LABORATORY OF BIOLOGICAL DIVERSITY
KAMADA Group
Assistant Professor:

KAMADA, Yoshiaki

Tor (target of rapamycin) is a Ser/Thr protein kinase
which is well conserved in organisms spanning from yeasts
to mammals. Tor forms two distinct protein complexes,
TORC1 (Tor complex1) and TORC2. On one hand, TORC1
is involved in amino acid sensing, regulation of protein
synthesis (especially the translation step), the cell cycle, andautophagy. On the other hand, TORC2 is responsible foractin organization and cell integrity. Up until now, it’s been
deemed unlikely that TORC2 can recognize nutrient signals.
The aim of our research group is to reveal the molecularmechanisms underlying how TORC1 receives amino acidsignals and how the TORC1/2 pathways regulate phenomena
they are associated with. We have been studying Tor signaling in the budding yeast Saccharomyces cerevisiae, and havefound three novel TOR signaling pathway branches (Figure 1).

product of ARS acts as an amino acid signal rather than anamino acid itself. Biochemical in vitro TORC1 assay also
revealed that tRNA directly inhibits TORC1 kinase activity,
suggesting that uncharged tRNA, produced under aminoacid-depleted condition, functions as a starvation signal.
Reducing cellular tRNA molecules desensitizes TORC1 inactivation by nitrogen starvation in vivo.
Based on these results, a TORC1 regulatory model was
proposed which contends that free tRNA released fromprotein synthesis under amino acid starvation inhibits
TORC1 activity. Therefore, TORC1 employs a tRNAmediated mechanism to monitor intracellular amino acids(Figure 2).

Figure 2. A schematic model of how amino acid is sensed by TORC1.
Cytosolic free tRNA inactivates TORC1 under amino acid limited condition.

Since tRNA directly inhibits TORC1 activity, TORC1
should have a tRNA-binding site(s). Thus, we have investigated further so as to determine the tRNA-binding site. So
far, we have obtained a good candidate for the tRNA-bindingsite in Tor protein, and we will now focus on this domain to
determine its function.

II. TORC1 phosphorylates Atg13, the molecularswitch of autophagy

Figure 1. Tor signaling pathway for budding yeast. Our group has foundthree branches of the Tor pathway.

I. How do amino acids regulate TORC1?

TORC1 is regulated by amino acids which in themselves
are fundamental nutrients. 20 species of amino acids thatbuild proteins cannot be interchanged with each other.
Therefore, each amino acid must be individually detected by
TORC1.
We have conducted genetic research and discovered the
involvement of (aminoacyl-) tRNA in TORC1 regulation.
For example, mutants of aminoacyl-tRNA synthetases(ARSs) exhibited inactivation of TORC1 even under aminoacid-rich condition, suggesting that aminoacyl-tRNA, a

TORC1 negatively regulates autophagy: a protein degradation system induced by nutrient starvation.
We have been consequently able to discover the TORC1mediated regulatory mechanism of autophagy. Under nutrient-rich conditions, TORC1 directly phosphorylates Atg13,
a component of the Atg1 kinase complex. Atg1 is a Ser/Thr protein kinase, the activity of which is essential for
autophagy and is largely enhanced in response to TORC1
inactivation. Activation of Atg1 requires formation of theAtg1 complex. Phosphorylation of Atg13 by TORC1 playsa pivotal role in Atg1 complex formation; phosphorylated
Atg13 loses its affinity to Atg1 resulting in disassembly of
the Atg1 complex and repression of autophagy. On the other
hand, dephosphorylation of Atg13 triggers formation of theAtg1 complex, activation of Atg1 kinase, and consequent
induction of autophagy.

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III. Relationships between TORC1 and eIF4Ebinding proteins

EIF4E-binding proteins (4EBPs), which binds to translation initiation factor 4E (eIF4E), are thought to negatively
regulate translation initiation, because their competition
infused binding to eIF4E prevents eIF4E-eIF4G association,
which is a primary and essential procedure in translation
initiation. In mammals, the inhibitory function of 4EBP1
is regulated by mammalian TORC1 (mTORC1). Under
nutrient-rich conditions, mTORC1 phosphorylates 4EBP1,
and the phosphorylated 4EBP1, lose affinity to eIF4E and let
eIF4E bind to eIF4G.
We examined whether two yeast 4EBPs, Caf20 and Eap1,
have properties in common with 4EBP1. Caf20, but not
Eap1, is phosphorylated in a TORC1-dependent manner, itbinds to eIF4E, and it never associates to eIF4G. However,Caf20 was not directly phosphorylated by TORC1, Caf20eIF4E binding was not affected by TORC1 activity, andCaf20-eIF4E complex was found in ribosome fraction, suggesting that the function of the yeast 4EBP is different fromthat of its mammalian counterparts.

Publication List:〔Original paper〕

T., Ishii, T., Kamada, Y., Funakoshi, M., Kobayashi, H.,• Sekiguchi,
and Furuno, N. (2022). Involvement of Gtr1p in the oxidative stressresponse in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res.
Commun. 598, 107–112. DOI: 10.1016/j.bbrc.2022.02.016

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LABORATORY OF BIOLOGICAL DIVERSITYHOSHINO Group
Assistant Professor:
Technical Assistant:

HOSHINO, Atsushi
TAKEUCHI, Tomoyo
ITO, Kazuyo
KAWADA, Saki

Figure 1. The duskish mutant of I. nil shows variable flower phenotypes
and produces variegated, fully pigmented, and pale grayish-purple flowers.
It segregates offspring that only show fully pigmented or pale grayishpurple flowers, which can be stably inherited by further generations.

Genomic structures and their genetic information are stablytransmitted into daughter cells and future generations during
cell division; however, they can vary genetically and/or
epigenetically. Such variability impacts gene expression andevolution. To understand these genomic dynamics in eukaryotes, particularly in plants, we are analyzing the flower
pigmentation of morning glories, including Ipomoea nil(Japanese morning glory), I. purpurea (the common morning
glory) and I. tricolor.

I. Flower pigmentation patterns

The wild type morning glories produce flowers with auniformly pigmented corolla. For our study, we collectedmutants displaying pigmentation patterns. Flower pigmentation patterns are easily observable and the molecular mechanisms underlying these phenomena provide useful model
systems for investigating genomic variability.
Recessive mutations, duskish of I. nil and pearly-v of I.
tricolor, confer variegated flowers caused by a stable insertionof a transposable element into a gene for flower pigmentation,
regulated by epigenetic mechanisms (Figure 1). We intend toanalyze detailed molecular mechanisms of these mutations.

II. Whole-genome analysis of various I. nil lines

The National BioResource Project (NBRP) Morning
Glory (described below) maintains ~3,500 lines, including 2 standard lines: the Tokyo Kokei Standard and Violet.
A high-quality draft whole-genome sequence of the Tokyo
Kokei Standard, which is accessible from our database, hasbeen published. The NBRP Genome Information Upgrading
Program supported whole-genome sequencing of 100 representative lines. We chose lines based on the needs of theresearch community and to provide information regarding
their polymorphisms and gene mutations. We included themultiple mutants called “Henka-Asagao,” which covered
the major mutations, and the wildtype plants isolated fromnatural populations outside Japan. From this, we discovereda total of 25,000 Tpn1 transposon-induced insertion polymorphisms and SNPs, Indels, and CNVs at 25 million loci.
Several of those polymorphisms were tightly linked to morphological mutations, including the mutations that “HenkaAsagao” carry.

Figure 2. The I. nil lines used for the whole-genome analysis. (a) Violet:
The most widely used standard line. (b) Africa: The wildtype line isolated in
Guinea in 1956. (c) Nepal: The wildtype line isolated by Dr. Sasuke Nakao
in Nepal in 1952. (d) Danjuro, one of the most popular cultivars, named after
the reddish-brown color of the costume used by Danjuro Ichikawa II in his
Kabuki play “Shibaraku.” (e) The tiny mutant has small organs. White spots
on the leaves are due to the variegated mutation. (f) A mutant line of “HenkaAsagao” showing double flowers with narrow flower petals and sepals.

III. Flower opening and time control

The time of flower opening is important for the reproductive strategy of plants, and many plants are known toflower at specific times during the day. However, little is
known about the molecular mechanisms that determine
flower opening time as well as flower opening itself. As
their name suggests, morning glories typically bloom in theearly morning. There is significant physiological knowledge
about the circadian rhythm that controls flower opening time.
To elucidate these molecular mechanisms, we have begun afunctional analysis of genes for petal opening and the circadian rhythm.

IV. Morning glory bio-resources

NIBB is the subcenter for the NBRP dedicated to morning
glories. In this project, we collect, maintain, and distributestandard and mutant lines for flower pigmentation and DNA
clones from EST and BAC libraries of I. nil and its related
species. I. nil is one of the most popular floricultural plants inJapan and has a 100-year history of extensive related genetic
studies. Our collection includes 241 lines and 177,000 DNA
clones. The whole-genome sequence, the transcriptome

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Evolutionary Biology and Biodiversity

sequences, and the end sequences of the DNA clones can beviewed via the I. nil genome database (http://viewer.shigen.
info/asagao/index.php).

Publication List:〔Original paper〕

K., Furuta, T., Yamaji, N., Kuniyoshi, D., Ishihara, M.,• Nagaki,
Kishima, Y., Murata, M., Hoshino, A., and Takatsuka, H. (2021).

Effectiveness of Create ML in microscopy image classifications: a
simple and inexpensive deep learning pipeline for non-data scientists.
Chromosome Res. 29, 361–371. DOI: 10.1007/s10577-021-09676-z

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LABORATORY OF BIOLOGICAL DIVERSITYTSUGANE Group
Assistant Professor:

TSUGANE, Kazuo

Although transposons occupying a large portion of the
genome in various plants were once thought to be junkDNA, they play an important role in genome reorganization and evolution. Active DNA transposons are important tools for gene functional analysis. The endogenous
non-autonomous transposon, nDart1, in rice is said togenerate various transposon-insertion mutants because
nDart1 elements tend to insert into genic regions undernatural growth conditions. The transpositions of nDart1
were promoted by an active autonomous element, aDart127, on chromosome 6. By using the endogenous nDart1/
aDart1-27 system in rice, a large-scale nDart-inserted
mutant population was easily generated under normal
field conditions, and the resulting tagged lines were freeof somaclonal variation. The nDart1/aDart1-27 system
was introduced into a rice variety, Koshihikari (Oryza
sativa subsp. japonica), and Basmati (Oryza sativa subsp.
indica). Various mutations caused by the insertion ofnDart have been screened for characteristic phenotypes.

bore slightly larger grains (Figure 1) as a dominant inheritance. Transposon-display identified the insertion site ofnDart1 in the Lgg mutant.

II. Analysis of Lgg mutants

The identified LGG gene shows similarity to RNA
binding proteins. To investigate the subcellular localization of LGG protein, green fluorescent protein (GFP)fused constructs driven by 35S promoter, 35S:LGGNPGFP was transformed into rice calli. GFP fluorescencespots were observed in the nuclei in calli. These results
suggest that LGG is localized to the nucleus. To verify
that LGG was responsible for the long grain phenotype, knockout (GE) and overexpression (OE) lines were
produced by transforming a CRISPR/Cas9 constructtargeting the RNA recognition motif region of LGG andan LGG construct with its WT promoter into into cv.
Nipponbare (NP), respectively (Figure 2(A)). Several
independent GE lines showed significantly increased
grain length, and three independent OE lines had shorter
grains than NP. Any significant differences of panicle
length, number of panicles per plant and number of spikelets per panicle were not observed among NP, GE andOE except for culm length. Observations of longitudinal
sections from lemmas of GE and OE lines revealed thatthe cell sizes of GE and OE lines were comparable to NP(Figure2 B-C), suggesting that LGG might regulate thelongitudinal cell number of spikelet hull and thus grain
length.

I. Large grain (Lgg) mutation in rice

Seed size and number were controlled by various genes
in the plants. It was reported that expression changes inhigh contribution genes for seed size, number and panicle
shape resulted in a decrease of the total yield. A strategy
for boosting rice yield based on molecular biology is tostack the finely tuned gene expressions. The Lgg mutant
which was isolated from Koshihikari-nDart tagging line

Figure 1. Phenotype of Large gain (Lgg). Harvested panicle and seeds.

Figure 2. The spikelets morphology and longitudinal sections and cell
length of the lemma of NP, GE, and OE, respectively. (A) The spikelets.
Bar = 5 mm. (B) The Section of lemma in transgenic plants. The red andblack arrows indicate adaxial epidermis and chlorophyll-contained parenchyma cell. (C) Cell length of lemma in NP, GE and OE. n = 3. mean ± SD.

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Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITY
KATO GroupSpecially Appointed Assistant Professor:
KATO, Kagayaki
Technical Assistant:
HYODO, Miwa

Computer programs that accurately capture and measure thefeatures from images that express the phenomenon become
an inevitable tool to objectively describe and evaluate abiological phenomenon obtained as digital imaging data.
From this point of view, we are developing image analysis
programs for each experimental case and analyzing image
data that captures various biological phenomena.

I. Cell segmentation/tracking system

To better understand organ formation mechanisms, it is necessary to analyze individual cells’ morphology and dynamics
quantitatively. However, it is difficult to achieve it due tothe large image volume size generated by such as time-lapse
microscopy and its ambiguity.
To unveil organogenesis from the point of view of distinct
cell behaviors, we are developing application software
capable of describing cell dynamics from time-lapse imaging
data sets by employing image processing techniques. To
observe this, we are developing a software pipeline which
will automatically recognize individual cell shapes out
of 3D space and tracks them through time. This system
extracts cell boundaries and reconstitutes cell shapes in theform of a skeletonized chain of voxels spanning 3D space.
This abstract form of cell visualization makes it possible todescribe morphometric quantities and kinetics of cells at asingle-cell resolution (Figure 1). These morphometric quantities allow us to perform comparative studies on shapes andbehaviors more precisely under several experimental conditions to gain a better understanding of the genetic programs
underlying organogenesis. We are now applying this system

Figure 1. Visualized apical cell surface of Drosophila embryonic epidermal cells. A time-lapse confocal microscopic data set expressing
E-cadherin-GFP was processed. Cell boundaries (green), the center ofgravity (blue), and normal vector (magenta) are indicated for each cell.

66

to several experimental models to determine the practicalityof the system.

II. Image processing pipeline for morphogenesis

In complex organs, methods for identifying and tracking
the initial positions of the cell groups consisting of the final
organ morphology are essential analytical methods for elucidating the developmental program.
Here, we analyzed the details of the pathways of individual
cells in a brain primordium throughout the brain morphogenesis in chick embryos in its early stages. We identified thecell positions out of randomly fluorescently labeled brain
primordium cells from a series of time-lapse images andconstructed a database of their time evolution. Based on thisdatabase, we developed a software system for automating thevisualization and quantification of the local migration patternof brain primordium constituent cells from several experimental conditions.

III. Quantification of fish body shapeTechniques for quantitatively analyzing the movements andbody forms of model organisms are considered indispensable for phenotypic evaluation in the process of elucidating
the molecular mechanisms underlying the behavior of living
animals.
To elucidate the mechanism of neuronal development ofzebrafish, we have developed a system that extracts the
body region from each frame of a video of free migrationof fish juveniles taken at a high frame rate and describes themorphology from the head to the tail as a free curve model.
From these curve models, we developed a system that classifies and simplifies the continuous bending forms of fish
as a small number of classes. This system was applied tothe movies acquired under various experimental conditions
and successfully described and statistical evaluation of thephenotype.

Publication List:〔Original paper〕

K., Kato, K., Iida, H., Teramoto, M., Kawamura, A., Watanabe,• Yoshihi,Y., Nunome, M., Nakano, M., Matsuda, Y., Sato, Y., Mizuno, H.,•

Iwasato, T., Ishii, Y., and Kondoh, H. (2022). Live imaging of avian
epiblast and anterior mesendoderm grafting reveals the complexityof cell dynamics during early brain development. Development 149,
dev199999. DOI: 10.1242/dev.199999
Kawano, K., Kato, K., Sugioka, T., Kimura, Y., Tanimoto, M., andHigashijima, S. (2022). Long descending commissural V0v neurons
ensure coordinated swimming movements along the body axis in larvalzebrafish. Sci. Rep. 12, 4348. DOI: 10.1038/s41598-022-08283-0

LABORATORY OF BIOLOGICAL DIVERSITYOHTA GroupSpecially Appointed Assistant Professor:
OHTA, Yusaku

Image analysis is an important component of understanding
life sciences. It enables us to quantify phenomena by extracting meaningful information from a large number of images
and to properly represent that information. I am developing
image analysis techniques aimed at analyzing the developmental process of early zebrafish embryo at whole embryo
scale and single-cell resolution. Currently, I am developing an analysis technique that can simultaneously extract
multiple pieces of information such as cell morphology, cell
motility, and cell dynamics by combining 3D cell tracking
and functional imaging.

I. Simultaneous multifunctional analysis of early
embryonic development at a whole embryo
scale and single cell resolution.

Three-dimensional remodeling of cell populations throughcell migration is essential in early embryogenesis. Cell
migration is highly coordinated by controlling cell-celladhesion. To elucidate the principles of such complex
embryogenesis, it is necessary to understand the cellular
dynamics of the whole embryo at single cell resolution. I am

Figure 1. Simultaneous multifunctional analysis of early embryonicdevelopment

developing image analysis techniques to analyze the developmental process of early zebrafish embryogenesis at wholeembryo scale and single-cell resolution.
With the development of microscopy, imaging of early
embryonic imaging has evolved from two-dimensional,
embryonic development has evolved from two-dimensional,
fixed specimen and partial embryo observation to threedimensional, living specimen and whole embryo scale.
This evolution has led to an explosion in the analysis ofcell migration during the development of early embryos inrecent years. However, conventional image analysis techniques could only extract information on cell migration inearly development. Therefore, I have developed an analysis
method that can simultaneously extract information on cell
migration, cell dynamics, and cell morphology at whole
embryo scale and single cell resolution.

II. Research support by image analysis

The development of imaging technology in life science
research has been remarkable, allowing many researchers toeasily acquire large and complex image data sets. However,image analysis is still a hurdle for researchers and can becomea research bottleneck. To solve this problem, I provide
research support based on the following three concepts.
The first concept is quantitative image analysis based on
extensive knowledge of image processing and statistics. For
most researchers, image evaluation is limited to qualitativeand subjective analysis. Correct analysis based on knowledgeof image and statistics supports quantitative and objective
analysis. The second concept is to support image analysis
through machine learning, including deep learning. Machinelearning has made remarkable progress in recent years,
and with a little learning, it is possible to simplify analysis,
which has been difficult with conventional image analysis
techniques. The third concept relates to providing researcherswith easy-to-understand explanations of image analysis techniques on the Web. The content ranges from the principles
of image analysis methods to how to use image analysis
software and plug-ins.

Figure 2. A website that explains how to use image analysis software

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National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYSHINOMIYA GroupSpecially Appointed Assistant Professor:
SHINOMIYA, Ai
Technical Assistant:
TSURUTA, Emiko

Figure 1. Differences in critical day length (right) were detected among
medaka populations that had originated in various latitudes (left) .

The natural environment, which incorporates phenomena such as day length, solar radiation, temperature, andprecipitation, generates seasonal changes that affect organisms. Although animals alter their physiology and behavior
in response to seasonal changes in their environment, themechanism of seasonal adaptation remains largely unknown.
Medaka (Oryzias latipes) provide an excellent model tostudy these mechanisms because of their rapid and obvious
seasonal responses. In addition, it is also possible to apply
transgenic and genome-editing approaches when researching
them, as well as reference genome sequences.
In keeping with this, we are currently using Medaka to
study the molecular mechanisms of seasonal adaptation, as
well as the relationship between seasonal information fromthe environment and the organism’s response.

Geographical variation critical day length was detected, andpopulations from lower latitudes indicated a shorter criticalday length (Figure 1). To identify the genes governing
critical day length, quantitative trait loci (QTL) analysis was
conducted using F2 offspring derived from crosses between
populations experiencing different critical day length. We
thus identified significant QTLs using Restriction-site
Associated DNA (RAD) markers (Figure 2).

Figure 2. Result of QTL analysis for critical day length.
A medaka habitat in summer (Miyazaki, Miyazaki Prefecture) (left). A
school of wild medaka (Toyohashi, Aichi Prefecture) (right).

I. Underlying mechanisms that define criticalday length and temperature

Most animals living outside the tropics reproduce only
during a particular season of the year. These are calledseasonal breeders, and it is well established that day length isa crucial cue for reproduction in many of them. In addition,it has been demonstrated that temperature changes are also
important for them in the how they detect seasons. However,it remains unknown how animals measure seasonal changes
in relation to these environmental factors.
Applying forward genetic approaches, we examined criticalday length in reproduction (i. e., the duration of lighting
time required to cause gonadal development) using various
Medaka populations, that originated in various latitudes
throughout Japan to elucidate these mechanisms.

68

Whole-genome re-sequencing in various medaka populations experiencing different critical day lengths were conducted. We have identified potential candidate genes thatdefine the critical day length by analyzing this genome
sequencing data.
We also performed experiments to identify critical temperature, and subsequently detected geographical variation
among Medaka populations. Significant QTLs for criticaltemperature have been detected from the genetic analysis
that was performed.

II. Analysis of the relationship between breedingrhythms and environmental information

Organisms exhibit various scales of rhythm, ranging inseconds to years. On the other hand, the natural environment provides the rhythmic changes concerning organisms.
However, the quantitative relationship between the information on environmental factors and biological rhythms ispoorly understood.

We conducted a linear regression analysis regarding annual
rhythms in gonadal development using data pertaining to
the annual changes in the gonadal size of the medaka andenvironmental information (day length, solar radiation, watertemperature) in the experimental field. The regression model
explained which environmental factors contributed to theseasonal change in medaka gonads and to what extent they
contributed to this change.

Publication List:〔Original papers〕

T., Okimura, K., Shen, J., Guh, Y.-J., Tamai, T.K., Shimada,• Nakayama,A., Minou, S., Okushi, Y., Shimmura, T., Furukawa, Y., Kadofusa,•N., Sato, A., Nishimura, T., Tanaka, M., Nakayama, K., Shiina, N.,
Yamamoto, N., Loudon, A.S., Nishiwaki-Ohkawa, T., Shinomiya,A., Nabeshima, T., Nakane, Y., and Yoshimura, T. (2020). Seasonalchanges in NRF2 antioxidant pathway regulates winter depression-likebehavior. Proc. Natl. Acad. Sci. U.S.A. 117, 9594–9603. DOI: 10.1073/
pnas.2000278117
Yassumoto I, T., Nakatsukasa, M., Nagano, A.J., Yasugi, M., Yoshimura,T., and Shinomiya, A. (2020). Genetic analysis of body weight in wild
populations of medaka fish from different latitudes. PLoS One 15,
e0234803. DOI: 10.1371/journal.pone.0234803

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Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYSAITO GroupSpecially Appointed Associate Professor

SAITO, Nen

Postdoctoral Fellow:

FUKUYAMA, Tatsuya

Measurement technologies, such as live imaging and nextgeneration sequencers, have been the recipients of rapid
development. We have entered a new era in which molecular
activities and gene expression levels in living tissues canbe measured at a single-cell resolution in a high throughput
manner. Our research group aims to elucidate the theoretical logic of dynamic living systems from such data by
combining mathematical modeling and machine learning.
The research topics in our group are diverse and cover subcellular scale phenomena as well as cellular and tissue level
phenomena.

Figure 1. Researches combining machine learning and mathematical modeling.

I. Simulations of amoeboid cells

We conducted mathematical modeling of the cell shape
dynamics of single cells. Migrating cells take on a varietyof complex shapes and sometimes dynamically deform toperform important biological functions. On one hand, during
the amoeboid movement of Dictyostelium discoideum, said
movement is driven by the dynamic formation and disappearance of pseudopods at the cell front. On the other hand,in the epithelial cells of fish, a network of actin fibers called
lamellipodia appear at the cell front, and migratory movements are performed while maintaining their overall shape.
Using the phase-field method, we have developed a mathematical model that can describe the two-dimensional shape
dynamics of these various forms of locomotion. We havealso applied deep learning to extract features that quantify
the shape of migrating cells, and systematically compared theresults between experiments and simulations. By combining
this type of modeling and data-driven analysis, it is possible70

to estimate which physicochemical parameters are responsible for the differences in cell shapes due to cell type andmutation.
We also performed three-dimensional modeling of cell
deformation in micropinocytosis. Macropinocytosis refers
to the nonspecific uptake of extracellular fluid, which plays
a ubiquitous role in cell proliferation, immune surveillance,and virus invasion. Although this phenomenon is widespread, it is still unclear how the cup-shaped cell membrane
is initially formed. We demonstrated that the protrusive
force localized to the edge of the patches can give rise to aself-enclosing cup structure, without further assumptionsof local bending or contraction. Furthermore, our model
exhibits a variety of cup morphologies self-organized via acommon mutually-dependent process of reaction-diffusion
and membrane deformation.

II. Mathematical Model of Microbial Symbiosis
via Metabolite Leakage

In microbial ecosystems, a wide variety of microbial
species sometimes can coexist even when there is only one
niche (e.g., one nutrient supply). In recent years, it has been
widely stated that nutrient symbiosis via metabolite leakage.
Said leakage is caused by cross-feeding, a phenomenon inwhich a cell leaks metabolites while simultaneously utilizing
another metabolites leaked from another cell, and is important for the formation of complex ecosystems. However, it
is unclear why cells secrete metabolites in the first place.
It makes sense if it is to dispose of unnecessary waste, butsometimes cells also secrete metabolites that are supposed tobe essential for their growth.
Based on analytical and numerical calculations, we haveillustrated that if the intracellular metabolism includes multibody (e.g., catalytic) reactions, leakage of essential metabolites can promote the leaking cell’s growth. We have also
demonstrated that mutualistic relationships among diverse
species can be established as a result of cell-level adaptationof metabolite leakage; each species cross-feeds others bysecreting essential metabolites for their own benefit, which
are usefully consumed by others, in a manner reminiscent ofgift giving. In this case, the exchange of metabolites becomesentangled, which in turn leads to the coexistence of diverse
microbes. The resultant ecosystems become resilient against
external perturbations including the removal of each coexisting species.

III. M athematical Model of collective cell
migration
Active matter physics, which is the study of collective
motions such as flocks of birds, has been well studied so far
in biology and physics. Considering the motion of a cell population in terms of active matter, the cells differ significantly
from existing active matter models in that the shape of individual cells can be deformed. How such deformability affects
cell collective migration is not fully understood.
This study proposes a mathematical model based on the
Fourier series expansion of cell contours to describe cell
shape. The shape is described as a superposition of Fouriermodes, and cell deformation is described as the dynamics ofeach Fourier coefficient. Unlike the cell-vertex model that
describes epithelial cells, the proposed model can describe
various shapes other than polygons. We performed a numerical analysis of a situation in which self-propelled cells with
only exclusion volume effects are densely packed using the
proposed model. We found that the cell population exhibitsa solidification-fluidization transition with only a change incell softness.

Publication List:〔Original papers〕

G., Saito, N., Fujimori, T., Hashimura, H., Nakamura, M.J.,• Honda,Nakajima, A., and Sawai, S. (2021). Microtopographical guidance of•••macropinocytic signaling patches. Proc. Natl. Acad. Sci. U.S.A. 118,e2110281118. DOI: 10.1073/pnas.2110281118
Imoto, D., Saito, N., Nakajima, A., Honda, G., Ishida, M., Sugita,
T., Ishihara, S., Katagiri, K., Okimura, C., Iwadate, Y., and Sawai, S.
(2021). Comparative mapping of crawling-cell morphodynamics indeep learning-based feature space. PLoS Comput. Biol. 17, e1009237.
DOI: 10.1371/journal.pcbi.1009237
Saito, N., and Sawai, S. (2021). Three-dimensional morphodynamic
simulations of macropinocytic cups. iScience 24, 103087. DOI:
10.1016/j.isci.2021.103087
Yamagishi, J.F., Saito, N., and Kaneko, K. (2021). Adaptation of
metabolite leakiness leads to symbiotic chemical exchange and to a
resilient microbial ecosystem. PLoS Comput. Biol. 17, e1009143. DOI:
10.1371/journal.pcbi.1009143

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Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYOHNO Group
Assistant Professor:

OHNO, Kaoru

The aim of this laboratory is to research reproductive
hormones in invertebrates, especially in echinoderms, and toanalyze the mechanisms by which they work. The comparisons of such molecules and mechanisms in various species
are expected to provide insights into the evolution of reproductive hormone systems.

I. Gonadotropins in the starfish, Patiria pectinifera

Gonadotropins play important regulatory roles in reproduction in both vertebrates and invertebrates. The vertebrate
gonadotropins, LH and FSH, are structurally and functionallyconserved across various species, whereas no such moleculehas been identified in invertebrates. Some gonadotropic
hormones have been found in invertebrate species. An insulin-like peptide was reported to be responsible for the regulation of egg maturation in the mosquito, Aedes aegypti, thus
demonstrating the involvement of insulin signaling in egg
maturation among invertebrates.

The gonad-stimulating substance (GSS) of an echinoderm,the starfish, was the very first gonadotropin to be identified
in invertebrates. GSS mediates oocyte maturation in starfish
by acting on the ovary to produce the maturation-inducing
hormone (MIH), 1-methyladenine, which in turn induces thematuration of the oocytes. In this sense, GSS is functionallyidentical to vertebrate LH, especially piscine and amphibian
LHs, and acts on the ovarian follicle cells to produce MIH
to induce the final maturation or meiotic resumption of theoocyte. Considering the functional similarity that GSS shareswith vertebrate LH, it is very important from an evolutionary
point of view to know the chemical and molecular structure of GSS. We cloned the gene encoding the amino acidsequence of purified GSS from radial nerves of the starfish,
Pateria pectinifera. Interestingly, phylogenetic analyses
revealed that it belonged to the insulin/insulin-like growth
factor (IGF)/relaxin superfamily and, more precisely, to thesubclass of relaxin peptides (Figure 1).

II. S earch for reproductive hormones inechinoderms

In a collaborative effort with Prof. Yoshikuni and AssociateProf. Kurita’s Laboratory at Kyushu Univ., we are searching for reproductive hormones in echinoderms, including
starfishes, brittle stars, sea urchins, sea cucumbers, andcrinoids. The collaborating parties have been able to purify
physiological materials which induce egg maturation fromnerve extracts and analyze them with a protein sequencer anda tandem mass spectrometer in the analytical center of ourinstitute. One of them, named cubifrin, which is an NGIWYamide peptide, is in the sea cucumber Aposticopus japonicus.
We have identified many neuropeptides from our EST
analysis of nerve tissues and many from RNA-seq and WGS
data of the NCBI database. In particular, relaxin like peptide
precursor genes and insulin/IGF like peptide precursor genes
were identified from many species. We are producing theseneuropeptides in a chemical synthetic manner to provide tocollaborating researchers for biological assays.

III. S earch for the lost mutants of female
hormones E2 in Oryzias latipes

Sequence analysis by capillary sequencer was carried out
from the tilling library of O. latipes in order to analyze thefunction of fish female reproductive hormone E2 as a part ofour collaborative research. Upon the discovery of aromatasemutants, a detailed analysis was carried out using these strains.

Publication List:〔Original papers〕

Y., Miyazoe, D., Togawa, R., Yokoyama Keiko and Nakasone,• Nishiike,K., Miyata, M., Kikuchi, Y., Kamei, Y., Todo, T., Ishikawa-Fujiwara, T.,

Figure 1. Amino acid sequence of starfish GSS. (A) Comparison of theheterodimeric structure of starfish GSS with those of various representative members of the insulin superfamily. The cysteine bridges are shownin red. (B) Coding DNA sequence and predicted amino acid sequencesof GSS. Sequences of the A and B chains are shown in the green andblue boxes, respectively. Characters shown in red boldface indicate basic
dipeptides that are the sites of proteolytic cleavage. The inverted triangle
shows the deduced cleavage site of the signal peptide.

72•

Ohno, K., Usami, T., Nagahama, Y., and Okubo, K. (2021). Estrogen
receptor 2b is the major determinant of sex-typical mating behavior
and sexual preference in medaka. Curr. Biol. 31, 1699-1710.e6. DOI:
10.1016/j.cub.2021.01.089
Yuhi, T., Nishimiya, O., Ohno, K., Takita, A., Inoguchi, T., Ura, K., and Takagi,
Y. (2022). Cloning of cDNA encoding a newly recognized apolipoproteinlike protein and its expression in the northern sea urchin Mesocentrotus
nudus. Fish. Sci. 88, 259–273. DOI: 10.1007/s12562-022-01584-3

LABORATORY OF BIOLOGICAL DIVERSITYSAKUTA Group
Assistant Professor:
Technical Assistant :SAKUTA, Hiraki
KODAMA, Akiko

The homeostatic osmoregulation of body fluids (such asplasma and cerebrospinal fluid (CSF)) is vital to life. This isbecause substantial changes in cell volumes due to hypertonicity or hypotonicity cause irreversible damage to organs andlead to lethal neurological trauma. Water deprivation (loss ofwater from the body) elevates the concentration of Na+ ([Na+])and osmolality in body fluids. Animals exhibit prominent andeffective responses to water deprivation, including behavioral
responses, such as inducing water intake and avoiding sodium(Na), along with vasopressin-induced reductions in urine
volumes. The aim of our research group is to reveal the brain
systems for body-fluid homeostasis.

body fluids are presented in Figure 1. When [Na+] in plasma
and CSF increases, Nax channels in glial cells in the OVLT
are activated, leading to the synthesis of epoxyeicosatrienoic
acids (EETs) in Nax-positive glial cells. EETs released from
Nax-positive glial cells function as gliotransmitters to activate
neurons bearing TRPV4 channels in the OVLT, which are
involved in the stimulation of water-intake behavior.

Figure 1. Schematic drawings of sensing mechanisms of body-fluid conditions in the OVLT responsible for the induction of water intake. AA,arachidonic acid.

II. Identification of novel sensors involved inwater intake control

Visual overview: Two postulated systems for sensing body-fluid conditions

I. Thirst control by Nax and TRPV4[Na+] is the main factor influencing osmolality in vivo,
and is continuously monitored in the brain to be maintained
within a physiological range. We have shown that Nax,
which structurally resembles voltage-gated sodium channels(Nav1.1–1.9), is the brain [Na+] sensor to detect increases in [Na+] in body fluids. Nax is preferentially expressedin specific glial cells of sensory circumventricular organs(sCVOs) including the subfornical organ (SFO) and organum
vasculosum lamina terminalis (OVLT). We have found that
Nax signals in these brain regions deficient in a blood-brain
barrier are involved in the control of salt intake.
We demonstrated that Nax signals are also involved in thecontrol of water intake behavior. The signaling mechanisms in
the OVLT for water-intake induction by increases in [Na+] in

Water intake by Nax-KO mice after an ICV injection of
hypertonic NaCl solution was small, but still approximately
half that of WT mice. It was noteworthy that this was significantly higher than that of Nax-KO and WT mice after an ICV
injection of an equimolar hypertonic sorbitol solution. Thesefindings suggest the existence of another unknown [Na +]sensor and osmosensor. In order to identify the novel sensors
involved in water intake control, we performed RNA-seq
analysis of OVLT and identified many candidates for said
sensors. As a result, we revealed that SLC9A4 in the OVLT
functions as a [Na+] sensor for the control of water intake
behavior among these candidates, and that the signaling
pathway originating from this sensor is independent of the
Nax/TRPV4 pathway, another [Na+]-sensing pathway for thecontrol of water intake (Figure 1). Our experimental results
suggested that SLC9A4-positive neurons are activated via
ASIC1a in a H+-dependent manner. Our experimental results
also revealed that water intake induced by the increase in[Na+] in CSF was completely lost in slc9a4-knockdown
Nax-KO mice. Thus, water intake induced by [Na+] increases
in body fluids may be explained by the Nax/TRPV4 and
SLC9A4/ASIC1a pathways. In addition to these two [Na+]dependent pathways that induce water intake, another signaling pathway originating from the osmosensor may be independently involved in the induction of water intake. We arenow examining the functional roles of remaining candidates
in water intake to clarify the osmosensing system.

73

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYWUDARSKI GroupSpecially Appointed Assistant Professor:
WUDARSKI, Jakub

Understanding the mechanisms of regeneration
using transgenic flatworms and IR-LEGO

Regeneration is the process of restoring lost or damaged
tissues and organs. Flatworms have long been considered
as model organisms for studying regeneration; some speciesof planarian flatworms can even restore all their body parts
from small pieces. In my research, I am using the new
powerful flatworm model organism, Macrostomum lignano,
to study how stem cells differentiate into various cell types
during regeneration and how body patterning is established.
The main advantage of M. lignano is the availability oftransgenesis methods which I have developed during my
PhD. It enables tracking specific cells and their progenitors
during development and regeneration.

Figure 1. ERK-KTR biosensor in Macrostomum lignano red arrow points
at the cytoplasm and yellow at the nucleus.

Positional control of regeneration in flatworms

Flatworms have remarkable regeneration capabilities. They
are able to regrow their whole body after amputation, including their reproductive organs. They can do this thanks to a
population of adult stem cells, collectively called neoblasts.
One of the fascinating aspects of flatworm regeneration is the
positional control of the process along the anterior-posterior
axis (head-tail). How cells know where specific body parts
need to be reconstructed is a question that still lacks a fullanswer. Our current state of knowledge is that Wnt pathway
and the mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK) signaling play major rolein this process. However, most of the research done on flatworms is based on information inferred from experiments
on gene knock-down via RNA interference (RNAi). Gene
activation and overexpression studies are absent in planarians, the more common flatworm model organisms, becauseof the lack of transgenic methods available for these animals.
I am trying to use the ERK-KTR biosensor in Macrostomum
lignano (Fig. 1), to track ERK signaling and test the functionof genes shown to be involved in positional control during
growth and regeneration. I am also adapting the infraredlaser evoked gene operator (IR-LEGO) technology to use
with the previously established HSP20 promoter (Fig. 2).
This will enable me to track the cell fate in vivo and overexpress selected genes even on a single cell level.
The research is financed by the Mistubishi Foundation
grant.

74

Figure 2. Expression of mScarlet under the HSP20 promoter 24 hours after
induction using IR-LEGO. The lightning bolts point at the targeted sites.

Publication List:〔Original paper〕

K., Wudarski, J., Sukhikh, I., Reinoite, F., Mouton, S.,• Ustyantsev,
and Berezikov, E. (2021). Proof of principle for piggyBac-mediatedtransgenesis in the flatworm Macrostomum lignano. Genetics 218,
iyab076. DOI: 10.1093/genetics/iyab076

NIBB-COS IINTERNATIONAL COLLABORATION RESEARCH PROJECTGUSE Group
Guest Professor:
Postdoctoral Fellow:

GUSE, Annika
KISHIMOTO, Mariko

diaphana) as a model for corals, we are conducting research
to reveal the molecular mechanisms of light sensing focusing
on the evolutionary conserved photoreceptor “opsin”.
Additionally, we are aiming to establish a method of genefunction analysis using genome editing technology or genesilencing techniques in said sea anemone which will inturn elucidate the mechanisms of light response by opsin.
Understanding how symbiotic cnidarians perceive light tosynchronize sexual reproduction and behavior will provide
us with key insights into its evolution and ecology; a prerequisite to combat the decline of corals through climate change
which threatens reef ecosystems worldwide.

I. Light sensing changes by symbiotic status

The gene expression of Aiptasia opsins changes dependingon their symbiotic status. This suggests that symbiotic andnon-symbiotic Aiptasia have different sensitivities to light.
This could be due to the different opsin types and the light
responses in algae.
In this study, we found that symbiotic and non-symbiotic Aiptasia have different phototaxis. Irradiation withhigh levels of light caused negative-phototaxis in symbiotic Aiptasia while non-symbiotic Aiptasia did not react. The
symbiosis-specific negative phototaxis was dependent on the
wavelength specificity, and peaked in the blue region. Thus,
symbiosis with algae changes Aiptasia’s light sensitivity.

Figure 1. Wavelength specificity of negative phototaxis in symbiotic Aiptasia.

Light dominates life for most organisms. On one hand,
sunlight acts as a key regulator for various functions including photosynthesis and circadian clock control. Similarly,
moonlight is important for synchronous gamete release inmany marine animals, such as cnidarians. Cnidarians, which
include corals and anemones, are basal, aquatic animals
with immense ecological importance. Notably, coral reefs
are the most biodiverse marine ecosystems. Their productivity depends on a functional symbiosis between reefbuilding corals and photosynthetic dinoflagellates of theSymbiodiniaceae family, which transfer nutrients to theircoral host to provide a source of fixed carbon in oligotrophic
environments.
In this COS-NIBB joint project, we aim to dissect key
molecular mechanisms underlying the sensing of light insymbiotic cnidarians and how this is used in environmental
adaptation. Using the sea anemone, Aiptasia sp. (Exaiptasia

II. Functional analysis of Aiptasia opsins

Aiptasia has 18 types of opsins, and 3 of them have higher
gene expression in their symbiotic state. In this study, we areaiming to analyze the light sensitivity of the distinct Aiptasia
opsins using a heterologous cell culture-based assay. To date,
we have successfully cloned 12/18 opsins including threesymbiosis-specific ones. Using cell culture-based assays
monitoring cAMP and Calcium signaling, we were able
to detect a light-dependent response of these three opsins.
We have determined the absorbance wavelength specificityof the three symbiosis-specific opsins. Moreover, we havefound that Aiptasia anemones exhibit negative phototactic
behavior in a symbiosis-dependent manner. Together, thesefinding provide the foundation to relating distinct opsins withlight-dependent behavior including phototaxis and spawningin Aiptasia.

75

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

Figure 2. Functional analysis of Aiptasia opsins cloned in this study.

Publication List:〔Original paper〕

N., Nakamura, S., Kishimoto, M., Moriyama, Y., Kawano,• Urakawa,S., Higashiyama, T., and Sasaki, N. (2022). Semi-in vitro detection of

Mg2+-dependent DNase that specifically digest mitochondrial nucleoidsin the zygote of Physarum polycephalum. Sci. Rep. 12, 2995. DOI:
10.1038/s41598-022-06920-2

76

LABORATORY OF BIOLOGICAL DIVERSITYYAMASHITA AND OTSUBO GroupSpecially Appointed Associate Professor

YAMASHITA, Akira

Specially Appointed Assistant Professor:
OTSUBO, Yoko
Technical Assistant:
NAKADE, Atsuko
Admin Support Staff:
SAKAGAMI, Mari
Cells sense the environment around them (e.g. the amountof nutrients and hormones present, as well as the temperatureand pressure), and decide what kind of activities to undertake
based on this information. In response to ambient conditions, germ cells producing sperm and eggs begin halving
their number of chromosomes during a special kind of cell
division called meiosis; a process for bringing forth genetically diverse progeny. In our group, we use the fission yeast
Schizosaccharomyces pombe, the simplest organism that
performs meiosis (Figure 1), to research the mechanisms bywhich cells switch from mitosis (a kind of cell division that
divides cells equally to create two identical cells) to meiosis.
In addition, we have started to study cellular responses to anovel stress: atmospheric pressure plasma.

tor2 mutants do. We designated these mutants as hmt, which
stands for hypermating and temperature-sensitive growth. We
cloned the responsible genes and found that several responsible genes encoded tRNA-related factors. We also found thatthe expression of tRNA precursors decreases upon nitrogenstarvation occurring. Furthermore, overexpression of tRNAprecursors prevents TORC1 downregulation in response tonitrogen starvation and represses the initiation of sexual differentiation. Based on these observations, we have proposedthat tRNA precursors act as key signaling molecules in the
TORC1 pathway in response to nitrogen availability (Figure2). We are also studying how S. pombe cells respond to othertypes of starvation other than that of nitrogen.

Figure 2. TORC1 regulation by tRNA precursors. TOR complex 1(TORC1) promotes vegetative growth and suppresses sexual differentiation under nutrient-rich conditions. tRNA precursors (pre-tRNAs)
positively regulate the TORC1 activity.

II. The molecular mechanisms that establish
the meiosis-specific gene expression profile

Figure 1. The life cycle of the fission yeast S. pombe. S. pombe cells
mitotically proliferate under nutrient-rich conditions. When starved ofnutrients, especially nitrogen, S. pombe cells arrest the mitotic cell cycle
and haploid cells conjugate with cells of the opposite mating type. The
resulting diploid zygotes undergo meiosis and produce spores.

I. Signaling pathways that regulate the onset of
sexual differentiation

We have been trying to elucidate how S. pombe cells switch
their cell cycle mode from mitotic to meiotic. To achievethis, we have focused on a highly conserved kinase, namely
Target of Rapamycin (TOR) kinase, which plays a key rolein the recognition of nutrition and the onset of sexual differentiation in S. pombe. TOR kinase forms two types ofcomplexes, TORC1 and TORC2. TORC1 contains Tor2 as
its catalytic subunit, and is essential in suppressing sexual
differentiation in the presence of nitrogen. TORC2 contains
Tor1 and, in contrast to TORC1, is required for the onset of
sexual differentiation under nitrogen starved conditions.
Temperature-sensitive tor2 mutants initiate sexual differentiation even on rich mediums under restrictive temperature
conditions. To gain an insight into the TORC1 signaling
pathway, we have isolated mutants that initiate sexual differentiation ectopically under nutrient-rich conditions, as

The expression of hundreds of genes is upregulated duringmeiosis. Expression of meiotic genes is strictly regulated,since untimely expression of gametogenic genes, including
meiotic genes, has a deleterious effect on somatic cells. We
have shown that specific control of the stability of meiotic
transcripts, which is orchestrated by the interplay between
RNA-binding proteins and a long non-coding RNA, contributes to the meiosis-specific gene expression in the fission
yeast S. pombe. Understanding precise mechanisms of thiscontrol will shed light on the regulation of timely gene
expression during meiosis.
A YTH-family RNA-binding protein Mmi1 plays a crucialrole in the selective elimination system of meiosis-specifictranscripts during the mitotic cell cycle. Mmi1 recognizesa region termed DSR (Determinant of Selective Removal)
in meiotic transcripts, which is enriched by repeated hexanucleotide motifs. Meiotic transcripts bound to Mmi1 aredegraded by the RNA-degradation nuclear exosome machinery. Mmi1 also induces the formation of facultative heterochromatin at a subset of its target genes. Furthermore, Mmi1
regulates the termination of transcription of its target genes.
Mmi1-mediated termination of an upstream non-coding RNA
ensures the expression of downstream genes, one of which
encodes a mitogen-activated protein kinase kinase kinase(MAPKKK) essential for the initiation of sexual differentiation.

77

National Institute for Basic Biology

Evolutionary Biology and Biodiversity

When S. pombe cells undergo meiosis, a meiosis-specificnuclear body, called Mei2 dot, blocks the Mmi1-mediatedelimination system. The Mei2 dot is composed of theRNA-binding protein Mei2 and a long non-coding RNA
species referred to as meiRNA. Mei2 physically interacts
with meiRNA and forms the dot structure at the chromosomal sme2 locus, which encodes meiRNA. The Mei2 dotlures Mmi1 through numerous copies of the DSR motif on
meiRNA and inhibits its function, so that meiotic transcripts
harboring DSR are stably expressed.
We have shown that Mmi1 prevents untimely expression of
meiotic proteins by tethering their mRNAs to nuclear foci.
Mmi1 interacts with itself with the assistance of Erh1. Mmi1
self-interaction is crucial for nuclear foci formation, target
transcripts elimination, their nuclear retention, and protein
expression inhibition. As such, multi-layered suppression of
meiotic genes by Mmi1 is vital for mitotic growth (Figure 3).
We have also demonstrated that a conserved complex called
MTREC (Mtl1-Red1 core) or NURS (nuclear RNA silencing) that consists of a zinc-finger protein, Red1, and an RNA
helicase, Mtl1, is required for the recruitment of the nuclear
exosome to Mmi1 foci.

< NINS CNIS Department of Plasma Sciences >gas temperature in collaboration with the National Institutefor Fusion Science (Figure 4).

Figure 4. Direct plasma irradiation to the fission yeast S. pombe on anagar medium.

Publication List:〔Original paper〕

H., Hatta, Y., Hayashi, K., Shimasaki, T., Otsubo, Y.,• Ohtsuka,
Ito, Y., Tsutsui, Y., Hattori, N., Yamashita, A., Murakami, H., andAiba, H. (2022). Cdc13 (cyclin B) is degraded by autophagy undersulfur depletion in fission yeast. Autophagy Reports 1, 51–64. DOI:
10.1080/27694127.2022.2047442

Figure 3. Mmi1-mediated multi-layered regulation to prevent mistimed
expression of meiotic genes. In mitotically growing cells, meiotic transcripts carrying DSR are recognized by Mmi1, and are degraded by
the RNA exosome. Mmi1 also induces heterochromatin formation.
Furthermore, Mmi1 prevents nuclear export and ectopic protein expression of its targets.

III. Cellular responses to atmospheric pressureplasmaPlasma, which is the fourth state of matter after solid,
liquid and gas states, is a form of ionized or electricallycharged gas. It has been known that plasma irradiationinduces a wide variety of effects on living organisms.
Recently, atmospheric pressure plasma has been used invarious fields including medicine and agriculture. However,detailed mechanisms underlying responses to plasma irradiation remains unknown. Accordingly, we are addressing
molecular mechanisms of cellular responses to plasma
by using the fission yeast S. pombe. To this end, we havedeveloped plasma irradiation devices that enable control of78

< NINS ASTROBIOLOGY CENTER >NINS ASTROBIOLOGY CENTER
The Astrobiology Center (ABC) in NISS was established in 2015 to promote interdisciplinary studies that
include astronomy, earth science, and biology by organizational adaptation between National Astronomical
Observatory of Japan (NAOJ) and National Institutefor Basic Biology (NIBB). In the 2021 fiscal year, ABC
stayed active to encourage interdisciplinary studies
and grow the research community as the hub institute of astrobiology despite social constraints under
the COVID-19 pandemic. ABC symposium was held
online on Jan 28th, 2022, inviting speakers from diverse
research fields including an observatory mission by
James Webb Space Telescope. Life in the Universe
Workshop was also held online on Feb 17th-18th,
2022 to present the latest results from ABC-subsidized
research.

LABORATORY OF BIOLOGICAL DIVERSITYTAKIZAWA GroupSpecially Appointed Associate Professor

TAKIZAWA, Kenji

The Takizawa group operating out of ABC is currently studying
the environmental responses of photosynthesis in order to predict
photosynthetic apparatus of so-called ‘Alien’ plants under
extreme conditions on the aforementioned ‘Another Earth’.

Light adaptation mechanisms of photosynthesis

We investigated the flexible molecular mechanisms of lightharvesting and following photosynthetic reactions in cooperation with the division of environmental photobiology. In
photosynthesis research, we are specialized in spectroscopic
analyses which can be applied to astrobiology studies.

Characterize hypothetical plants in exo-planets

Figure 1. ABC mascot characters: “Exoplanet Friends” (Credit:
Hayanon Science Manga Studio)

Three project offices in ABC are working together
to find a habitable exo-planet and a sign of life on
said planet. Exo-Planet Search Project Office andAstrobiology Instrument Project Office which arelocated in the Mitaka campus of NAOJ are preparing
direct-imaging surveys of “Another Earth” by next-generation, 30m-scale telescopes. To support future observation projects, three groups from NIBB in Okazaki, theTakizawa, Johzuka, and Fujita groups, are participatingin the Exo-Life Search Project Office to investigate
life on Earth utilizing three different approaches: 1)
assessing the biosignature of various photosyntheticorganisms to predict biosignatures of hypothetical lifeon exoplanets, 2) evaluating the effects of cosmic radiation on living cells, and 3) elucidating mathematicalprinciples in the formation of self-organizing structuresin organisms.

One of the most plausible biosignatures detected by the
exo-planet observations is a specific reflection pattern on
the land surface named ‘vegetation red edge’ or VRE. VRE
arises from strong absorption of red light by photosynthetic
pigments and high reflectance of near-infrared radiation(NIR) by the developed leaf tissue structure. The wavelength
position and amplitude of VRE could be different on anotherplanet. We studied the light reflection spectrum of various
plants and predicted that substantially large VRE could beobserved even on the ocean planets if floating plants cover
the water surface (Figure 2). We also predicted the possibilityof red-sift in VRE on the planets exposed to NIR rather thanvisible light. We have revealed that several metal-containing
pigments can be functional in reaction centers under NIR
radiation conditions via quantum chemical calculations.

Figure 2. Remote sensing of light reflectance of floating leaf of waterlily.
Light reflectance in visible light (RGB) and NIR were obtained by thedrone-based multiband sensor.

Publication List:〔Original papers〕

Y., and Takizawa, K. (2021). A quantum chemical study on the• Komatsu,
effects of varying the central metal in extended photosynthetic pigments.

•

Phys. Chem. Chem. Phys. 23, 14404–14414. DOI: 10.1039/d1cp00760b
Pan, X., Tokutsu, R., Li, A., Takizawa, K., Song, C., Murata, K.,
Yamasaki, T., Liu, Z., Minagawa, J., and Li, M. (2021). Structural basisof LhcbM5-mediated state transitions in green algae. Nat. Plants 7,1119+. DOI: 10.1038/s41477-021-00960-8

79National Institute for Basic Biology

Evolutionary Biology and Biodiversity

LABORATORY OF BIOLOGICAL DIVERSITYFUJITA Group
Assistant Professor:
Technical Assistant:

FUJITA, Hironori
EGAWA, Akane
TAKEKAWA, EikoIn nature, a variety of self-organized patterns, such as thegalaxy and the snowflake, are found on a wide range ofspatiotemporal scales. Particularly in living organisms, suchself-organization of spatiotemporal patterns is both remarkable and essential. Therefore, we aim to elucidate the mechanism of generation and control of self-organized patterns inliving systems with a particular focus on plants using boththeoretical and computational approaches (Figure 1).

formed under the condition of “spot inoculation”, in whichE. coli cells are inoculated in the center of soft-agar plates(Figure 2B). In order to further develop the research, wehave newly developed an assay system “uniform inoculation”, which causes a more dynamic self-organization inwhich colony spots initially emerge from the entire surfaceof agar medium, and they move and fuse with each otherrepeatedly (Figure 2C). Whereas experimental conditions
in biological experiments are usually greatly restricted, thesynthetic approach is a powerful research method that cancontrol and modify the experimental conditions in a more
flexible manner compared to the method used in standard
experiments. Therefore, we try to apply the synthetic
approach to E. coli to understand the principle of spatiotemporal self-organization of cell populations.

A

Desktop darkroom

Side angle LED
ring lights

DigitalcameraE. coliSoft-agar medium

B Spot inoculation






 


0 hr

24 hr

48 hr

72 hr

96 hr

E. coli (Soft-agar medium)

C Uniform inoculation

Hard-agar medium

Figure 1. (Left) Phyllotaxis pattern is self-organized by the feedback
regulation between auxin concentration and PIN1 polarization. (Middle)
Leaf venation pattern is self-organized by the feedback regulation
between auxin flux and PIN1 polarization. (Right) Shoot apical meristem(SAM) is self-organized by the interaction between WUS and CLV3.

0 hr

I. S patiotemporal self-organization of cell
population

One well-known example of spatiotemporal self-organization in living systems is Dictyostelium discoideum, known
as cellular slime mold. D. discoideum usually exists as aunicellular organism, but when stimulated by starvation, cells
aggregate while forming a spiral pattern and develop a sluglike multicellular body. This remarkable self-organization is
induced by chemotaxis to the signal molecule cAMP.
On the other hand, it is reported that Escherichia coli,
which is a unicellular model organism, can self-organize
spotted colony patterns (Figure 2B). This pattern formation
is induced by the positive feedback between chemotaxis toaspartate (Asp) and Asp synthesis, and can be an excellent
experimental system in the study of self-organization ofcell populations. This stationary pattern of E. coli colony is

80

2 hr

3 hr

4 hr

5 hr

Figure 2. (A) A device system for photographs and time-lapse videos ofspatiotemporal patterns of cell populations. (B) Spot inoculation; E. coli
cells are inoculated in the center of soft-agar plates. The cell population
spreads outward while forming a stable spot pattern. (C) Uniform inoculation; E. coli cells are evenly inoculated in soft-agar medium. The cell population shows a dynamic spatiotemporal self-organization in which colony
spots initially appear from the entire surface of soft-agar medium (2 hr),and they move to fuse with each other repeatedly (2–5 hr).

Publication List:〔Original paper〕

K., Fujita, H., Isa, M., Gotoh, S., Arasaki, A., Ishida, H.,• Kataoka,
and Kimura, R. (2021). The human EDAR 370V/A polymorphism

affects tooth root morphology potentially through the modification ofa reaction–diffusion system. Sci. Rep. 11, 5143. DOI: 10.1038/s41598021-84653-4

< NINS ASTROBIOLOGY CENTER >LABORATORY OF BIOLOGICAL DIVERSITYJOHZUKA Group
Assistant Professor:
Technical Staff:changes from a simple loop into a complicated twisted shapeas the cell cycle progresses from metaphase to anaphase.

JOHZUKA, Katsuki
ISHINE Naomi

I. Condensin-dependent chromatin folding

Chromosome condensation is a basic cellular process that
ensures the faithful segregation of chromosomes in bothmitosis and meiosis. Any abnormality in this process leads tosegregation errors or aneuploidy, which results in cell death.
Chromosome condensation is mainly achieved by condensin, a hetero-pentameric protein complex, widely conserved
across a variety of organisms ranging from yeast to humans.
Despite its conservation and importance in chromosome
dynamics, it is not fully understood how condensin works.
Our aim is to understand the mechanism and regulation of chromosome condensation. To this end, we havebeen studying the role of condensin in the budding yeast
Saccharomyces cerevisiae. We have found that condensin
specifically binds to the RFB site located within the rDNA
repeat. To date, the best characterized condensin binding
region is the rDNA repeat on the right arm of chromosome
XII in budding yeast. We further discovered the multiple
protein network required to recruit condensin to the RFB
site.
The RFB site, which consists of a ~150bp DNA sequence,
functions as a cis-element for the recruitment of condensin
to chromatin in the yeast genome. If the RFB site is inserted
into an ectopic chromosomal locus, condensin can associatewith the ectopic RFB site. To explore the role of condensin
in chromosome organization, we have constructed a strain
in which two RFB sites are inserted into an ectopic chromosome arm with an interval of 15kb distance in the cell with acomplete deletion of chromosomal rDNA repeat. Using thisstrain, condensin-dependent chromatin interaction between
two RFBs was examined by chromosome conformation
capture (3C) assay. We discovered the condensin-dependent
chromatin interaction between the two RFB sites on the chromosome arm. This result indicates that condensin plays a rolein chromatin interaction between condensin binding sites,
and this interaction leads to the creation of a chromatin loop
between those sites (Figure 1). It is thought that condensindependent chromatin folding is one of the basic molecular
processes of chromosome condensation. During the cell
cycle stages, the RFB - RFB interaction signal increases in
metaphase and reaches its maximum level in anaphase. In
addition to the RFB - RFB interaction, the chromatin interactions between the internal regions of the two RFBs increases
in anaphase. Thus, the configuration of chromatin fiber

Figure 1. A Schematic model of chromosome condensation. Condensin
makes chromatin interactions between adjacent binding sites (RFB, forexample). This leads to a folding of chromatin fibers between the sites, asa basic process of chromosome condensation.

II. Plasma-induced cellular responses

Plasma is an ionized gas, which consist of ions and electrons and is extremely reactive. Our question is to understandthe effect of the plasma on living organisms.
We have developed a new plasma irradiation device that cancontrol the temperature to the optimal growth temperatureof cells, and have developed a system to study the effects ofdirect plasma exposure to cells without heat stress (Figure2). Using yeast, S.cerevisiae, it was found that direct plasma
exposure treatment increases lethality as the processing timeincreases (Figure 3). Furthermore, a plasma-resistant mutant
was isolated and the causative gene responsible for plasmaresistance was identified. We are studying functional analysisof the gene to understand the molecular mechanisms of thecellular responses to plasma.

Figure 2. Schematic diagram of atomosperic pressure plasma irradiation
device (left). Direct plasma irradiation experiment (right).

Figure 3. wild-type (upper panels) and plasma-resistant mutant (lower panels)

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