-to-the-public symposium. Many people from inside

Assistant Professor). We would like to increase the employment

and outside the University have participated in the symposium,of female and foreign researchers in the future.

which has become a valuable forum for the exchange ofintellectual information. In addition to the annual symposium, we
have invited renowned researchers from Japan and abroad in

Education
The Graduate School of Biostudies aims to accomplish theDecember 2002 and 2003 (three from abroad, four from Japanfollowing objectives to establish day-to-day operations, provide

in 2002; two from abroad, five from Japan in 2003) and held

outstanding education, and promote research studies:

research conferences. We received meaningful opinions andcomments as well as evaluations from the outside the University.

(1) To develop human resource who are equipped withknowledge of next-generation, advanced life sciences.
We aim to develop a new type of human resource who

Organization and Management
At the Graduate School, Faculty Council meetings, which
include the professors from both divisions, and Department
meetings, which include faculty members who hold positions

4

possesses unique and creative qualities to deal withvarious unknown issues that the next generation ofhumanity must face.
(2) To develop human resources who establish a sense ofabove lecturer in all participating labs, are held monthly. The“self” in society.

dean presides over both meetings. At Faculty Council meetings,

We value the unique academic background and future

most issues regarding activities at the graduate school are

vision of each member of the Graduate School, and weclosely and carefully examined, and decisions are made.

aim to establish a system to assess the effects of an

Department meetings deal with educational issues, such as theunconventional diversified education and help students

acquire healthy and fair critical minds.

classes. Furthermore, we designed a special opportunity for the(3) To encourage active and flexible personnel management.

students to learn bioscience in English: we offered intensive

We encourage active information exchange between

lectures on molecular biology in English. To refine the students’labs, and aim to advance and develop unique researchlinguistic ability, we supported an International Student Seminarprojects to explore novel fields of life sciences.

that was hosted by the graduate students. We also invited(4) To maximize the post-doctoral program and establish arenowned researchers from abroad to present in the lecture

method to assess and evaluate achievement.

series. We hosted an open forum with guest speakers twice.

In order to intensively develop internationally active life

Through these experiences, we have provided opportunities forscientists, we aim to maximize the existing system, andthe students to be exposed to the most advanced research. In

maintain a faculty to student ratio lower than at the2004, at the third International Student Seminar, two native

former graduate school.

English speaking researchers and four outstanding Japanese

The Graduate School of Biostudies offers a two-year

researchers from other research institutions were invited andMaster's degree program, and a three-year doctoral degree

asked to evaluate the students for their oral presentation skills inprogram. Students require 30 units to graduate from theEnglish. Ten students will be given the opportunity to give oral

Master’s program; attending lectures, seminars, submitting apresentations at the Kyoto-Singapore International Symposium,

Master thesis based upon the student’s research project, andwhich will be held in January 2005. The 21st century COE

passing the comprehensive examination and thesis defense areprogram provides further financial support to the graduate

required for graduation. Doctoral candidates are obligated to

students; the students are employed as research and education

attend seminars that credit more than 8 units per year, conduct a

assistants. Also, in certain research fields where further

unique research project, publish original academic papers, andpromotion is thought to be necessary, we are actively involved insubmit a Ph.D. dissertation. A comprehensive examination andemployment of young researchers as “post-doctoral fellows.”

thesis defense are mandatory to qualify for the Ph.D. degree.
It has been six years since the Graduate School of Biostudies

To date, accounting for some fluctuation in each academic
year, 80 to 85 Master’s students and 45 to 55 Ph.D. candidates

was founded. As expected, many important outcomes haveenroll in the Graduate School of Biostudies each year. As aalready been produced through our research activities. The

result, the Graduate School of Biostudies has a total enrollmentstselection of our institute for the 21 century COE program inof 326 hard working graduate students, 162 Master’s students

2002 has further enhanced our programs and broadened and(of whom 53 are women), and 164 Ph.D. candidates (of whom

deepened the horizons of our research activities. In this new

37 are women). In April 2004, 17 students, who were among theenvironment, graduate students are given more opportunity to

first graduates of the doctoral program at the Graduate School ofclosely work with the instructors; this results in an encouraging

Biostudies, received their Ph.D. degrees. More students haveatmosphere of cooperativity and helps foster a unique character

applied to defend their Ph.D., and about 25 students will receivethat values diversity and the frontier spirit.

their degrees at the end of this academic year.

st

We make the most of the 21 century COE program funds to

In March 2001, at the time of the first graduation from theeducate graduate students and young researchers. For example,

Master's program, we initiated an alumni association “Ibuki” ofto be active in the global research community, researchers need

the Graduate School of Biostudies. Thereafter, two issues of thethe linguistic skills for oral presentation and writing manuscripts

alumni bulletin have been published. We hope the alumni

in English; therefore, we have designed special classes where

association will become an open forum where the graduates andstudents can learn English conversation directly from native

students can freely exchange opinions and information.

instructors. Reduced class size allows active involvement. In
2002 and 2003, almost 400 students were enrolled in these5

Laboratoryof Plant Molecular Biology

Laboratory of Plant Molecular Biology

Takayuki Kohchi (Professor, D.Agr.)
Hideya Fukuzawa (Associate Professor, D.Agr.)
Katsuyuki Yamato (Instructor, D.Agr.)
http://www.lif.kyoto-u.ac.jp/labs/plantmb/

6

Takayuki Kohchi

Molecular Mechanisms of Plant Growth and Development
All organisms have to acclimatize themselves totheir environment that undergoes continuous changeover both short and long time periods. Plants are
exquisitely sensitive to their environment, and havedeveloped well-refined sensing and signaling systems to modulate their growth and development.
How do plant cells recognize and integrate ambientconditions such as light and CO2? The research inthis laboratory focuses on the molecular basis ofdevelopmental plasticity and genetic control of
Figure 1 Model photosynthetic organisms used in thegrowth and differentiation using Arabidopsis, moss
laboratory
Marchantia, and green alga Chlamydomonas as
Top left; Arabidopsis thaliana is a small weed of no economic use,
but the primary model organism for genetic studies of higher
model photosynthetic organisms (Figure 1). Topics
plants. Top right; Chlamydomonas reinhardtii has developed ainclude 1) photo-sensing and signal transduction bycarbon-concentrating mechanism (CCM) to acclimate to CO2limiting or high-light stress conditions to maintain photosynthesis.
phytochrome; 2) CO2-sensing and signal transducBottom; Marchantia polymorpha is dioecious plants with sextion; 3) hierarchical regulation of plant development
chromosomes - male (left) and female (right) plants.
by transcription factors; 4) sex chromosome andsexual development of plants; and 5) polyunsaturated fatty acid metabolism.

Structure-function assays of phytochrome
chromophore for photo-sensing in plants
The phytochrome family in plants is a major photoreceptor
that has the linear tetrapyrrole molecule, phytochromobilin
as its chromophore. Genes for tetrapyrrole metabolism
were identified by a molecular genetic approach with

Arabidopsis photomorphogenic mutants (Figure 2) and by
a comparative genomics. We have developed a genetic
modification system that is suitable for evaluating the

Figure 2 Phytochrome and chromophore biosynthesis
pathway in plantseffects of chromophore structure on photobiological

Structure of sex chromosomes and sexual devel-

activities in planta. We found that the structural require-

opment in liverwort

ment of phytochrome chromophore is diverged in the modes

We determined the draft sequence of the Y chromosomeof action and that the selection of phytochromobilin as afrom the dioecious liverwort, Marchantia polymorpha.

chromophore might have an ecological significance for red/

Currently we are investigating its genetic program of sex

far-red light responses in land plants (Kami et al 2004).

determination and differentiation by functional genomics
utilizing genomic information.

Molecular mechanisms of CO2-sensing and acclimation to CO2-limiting conditions

Lipid metabolism in liverwort and application to

Aquatic photosynthetic organisms adapt to CO2-limiting

plant molecular breeding

conditions by inducing a set of genes for carbon-concen-

Liverwort contains high levels of polyunsaturated fatty

trating mechanisms (CCM). The Ccm1 gene encoding a

acids (PUFA), such as arachidonic and eicosapentaenoic

protein with a novel zinc-finger motif is a master regulator

acids. We have isolated the PUFA-synthesizing enzyme

of CCM in Chlamydomonas. Transcriptome and genetic

genes encoding fatty acid elongase and desaturase fromanalyses of CO2-insensitive regulatory mutants revealed

liverwort, and applied to PUFA production in higher plantsthat cells acclimate to low-CO2 conditions by inducing(Kajikawa et al 2004).

CCM-related genes through CCM1 and MYB-transcriptional
factor LCR1, which binds to CO2-resposive DNA elements(Yoshioka et al 2004, Miura et al 2004).
Hierarchical regulation of growth phase transition
by receptor kinase and transcription factors
Intercellular communication mediated by receptor-likekinases (RLK) is important for diverse processes in plantdevelopment. A MADS-box protein, AGL24, that plays arole in promotion of floral transition was identified as a
substrate for mersitematic receptor-like kinase, MRLK
(Fujita et al 2003).

Kajikawa, M. et al (2004). Isolation and characterization of ∆6-desaturase, an ELO-like enzyme and ∆5-desaturase from theliverwort Marchantia polymorpha and production of arachidonic and eicosapentataenoic acids in the methylotrophic
yeast Pichia pastoris. Plant Mol. Biol. 54, 335-352.
Kami, C. et al (2004). Complementation of phytochrome chromophore-deficient Arabidopsis by expression ofphycocyanobilin:ferredoxin oxidoreductase. Proc. Natl. Acad. Sci. USA 101, 1099-1104.
Yoshioka, S. et al (2004). A Novel Myb Transcription factor LCR1 regulates the CO2-responsive gene Cah1 encoding aperiplasmic carbonic anhydrase in Chlamydomonas reinhardtii. Plant Cell 16, 1466-1477.
Miura, K. et al (2004). Expression profiling-based identification of CO2-responsive genes regulated by CCM1 controlling acarbon-concentrating mechanism in Chlamydomonas reinhardtii. Plant Physiol. 135,1595-1607.
Fujita, H. et al (2003). An Arabidopsis MADS-box protein, AGL24 is specifically bound to and phosphorylated by meristematic
receptor-like kinase. Plant Cell Physiol., 44, 735-742.

7

Laboratory of Science
Communication and Bioethics

Laboratory of Science Communication and Bioethics

Kazuto Kato, (Associate Professor, D.Sc.)

8

Kazuto Kato
Biostudies and society
Due to the rapid progress of biostudies over the last few decades, our society faces many problems in
the interface between scientific research and society. First, many areas of biostudies are highly specialized so that it has become difficult for non-specialists to understand current state of research. Second,
there are increasing numbers of socially controversial issues, such as cloning, stem cell research, genetically modified food etc. The aim of our research is to analyze these problems through practical andtheoretical approaches. We also try to propose possible actions and solutions. Current research topics
include 1) communication of biostudies to non-specialists, 2) analysis of ethical and social issues in theareas of biostudies, and 3) modern history of biostudies.

Communication of research to non-specialists

about aims and meanings of their own research.

Today, science communication is considered to be one of
the most important activities in the interface between

Analysis of ethical and social issues

biostudies and society. It contains communication among

For the balanced development of biostudies, it is important

and within various groups in the society such as commu-

to identify ethical, legal and social issues (ELSI) and findnities of scientists, private companies, mass media,

appropriate ways of dealing with them. Our researcheducational institutes, policymakers, and the general

focuses on the ELSI surrounding recently advanced

public. We are particularly interested in activities orga-

research fields including genomics, human ES cells andnized by scientists or scientific communities. We organize

others. In particular, we have been analyzing ethical andprograms or seminars in which scientists meet non-social issues arising from International HapMap project.

specialists and explain their research. Then effects of theWe have also conducted survey of how various countries

activities on the participants are analyzed. One example isparticularly in Asia are handling ELSI problems and foundthe “Genome Square” organized by us together withthat many countries in Asia are rapidly developing frame-

genome scientists in Japan. The event was held in two to

works for bioethics. Analyses of international projects andthree cities every year from 2002 to 2004. It contained

situations in other countries would facilitate reflections on

poster exhibitions by 30 to 40 laboratories for the general

the current system in Japan.

public. We have analyzed the effect of the events on
visitors and researchers. One of the findings is that

Modern history of biostudies

having conversations with members of the general public

Our third research topic is the analysis of modern history

provides researchers with opportunities to think deeplyof biostudies. We plan to focus on several research fields

that were important for the development of current

graduate students of the School of Biostudies. Topics ofbiostudies. To carry out the research, we conduct

current lecture courses include science communication,

literature surveys and interviews with scientists.

bioethics and other issues on social and cultural aspectsof biostudies. These lectures are expected to help

Lectures for graduate students

students to consider issues around their research and takeWe organize lectures on biostudies and society for

responsible actions.

Mitsuishi, S., Kato, K. and Nakamura, K. (2001). A study of a new way to communicate science to the public - Through thecreation of the Scientist Library. Public Understanding of Science 10, 231-234.
Kato, K. (2002). The Language of Present-day Biology and Biomedicine - Globalized Science meets Cultural Diversities. In“The Kyoto/Seoul Symposium on Linguistic Challenges in the Modern Sciences: First Movement” (T. Yokoyama and K. Y.
Sik ed.), Occasional Seminar Reports of the Institute for Research in Humanities No.7, 37-42.
The International HapMap Consortium (including K. Kato). (2004) Integrating ethics and science in the International HapMap
Project, Nature Reviews Genetics 5, 467 -475.
Kato, K. (2004). The ethical and political discussions on stem cell research in Japan. In W. Bender, C. Hauskeller, and A.
Manzei (ed.) CROSSING BORDERS: Cultural, religious and political differences concerning stem cell research, in press.

9

Laboratory of Cell Regulationand Molecular Network

Laboratory of Cell Regulation and Molecular Network

Masahiko Sugita (Professor, D.Med.Sci.)

10

Masahiko Sugita
CD1: A new paradigm for antigen presentation
A salient feature of our immune system is its ability to respond specifically against invading pathogens
and cancer cells. The molecular and cellular basis for the antigen-specific immune response appeared
fully established by studies carried out for the past two decades, focusing on the organization and thefunction of major histocompatibility complex (MHC)-encoded molecules. MHC class I and class II molecules bind peptide antigens and present them to specific T lymphocytes. We now know, however, thatthe universe of antigens recognized by the immune system includes non-peptide lipid antigens, which arepresented by CD1, a novel lineage of antigen-presenting molecules. Our recent studies indicate that CD1
mediates MHC-independent pathways for host defense against pathogens and cancer cells, thus encouraging us to develop a new class of lipid-based vaccines.

Dendritic cells and CD1 immunity
Dendritic cells (Figure 1) comprise the major cell population in our body that expresses CD1 molecules. Lipid
antigens taken up by dendritic cells are transported to
endocytic compartments where CD1 molecules capture
antigens. Subsequently, lipid antigen bound CD1 molecules traffic to the plasma membrane and activate lipid
antigen-specific T cells. Most of these T cells are CD8+cytotoxic killer T cells bearing αβ T-cell receptors, but afraction of γδ T cells also responds to lipid antigens
presented by CD1 molecules. These CD1-restricted T
cells are likely to mediate elimination of microbe-infected
cells and cancer cells.

Cell biology of CD1
Molecules of the human CD1 family (CD1a, CD1b, CD1c,
CD1d) are differentially expressed in endocytic compartments of dendritic cells (Figure 2). CD1b molecules areinternalized from the plasma membrane via clathrin-coatedFigure 1
Dendritic cells isolated from human epidermis

pits and then interact with a cytosolic adaptor protein
complex, AP-3, which directs their trafficking to lysosomes. In contrast, CD1a molecules fail to bind AP-3 andfollow a recycling pathway of the early endocytic system.
The differential distribution of CD1a and CD1b is importantfor efficient sampling of lipid antigens because lipids withshort carbon chains and those with long carbon chains aretransported to recycling endosomes and lysosomes,
respectively (Figure 3).

Identification of a human genetic disease with
impaired CD1 function
Patients with Hermansky-Pudlak syndrome type 2 (HPS-2)
carry mutations in the AP-3 genes. In cells derived from
HPS-2 patients, CD1b molecules are unable to gain
access to lysosomes, resulting in a profound defect in lipid
antigen presentation. Since MHC molecules function
normally in AP-3 deficient cells, defects in CD1b antigen

Figure 2
Distinct intracellular localization of human CD1 molecules
CD1a-containing vesicles (upper panel) and CD1b-containing
vesicles (lower panel) represent recycling endosomes (arrowheads) and lysosomes, respectively.

presentation may account for recurrent bacterial infectionsin HPS-2 patients.

Figure 3
Intracellular trafficking of CD1 molecules and lipid antigens

Sugita, M. et al (2004). New insights into pathways for CD1-mediated antigen presentation. Cur. Opi. Immunol. 16, 90-95
Winau, F. et al (2004). Saposin C is required for lipid presentation by human CD1b. Nat. Immunol. 5, 169-174
Sugita, M. et al (2002). Failure of trafficking and antigen presentation by CD1 in AP-3-deficient cells. Immunity 16, 697-706
Sugita, M. et al (1999). Separate pathways for antigen presentation by CD1 molecules. Immunity 11, 743-752
Sugita, M. et al (1996). Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs. Science 273,
349-35211