Department Members and their Research Activities

Over 25 years of research, we have discovered five novel groups of secreted peptide hormones involved in plant growth and environmental responses, including RGF, which is involved in root development, CEP, which communicates root nitrogen deficiency to the aboveground, and PSY, which regulates growth and stress response switching at the cellular level (Figure 1). We have also discovered the existence of long-distance-mobile non-secreted peptides such as CEPDL2, which communicates leaf nitrogen deficiency to the roots via phloem (Fig. 2, left). Although only a few plant hormones are listed in high school textbooks, the number of peptide hormones found in the past 25 years exceeds 20. By examining each peptide hormone in detail, we are amazed at how sophisticatedly plants engage in hormone- and receptor-mediated intercellular signaling during growth and environmental adaptation.

Fig. 1. Expression pattern of peptide hormone RGF required for root development (left). Systemic nitrogen demand signaling mediated by the peptide hormone CEP (right). Nitrogen deprivation of the right root causes a complementary upregulation of nitrate transporter expression in the left root. The peptide hormone CEP, which communicates nitrogen deficiency from roots to leaves, is involved in this process.

Various post-translational modifications are observed in extracellular secreted peptides and proteins, including peptide hormones, such as sulfation of tyrosine and hydroxylation of proline, as well as arabinosylation and galactosylation of hydroxyproline. All of these modifications are necessary to change the structure of peptides and proteins to achieve specific functions. We have succeeded in identifying the enzymes involved in these modifications and are investigating the physiological significance of post-translational modifications by observing the phenotypes of mutant plants deficient in these enzymes in detail (Fig. 2, right). In addition, protein phosphatases often play important roles downstream of peptide hormone receptors. Based on the identification of the substrates of protein phosphatases, we aim to discover new signaling pathways mediated by protein dephosphorylation.

Fig. 2. Peptide CEPDL2 communicates aboveground nitrogen deficiency to the roots via phloem (left). The green GFP-CEPDL2 signal is seen in the phloem in a transverse section of a root. Physiological function of galactosylation of hydroxyproline (right). In a mutant plant deficient in galactosyltransferase, the permeability of the plasmodesmata is increased and molecular movement between cells is facilitated.

Differentiating ylem vessel elements deposit thick, hydrophobic secondary cell walls and undergo programmed cell death, forming a hollow tubular structure. Prior to the secondary cell wall deposition, cortical cytoskeletons are rearranged into distinct patterns to direct the deposition of the cell walls in the annular, spiral, reticulate, and pitted patterns. We study the molecular signaling that governs the dynamic behavior of cytoskeletons in differentiating xylem vessel elements. (Fig. 1).

Fig. 1. Microtubules (left) and cell walls (right, magenta) in differentiating xylem vessel elements.

Plant cells undergo cytokinesis by forming a cell plate. The position and direction of the cell plate formation determine the shape and subsequent cell fate of the daughter cells. The plant-specific cytoskeletal structures, such as prophase microtubule bundles (PPBs) and phragmoplasts, direct cell plate formation (Figure 2). We study the mechanisms underlying the assembly and action of the PPB and phragmoplasts and their evolutionary aspects.

Fig. 2. Phragmoplast microtubules (magenta) and microtubule-associated proteins (green).

We have been revealing that sex is determined in the balancing of feminization and masculinization. Once the sex looks determined, the body still retains the potential to develop into female or male and the tug of war between the two sexes continue. This principle leads to sex changes (reversal). We have found that germ cells are critical for feminization and in the absence of the germ cells the somatic cells are predisposed to male development (PNAS 2007, 2020).

Figure1 Left: Sex reversed female medaka from male with an excess amount of germ cells. Right: male wild type medaka

triggers modules in germline stem cells that promote eggs or sperm development.
Eggs and sperm are continuously produced from sexually indifferent germline stem cells in ovary and testis (Science 2010). Then how do germline stem cells determine the sexual fate of descendant germ cells? We have identified the sexual switch gene in germ cells, foxl2l (foxl3) (Science 2015). We have also revealed that foxl2l initiates several modules that correspond to female-specific characters. During the analyses, we have found the evidence suggesting evolutional driving force may be different between eggs and sperm.

Figure 2 medaka chromosomes during meiosis

Several modules functioning during oogenesis and spermatogenesis are genetically independent, which allows combination of different modules to function in germ cells. This raises hypothesis that combination leads to different types of reproduction systems. Our lab is addressing a critical event that creates parthenogenetic species by investigating the change of modules using medaka and Poecillia. We are investigating a variety of reproduction in terms of change of modules (Front Cell Dev Biol 2022).

We have pioneered the discovery that the plant-specific blue light receptor, phototropin, serves as a light receptor for stomatal opening. The blue light signal received by phototropin induces the activation of the plasma membrane H⁺-ATPase, leading to the formation of the driving force for stomatal opening. (Figure 1). Currently, we are actively conducting physiological, biochemical, and genetic analyses, as well as synthetic biology research using guard cells around stomata, aiming for a comprehensive understanding of the signaling mechanism controlling stomatal aperture. As member of the Institute of Transformative Bio-Molecules (ITbM), we also explore research from a chemical biology perspective. Moreover, we are advancing research on stress-resistant plants and increased crop yield through the manipulation of stomatal aperture based on the results from basic research.

Figure 1. Localization of plasma membrane H⁺-ATPase, a key enzyme for stomatal opening, in guard cells (Left). Green fluorescence represents GFP-H⁺-ATPase and red fluorescence represents chloroplasts. GUS-staining of stomatal guard cells using guard cell-specific promoter (Right).

Land plants prevent excessive water loss through transpiration by closing stomata. Stomatal closure in response to an increase in CO₂ concentration is crucial for improving plant water use efficiency. The CO₂ sensor complex within stomatal guard cells detects this increase (Figure 2) and then initiates intracellular signal transduction, leading to stomatal closure. Our goal is to understand the function and molecular mechanisms of plant CO₂ sensing and signal transduction. We’re employing biochemical, genetic, and cell biological approaches, collaborating with experts in structural biology, chemical biology, mass spectrometry, and genome evolution to explore the principles of cellular function.

Figure 2. Structure model of Arabidopsis CO₂ sensor complex.

As part of courtship behavior, many animals engage in communication using sound. In this context, the receiver animal evaluates species-specific acoustic signals, often termed ‘courtship songs’, to determine whether to accept the sender as a potential mate. It remains unclear how the brain recognizes and processes these distinct courtship song signals emitted by conspecifics. We aim to address this topic by elucidating the properties of neurons responsible for processing auditory information and unraveling the associated neural circuitry (Figure 1). Our research, employing molecular genetics in studies using Drosophila, aims to comprehend the neural mechanisms that evaluate sound. Additionally, by applying these findings to mosquitoes, we aim to control populations of various disease-transmitting mosquitoes, contributing to disease prevention.

Figure 1 Auditory sensory neurons in the fly ear (Left) and neurons responsible for processing auditory information in the fly brain (Right)

Animal behaviors, and the signals used in communication, have diversified across species. How has the brain of animals evolved to efficiently perceive and extract signals from conspecifics? We focus on the diversification of signals such as sound and pheromones used in Drosophila courtship behavior across different species. By comparing the neurons and neural circuits of species utilizing different signals, we aim to unravel this mystery. Furthermore, our recent investigations involve studying how insect brains, particularly those of Drosophila breeding within flowers, have evolved to perceive signals from flowers (Figure 2).

Figure 2 Courtship behaviors of Drosophila elegans that breed within flowers

Despite having simple nervous systems, insects thrive and reproduce in diverse environments across the globe. To understand how insects have diversified their instinctive behaviors to ensure successful reproduction and survival, we study three important behaviors within the Drosophilidae family: courtship, mating, and social aggregation. We seek to uncover the neural mechanisms behind these behaviors, exploring how they have evolved and diversified, with diversification of instinctive behaviors evident even among Drosophila species. By comparing genes and neural circuits between the model species Drosophila melanogaster and other Drosophila family members, we aim to shed light on the mechanisms driving the evolution of instinctive behaviors.

The structure of the cerebellum and cerebellar neural circuits in vertebrates is relatively conserved from fish to mammals. During development, the major neurons of the cerebellum, granule cells and Purkinje cells (Figure 1 left panel), are generated from neural precursor cells located in the dorsal and ventral parts of the cerebellar primordium, respectively. Granule cells and Purkinje cells receive two different types of input fibers from outside the cerebellum (mossy fibers and climbing fibers). These two types of information are integrated in the Purkinje cells and eventually output to the outside of the cerebellum. We believe that this simple scheme is a good model for understanding the mechanisms of neural circuit formation in the brain. We are using transgenic zebrafish to visualize cerebellar neural circuits (Figure 1 right panel) and creating mutants using genome editing techniques to elucidate the molecular mechanisms of differentiation of cerebellar neurons and neurons that input to the cerebellum, as well as the formation of cerebellar neural circuits.

Figure 1. Major neurons in the zebrafish cerebellum (left). Green represents the axons of granule cells and magenta represents Purkinje cells. Visualization of cerebellar neural circuits (right). Green represents climbing fibers, and magenta represents Purkinje cells.

The cerebellum has been thought to be involved in coordinated motor control and motor learning. Recent studies have revealed that the cerebellum is also involved in higher brain functions such as emotions like anxiety and fear, as well as cognition. Furthermore, abnormalities in cerebellar neural circuits are thought to be related to human mental disorders such as autism spectrum disorder (ASD). We are using zebrafish to analyze the role of cerebellar neural circuits in motor learning, fear response learning, and social behavior. We aim to clarify the higher functions of cerebellar neural circuits by performing functional imaging of these circuits and manipulating their activity by expressing neurotoxins or optogenetic tools in cerebellar neural circuits.

Pigment cells are specialized cells in vertebrates that create body color and patterns. In mammals and birds, pigment cells consist of only one type, melanocytes, which produce melanin. However, other vertebrates have melanophores (pigment cells that exhibit black color) homologous to melanocytes, as well as iridophores and xanthophores. Additionally, some teleost fish have a variety of pigment cells, including leucophores and cyanophores. These pigment cells, like melanocytes, differentiate from a common pigment stem cell derived from neural crest cells. Therefore, teleost fish have more types of pigment cells differentiating from pigment stem cells compared to mammals and birds. We are using fish, particularly medaka, as a model for pigment cells, and using mutants (Figure 2) to elucidate the mechanism by which a variety of cell types are produced from stem cells.

Figure 2. Medaka with wild-type body color (left) and mutant medaka that do not develop melanophores, xanthophores, or leucophores (right).

Large number of animals have evolved sexual weapons and ornaments via sexual selection. Beetle weapons (e.g. horns and mandibles) are one of the best examples of sexually selected exaggerated traits. These weapons are effective in male-male combat but costly in normal life. Therefore, large weapons only develop in well-nourished large males, and small males that have grown up in poor conditions develop tiny, rudimental weapons. This striking plasticity is a common characteristic of sexual weapons. By using broad-horned flour beetle (Gnatocerus cornutus), we clarified that histone deacetylases (HDACs) regulate the mandible size plasticity. As an internal physiological signal of nutritional state, a growth factor called insulin-like peptide 2 (ILP2) is shown to control the nutrition-dependent weapon growth in broad-horned flour beetle.

Fig. 1 Broad-horned flour beetle (Gnatocerus cornutus). RNAi-mediated gene knockdown (KD) of insulin-like peptide 2 (ILP2) generates rudimentary mandible in males. Left: control (wild type), Right: ILP2-KD phenotype.

he colony of social insects such as ants consists of various castes. Queen is a specialized egg-layer, whereas intranidal workers perform nursing and extranidal workers are engaged in foraging and defense. These behavioral and physiological differences of castes are basically driven by developmental plasticity. We aim to clarify how caste differentiation occurs in terms of social interaction, gene expression and physiological state. In case of monomorphic ponerine ant Diacamma cf. indicum, insulin-signaling is up-regulated in queen to rapidly produce eggs. In contrast, intranidal workers has lowered metabolic state and they store energy inside their body (e.g. storage protein hexamerin). By focusing on social interaction, gene expression and physiological heterogeneity, we are trying to elucidate the evolutionary mechanism of social behavior.

Fig. 2 Ovary of Diacamma ant. Socially dominant individuals develop ovarioles and become functional queen (left). In contrast, ovarian development is arrested in subordinate individuals (right).

Humans cycle through Rapid Eye Movement (REM) sleep and non-REM (NREM) sleep approximately five times in 90-minute intervals during a night. Recently, it has been discovered that Australian dragons Pogona vitticeps also experience REM and NREM sleep. Remarkably, the cycle of REM/NREM sleep in dragons occurs roughly every 90 seconds, repeating over 400 times a night. While the cycle lengths vary greatly among animal species, the underlying “pattern generator” between REM and NREM sleep may be conserved across species. Revealing this entity is one of our major goals. We utilize various methods such as cutting-edge electrophysiological techniques, pharmacological methods, molecular biology techniques, and behavioral observations to advance our research.

Fig.1 Left, a lizard’s hippocampus exhibits a layered structure of neurons closely resembling that of the mammalian hippocampus. Right, neurons in the claustrum of a lizard. The claustrum is a mysterious brain region thought to be involved in consciousness. Recently, we have revealed that the claustrum is involved in generating neuronal activity during NREM sleep.

Our daily experiences are consolidated into memories in the brain during sleep. In recent years, it has been found that a brain oscillation called sharp wave ripple (SWR), generated in the hippocampus during sleep, “replay” neural activity that occurred during wakefulness, thereby consolidating memories. Furthermore, we have recently discovered that SWRs during sleep not only replay important memory information but also erase unnecessary information. This mechanism is believed to efficiently consolidate memories into the brain. Currently, we are diligently researching to further elucidate the function and dynamics of SWRs.

Fig.2 Hippocampal sharp wave ripples (SWRs). The high frequency component called “ripple (140-250 Hz)” play a role in memory consolidation.

A human genomic locus associated with our intelligence (IQ) contains SEPT3, a septin gene whose physiological function is unknown. Our systematic behavioral screening pinpointed premature memory decay in mice that lack Sept3. We discovered a unique abnormality in the hippocampal dentate gyrus (DG) synapses, which was recapitulated in cultured DG neurons. These mice and cultured neurons can be regarded as in vivo and in vitro models for mild cognitive impairment (MCI, or “forgetfulness,” the earliest symptom of dementia) in humans. Interestingly, the memory decay and morphological abnormalities of DG synapses in Sept3 knockout mice were ameliorated by rearing them in an enriched environment that promotes physical and social activities. Currently, we are searching for the metabolic and endocrine changes induced by environmental factors (exercise, dietary restriction) that are known to improve cognitive function including memory, and the mechanism by which their memory decay/synaptic abnormalities are improved.

Figure1.
 Glial processes (red: CDC42EP4, a major binding partner of septin) enwrap synapses of Purkinje cell dendrites (green: calbindin) in the cerebellum. In mice that lack CDC42EP4, which serves as a scaffold for glutamate transporters, reuptake of glutamate is delayed and glutamate remains in the perisynaptic spaces, resulting in motor learning defects.

Low molecular weight G proteins such as Rac/Rho/Cdc42 and their effectors regulate diverse cellular signaling pathways and cytoskeletal dynamics. The regulations are crucial for the brain development; ensuring progenitor cell division and migration, neurite protrusion and projection, and synaptogenesis. Countless genetic variations are found in pediatric patients with structural brain abnormalities, intellectual disabilities, and epilepsy, but only a few of them have been proved to be responsible for the pathology. Thus, we aim to screen pathogenic variants and elucidate the underlying molecular mechanisms by analyzing the brain structure and function in mosaic transgenic mouse fetuses/newborns.

Figure 2.
 A glial cell process (blue) enwrapping a synapse between a granule cell axon (the parallel fiber) terminal (uncolored) and a dendritic spines of Purkinje cell (light brown) in the cerebellar cortex. The black dots are SEPT5, which accumulates beneath the plasma membrane.

We have discovered that active proteolysis by the ubiquitin system plays an important role in many aspects of life phenomena. For example, we found that when the quantity and quality of nutrient sources available to cells change, proteins, which are switches for substance metabolism, are actively degraded to maintain the nutritional balance in cells. We have clarified that protein degradation by the ubiquitin system is important in order to respond flexibly to various environmental stresses that attack cells. In addition, when cells proliferate, organelles such as mitochondria are distributed to new cells (Fig. 1 left), and we found that the ubiquitin system plays an important role in this process as well. In this way, we are discovering new life phenomena controlled by the ubiquitin system and elucidating their mechanisms.

Fig1 Mitochondria passed to new cells (green)(left). Yeast cell membrane (green), vacuolar membrane (red), and mitochondrial DNA (blue)(right)

The cell, which is the basic unit of life, is a closed space surrounded by a cell membrane (Fig.1 right). By closing the space of the cell membrane, the substances necessary for vital activity are concentrated without diffusion. It is no exaggeration to say that it is this work that brought about the birth of life. However, biological membranes not only act as “partitions” but also have many functions, such as the exchange of substances and information inside and outside the cell, the scaffold for chemical reactions that are indispensable for sustaining life, and the interaction with other cells. In many situations, the ubiquitin system is involved as an important mechanism. We are studying the origins and various functions of biological membranes such as cell membranes.

The ubiquitin system is responsible for the degradation of various proteins as a mechanism for protein degradation in cells. Using this system, we have developed an auxin-inducible degron (AID) method that breaks down proteins that we want to destroy using a substance called auxin, which is a plant hormone (Fig. 2). We are improving this proteolytic system so that it can be used more easily, and we are analyzing the function of target proteins using this proteolytic system.

Figure 2. Degradation of centromere protein (red) in the nucleus of human cells by auxin: before auxin treatment (left) and after auxin treatment (right)

We discovered that Gm9999, a noncoding RNA expressed in sperm, encodes two small proteins, which we named Kastor and Polluks derived from the names of stars in the constellation Gemini (Figure 1 left).
The male mice lacking Kastor and Polluks were infertile. Kastor and Polluks were localized in the mitochondria of sperm, and electron microscopy revealed that sperm from mice lacking Kastor and Polluks had an abnormal mitochondrial morphology (Fig. 1, right). The small twin genes are essential for normal sperm development and may be one of the causes of male infertility in Human.

Figure 1. Expression of Polluks proteins in mouse testis (left). Nuclei of all cells (blue), sperm heads (green), and Polluks proteins localized in sperm mitochondria (red). Scanning electron microscope images of sperm (right). Mitochondria of normal sperm (upper right) and those of sperm lacking the twin gene (lower right).

The ribosome, which is responsible for protein translation, is composed of about 80 protein subunits. We discovered that the heart has a specialized ribosome in which RPL3, one of the ribosome subunits, is replaced by RPL3L. Echocardiography of mice lacking the Myo-ribosome revealed reduced contractility of the heart (Figure 2). Given that genetic mutations of RPL3L have been reported in patients with dilated cardiomyopathy, research on the Myo-ribosome may lead to the development of a new therapeutic approach for these patients.

Figure 2. Echocardiography of mice. Normal heart (upper) and heart deficient in Myo-ribosome (lower).

 High-resolution structural analysis is vital for understanding the assembly and disassembly of actin filaments at a structural level. In cells, older actin filaments are preferentially disassembled. Using cryo-electron microscopy and X-ray crystallography, we have clarified how actin filaments become aged and more prone to disassembly. Furthermore, the lifespan of the actin filaments is extended when they are pulled or twisted, and the mechanism behind this has been understood through mutant experiments and analysis of filament fluctuations using electron microscopy on actin filaments that are actually pulled or twisted.

Figure 1: Actin-cofilin complex structure (left) and ParM filament structure from Clostridium botulinum (right).

Actin filaments and microtubules, the main components of the cytoskeleton, are unique to eukaryotes, yet similar families exist in bacteria and archaea. For many years, we have studied ParM, a bacterial actin family protein, in collaboration with the Robert Robinson group. Although ParM subunits are structurally similar, they assemble into structurally diverse filaments, serving as a model for studying protein filament dynamics. In 2019, we elucidated that the ParM of Clostridium botulinum forms a complex structure consisting of 15 strands (Figure 1, right). It is surprising that such a structure can be formed by the assembly of just one protein. Additionally, recent collaborative work with the Robinson group has enabled us to successfully analyze the structure of archaeal microtubules. While individual subunits of archaeal microtubules closely resemble those of eukaryotes, their filament structure significantly differs. Our goal is to elucidate the evolutionary transformation of these archaeal microtubules into eukaryotic microtubules through the course of evolution from archaea to eukaryotes.

Figure 2: Cryo-electron micrograph of archaeal microtubules (left) and its three-dimensional structure (right).

In our laboratory, we conduct groundbreaking life science research, combining cutting-edge model-driven approaches with emerging data-driven approaches to bridge diverse research fields. The core of our research methodology is creating a comprehensive approach that maximizes the strengths of each method and effectively compensates for their respective weaknesses. For example, by combining different types of data, we can carry out research that can quantitatively and accurately predict disease progression or treatment outcomes at an individual level. Moreover, our investigations extend to the application of insights derived from the digital realm to real-world scenarios. This is accomplished by generating extensive virtual data that replicates the characteristics of real-world data, allowing for the evaluation of diverse scenarios. Our research delves into these themes by seamlessly transitioning between the physical and digital realms (see Figure 1).

Figure 1. Research that integrates various fields

In recent years, various infectious diseases such as COVID-19 and Mpox have spread globally and been successively introduced into Japan. The risk of importing these emerging and re-emerging infectious diseases is increasing due to global outbreaks of viral hemorrhagic fevers and zoonotic diseases like Ebola, Lassa fever, and Crimean-Congo fever. The COVID-19 pandemic has left a significant impact on the world, yet it has also generated an unprecedented volume of high-quality data, unparalleled in human history. To prepare for a potential future pandemic, it is crucial to analyze these datasets. Our group is committed to establishing an interdisciplinary platform using the latest data science technology to robustly support drug and vaccine development, as well as the formulation of appropriate countermeasures against infectious diseases and treatment strategies for severe cases.

Figure 2. Infectious disease study using mathematical models, computer simulations, and AI technologies.

The phenomenon of “Cell competition”, where cells compete for survival, is increasingly recognized as playing a significant role in tissue growth, homeostasis, and cancer development. Our goal is to elucidate the mechanism by which cells recognize each other using chemical and mechanical signals, thereby determining ‘losers’ and ‘winners’. Furthermore, we are exploring the mechanism by which ‘winners’ expel ‘losers’ from the tissue. Simultaneously, we are aiming to reveal new physiological functions associated with this process (Fig.1).

The diverse external morphologies of insects such as wings, legs, and exoskeletal horns are shaped through the three-dimensional deformation of the adult primordium, which is a ‘folded epithelial sheet’ structure. By integrating experimental approaches with mathematical and physical modeling, we aim to elucidate the fundamental principles governing the deformation of these folded epithelia (Fig.2).

Fig.1 Perturbation of tissue stress caused the cell compaction, resulting in mechanical cell competition (Red: cell area large, Blue: cell area small). In mechanical cell competition, “mechanical compaction” changes “cell shape”, leading to the determination between cell’s life and death.

Fig.2 “Folded” wing imaginal disc (left) and the adult wing (right)

It has become apparent that the interaction between oncogenic cells and the surrounding cells plays a crucial role in the development and progression of cancer. We utilize a Drosophila tumor malignancy model to analyze the mechanisms behind tumor development and progression, focusing on the interactions between oncogenic cells and neighboring normal cells, such as epithelial cells and immune cells, as well as adjacent normal tissues (Fig. 3).

Fig.3 The eye-antennal imaginal disc bearing oncogenic mutant cells (green). Immune cells (magenta) exhibit a substantial accumulation on the oncogenic mutant cells.

The nematode C. elegans is a 1-2 mm long worm that feeds on soil bacteria. It is an excellent model animal that has been in the spotlight in recent years because it has many genes homologous to those of mammals, including humans, and is easy to study genetically and molecularly. In our lab, we mainly study the signaling mechanisms that control axon regeneration in C. elegans. In particular, we aim to understand the role of signaling mechanisms conserved from worms to humans in regulating the regeneration of severed nerves in order to provide a new basis for the study of therapies for nerve injury (Fig. 1).

Figure 1   Regenerating nerve axon. The severed nerve is visualized with red fluorescent protein and the collagen receptor DDR-2 protein is visualized with green fluorescent protein.

Parkinson’s disease is a neurodegenerative disorder characterized by the accumulation and propagation of a-Synuclein in the brain. This process begins as early as 10 to 20 years before symptoms appear (prodromal stage), and leads to progressive neural synapse dysfunction. Research is currently investigating the mechanisms of a-Synuclein aggregation and propagation during the prodromal phase of the diseases, as well as the synaptic degeneration caused by these processes at the molecular and cellular levels. The study is utilizing primary cultured hippocampal neurons and glial cells (Fig. 2).

Figure 2   Neuron-astrocyte co-culture (left) and a-Synuclein aggregates (right)
Lewy neurite-like aggregates (rod-like structures) and Lewy body-like aggregates (spherical structures) of a-Synuclein (green) are formed in primary cultured neurons derived from mouse hippocampus (right).

Chloroplasts not only conduct photosynthesis but also synthesize essential substances for plant survival, such as plant hormones, amino acids, and lipids. Additionally, they are involved in the regulation of nuclear genome gene expression, plant morphogenesis, and cell proliferation. We have discovered chloroplast-localized proteins that, when their function is impaired, lead to the emergence of cells without chloroplasts and abnormal leaf shapes or plant growth (see Figure 1). By conducting functional analyses of these proteins, we aim to elucidate chloroplast functions involved in plant morphogenesis and cell proliferation. Furthermore, we are working on the development of genome editing techniques for the orchid species and are also engaged in breeding new varieties of P. aphrodite.

Figure 1. Nucleus, chloroplasts (green), mitochondria (red), and DNA (blue) in leaf mesophyll cells of wild-type Arabidopsis thaliana (left) and crl mutant (right).

Another characteristic of Drosophila sperm is the enlargement of mitochondria. Shortly after meiosis, the mitochondria, which were dispersed throughout the cell until recently, aggregate and become spherical (see Figure 2). These mitochondria have a diameter of approximately 6 micrometers, larger than those found in typical cells, and can be described as “giant” mitochondria. Over time, the shape of these mitochondria changes to elongated forms, pushing and stretching the cell from the inside, reaching lengths of up to 1.8 mm in D. melanogaster. Interestingly, a strong correlation has been found between the volume of giant mitochondria and the extreme length of the sperm. This suggests that the size of giant mitochondria may determine sperm length. We are aiming to prove this causal relationship and elucidate the evolutionary background of this intriguing phenomenon.

Figure 2. Giant mitochondria (indicated by arrowheads) greatly enlarged and developed, identifiable even in photographs of the entire testis. The green structures are bundles of elongated sperm.

One of our main interests lies in how plant cells divide, a process called cytokinesis. During this process, plants partition a mother cell by forming the cell plate between two daughter nuclei. This unique cytokinetic system is widely conserved among land plants. However, because of its uniqueness, there are still many mysteries about plant cell division, such as how the cell plate is positioned and how it is formed. We aim to uncover the molecular mechanisms underlying plant cell division by focusing on proteins involved in cell plate formation.

Figure 1. Gametophores in P. patens and the early gametophore visualized by microtubules (green) and DNA (magenta).

Plant cell division, while sharing the main stages of eukaryotic mitosis, is distinguished from animal mitosis by lack of centrosomes, preprophase band, cytokinesis via phragmoplast. We work to discover unique plant-specific molecules and pathways involved into cell division and potentially harness those towards practical applications. There are several ongoing projects, including CRISPR/Cas9 genetic screening to discover novel cell division genes, investigating relationship between plant kinetochore and polyploidy and mechanisms of spindle positioning in plants (Fig. 1 left).

Fig. 1. Colony of moss Physcomitrium patens (left). Dividing moss cell expressing microtubule (magenta) and kinetochore (green) markers (right).

Microfluidic chip or device is a set of micro-channels molded into a polymer material (PDMS) in which plant cells can be grown without any adverse effects. We can precisely modulate the channel environment by controlling liquid flow in the channels, introducing or wash-out chemical compounds, and observing the cellular response. Furthermore, microdevice offers excellent properties for high-resolution imaging and can be used to visualize protein dynamics at the molecular level. We use microdevices for high-resolution cytoskeleton imaging in the cells of moss P. patens and testing the effect of various chemical compounds on cytoskeleton dynamics (Fig. 1 right).

Epithelial tissue is a sheet-like structure that covers the body surface and lumen of multicellular organisms. Epithelial tissue protects the body of multicellular organisms from various external stresses, while at the same time mediating or restricting the exchange of substances between the inside and outside of the body. It corresponds to the cell membrane of single-celled organisms. In order for epithelial tissue to remain attached to the body, it must be firmly attached to an extracellular matrix structure called the basement membrane, which lies directly beneath the epithelial sheet. Hemidesmosomes are adhesion protein complexes that bind epithelial tissue to this basement membrane (Figure 1 left). It is known that in patients with a congenital inability to form hemidesmosomes or an acquired disease that prevents the proper functioning of hemidesmosomes, the epidermis of the skin becomes detached from the basement membrane. We are investigating the mechanisms of hemidesmosome formation and disassembly, including their relationship to disease, mainly using cultured cells derived from epidermis (Fig. 1, right).

Figure 1. Electron micrograph of esophageal epithelial basal cells (left). Electron-dense hemidesmosomal structures are localized at the basal side of the epithelial cells, facing the basement membrane. Visualisation of hemidesmosomes in cultured cells derived from rat epidermis (right). Hemidesmosomes (green) and keratin filaments (red) are shown.

Amphipathic phospholipids spontaneously assemble into bilayer membranes in aqueous solution and necessarily form liposomes, which are closed-membrane vesicles. Liposomes have been used as the most simplified models to study biological membranes.
Real-time observation of liposomes using optical microscopes demonstrated that lipid membrane itself has the ability to cause large deformations that even involve topological changes such as fusion, fission, opening pore, or inside-out inversion (Figure 1). In addition, it has been revealed that such dynamic behaviors of lipid membrane occur under the collaboration with various biological factors. Typical examples are macromolecules including proteins and nucleic acids, and cytoskeletons such as actin and/or molecular motors.
Our group observes the phenomena that are induced when liposomes are exposed to such various factors that are candidates to be involved in the morphogenesis and movement of cells, and analyzes the dynamic mechanisms.
In addition to the study for membrane dynamics, we have also progressed research focusing on the features of biological factors as natural soft matter. For examples, nematic liquid crystal formation of actin filaments and the dynamics of cytoskeletons, nucleic acids, or membranes when the liquid-liquid phase separation takes place (Figure 2).

図1．リポソームのトポロジー変化も伴う劇的な変形例．上から順に，融合，分裂，穿孔，表裏反転．全て暗視野顕微鏡像．

図2．液液相分離を起こしているポリエチレングリコールとデキストランの二成分水溶液にアクチン線維(赤)とDNA(緑)を同時に加えた場合に観察される微小液滴内分布．

Fungi, especially several model species like S. cerevisiae (budding yeast), S. pombe (fission yeast), and A. nidulans (filamentous fungus), have made significant contributions to the fields of biology and medicine, playing a crucial role in elucidating fundamental cellular mechanisms. However, recent research has unveiled a diverse range of fungal species inhabiting marine environments, many of which exhibit distinct cell growth and division patterns compared to model fungi. For example, several black yeast species collected at Sugashima MBL have shown the ability to adapt their growth and division strategies in response to environmental cues (a phenomenon known as phenotypic plasticity). We aim to conduct cutting-edge cell biology research on fungi originating from marine sources.

Fig. 1. Black yeast collected at Sugashima MBL (colonies and cells).

Sugashima is renowned for its diverse array of seaweeds, including nori (a seaweed commonly used in Japanese cuisine). Our research delves into the cellular intricacies of these seaweeds. Many macroalgal species exhibit cellular characteristics that markedly differ from those of terrestrial organisms. For example, while cell division is a fundamental process for all living organisms, seaweeds showcase unconventional patterns even in this activity, challenging conventional understanding. When seeking to understand these phenomena, it is not possible to directly apply insights gleaned from research on model land plants. Presently, our focus is centred on the study of the green alga Bryopsis. This macroalga, which grows to ~10 cm, resembling bird feathers, is remarkably a single-celled organism containing multiple nuclei (called coenocyte). We are intrigued by how it forms intricate shapes without undergoing cell division.

Fig. 2. Green feather alga Bryopsis sp. collected at Sugashima MBL (right; magnified image).

The systematics of marine invertebrates is crucial due to their significant role in ecosystems, yet the field remains in a state of early development, with many species yet to be described. Our efforts span from the Arctic to the Antarctic, from intertidal zones to the hadal depths, utilizing research vessels and diving expeditions to collect and classify organisms from every corner of the globe. Despite the heavy reliance on fieldwork, substantial deskwork is also integral to our research process. When a collected specimen does not match any previously described species, it is classified as a new species. The thrill of discovering unknown creatures based on historical records is akin to the excitement felt by Indiana Jones, making this field especially appealing to those who cherish fieldwork.

Exploring the morphological and ecological evolution of “strange” creatures raises questions like why the males of certain anglerfish are smaller, or why clownfish cohabitate with sea anemones. By elucidating their evolutionary history, we delve into the origins of such unusual traits. While our primary focus is on symbiotic ecologies and morphological evolution, our interests extend to any “strange” creatures, including the advanced evolution of eyes in annelids.

図1．「変な」生き物達の紹介．高性能な眼を獲得したウロコムシ(左)．八放サンゴに共生するシリス(右)．

The mechanisms behind the creation and maintenance of biodiversity are exemplified by coral reefs, which support a vast array of life, contributing to their biodiversity. Organisms that play a pivotal role in shaping these ecosystems are known as ecosystem engineers. Recent diving expeditions around Sugashima have revealed the role of polychaetes as ecosystem engineers. Our ongoing research aims to understand the role of these “polychaete forests” within marine ecosystems, including the variety of organisms that utilize them.

図2．樹上の巣で構成されるゴカイの森．深場の岩礁帯において藻場に代わって多くの生き物を育んでいる．

Bioactive natural products have been used as pharmaceuticals and research reagents. Particularly, diverse bioactive substances are found in sessile marine organisms. Recent studies suggested that such compounds are involved in their defense and communication. We are working on discoveries of new MNPs, starting from collecting marine organisms in the field (Figure 1) and elucidating the structures using various chemical analysis instruments (Figure 2).

Figure 1. Collecting marine organisms.

Who biosynthesizes MNPs? In the environment, marine organisms and microorganisms live together in communities, where symbiotic microbes play crucial roles in MNPs production. Understanding the biosynthetic processes may provide a means of supplying therapeutically important small molecules, leading to discovery of unique enzymes that perform new chemical reactions, and thus yielding improved natural products through engineered biosynthesis. Our goal is to identify biosynthetic genes and understand the mechanisms of biosynthetic enzymes, using genetic analysis and biochemical experiments.

Figure 2. Collected samples (left) and an experiment scene (right).

Plants possess innate immunity as a defense system. Upon perception of microbe-associated molecular patterns (MAMPs), such as microbial cell walls and flagellar components as well as toxic effectors secreted by pathogens, through specific receptors, diverse signal transduction pathways are activated to stimulate the immune system. Induction of local innate immunity, plants release long-distance signals from infected leaves, leading to the activation of the immune system even in non-infected leaves. This plant-specific immunity is known as systemic acquired resistance (SAR). In SAR, salicylic acid (SA) accumulates in non-infected leaves and induces SA-responsive genes. We aim to elucidate the signaling pathway of SAR, a key immune system in plants.

Figure 1. Mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes on leaf surfaces. Ca2+ imaging using 35Spro:GCaMP3 (Col-0). The leaf surface of a 4-week-old plant was gently brushed. Scale bar, 0.1 mm.

The successful activation of SAR depends not only on the plant’s immune capacity but also on diverse environmental factors such as temperature, light, water, and herbivores. Analyses of crosstalk systems between signaling pathways activated by environmental factors and immunity are challenging, and most of the network remains unclear. We developed a cell-free protein synthesis system that comprehensively synthesizes candidate regulators of each signaling pathway, and investigate their regulatory mechanisms in vitro and their roles in vivo. Currently, we are studying the crosstalk between 1) jasmonic acid signaling involved in resistance against herbivores and necrotrophs, 2) light signaling, and 3) abscisic acid signaling involved in drought stress responses.

It is estimated that there are several hundred types of receptors in each plant that recognize microorganisms, but it is uncertain whether they identify all potential pathogens in nature. Recently, we discovered that in plants, trichomes, which are hair-like structures on leaf surfaces, function as sensors for mechanical stimuli and temporarily activate the immune system by detecting stimuli such as rain (Figure 1). Since many pathogens spread and infect through raindrops or wind, plants seem to perceive these mechanical stimuli as warning signals, preparing for potential infections. Currently, we are studying the downstream signaling mechanisms involved in the perception and signal transduction of mechanical stimuli (Figure 2).

Figure 2. Mechanosensory trichomes activate an immune response. Mechanosensory trichome cells initiate intercellular calcium waves in response to mechanostimulation. [Ca2+]cyt derepresses CAMTA and activates the phosphorylation of MPK3 and MPK6, thereby inducing WRKY-dependent transcription.

The advent of next-generation sequencing has made genome analysis relatively easy, and forward genetics, which works by creating mutants and searching for their causative genes, can be performed very efficiently. To effectively advance forward genetics, we have devised an efficient nucleic acid purification system using 96-well deep wells for concurrent culture and 96-well PCR plates and magnetic beads to efficiently advance mass genome analysis. In addition, we have established a data analysis workflow to process data from 300 to 1000 microbial genomic DNAs in a single analysis.

Extremely halophilic archaea (hereafter referred to as halobacteria) are a type of archaea that grow in a medium containing nearly saturated salt (25%) (Figure 1, left). Most halophilic bacteria have a protein similar to rhodopsin, which is used in human vision. Some species of bacteria have only a chloride ion pump, called halorhodopsin (hR), and prefer to grow in alkaline environments with a pH of 9~11. hR is purple in the presence of chloride ions and blue in the absence of ions, and X-ray crystallography has revealed its conformational changes (Fig. 1, right). To perform this structural analysis, we used a mutant strain expressing a large amount of hR, and genomic analysis revealed a mutation (D324N) in the protein NP0654A. We expect that the mutation in NP0654A probably caused a significant increase in hR gene expression. Why not use the mutants to conduct a treasure hunt for genes that microorganisms possess?

Figure 1. Dark-field micrograph of an extremely halophilic archaeon (left) and the three-dimensional structure of halorhodopsin (right).

Plants comprise a variety of cell types, spanning from small to large. These cells are meticulously arranged in aesthetically appealing patterns, like geometric expressions (Figure 1, left). Such exquisitely ordered cell patterns can be abundantly observed throughout the plant body. We are captivated by the mechanisms responsible for generating these orderly and beautiful patterns within the plant body. Our focus lies in understanding the role of molecules, specifically signaling molecules, involved in transmitting information between cells. We emphasize the significant role played by signaling molecules that originate exclusively in specific cells within the plant body (Figure 1, right).

Figure 1. (Left) Cross-sectional photograph of an Arabidopsis thaliana stem. Cell walls are stained. (Right) Photograph at the root tip. The nuclei of cells producing the signal molecules we focus on show yellow fluorescence protein. Cell walls are stained with blue fluorescence.

Within the intercellular communication in the plant body, there exists information that can alter the fate of the entire body. For instance, a failure to transmit even a single piece of information can lead to rapid senescence and deterioration of the entire body (Figure 2). We are interested in unraveling the mechanisms underpinning the well-regulated and organized order as an individual organism.

Figure 2. A normal Arabidopsis thaliana (left) and an instance where the individual undergoes rapid senescence (right).

Unlike normal plants, seeds of Striga stay dormant and never germinate unless host plants growing around (Fig. 1). When host plants grow nearby, the seeds sense host-exuded strigolactones and initiate germination. We developed a fluorogenic probe called yoshimulactone green which reacts with strigolactone receptors and emits fluorescence. The probe enabled us to identify strigolactone receptors and visualized wave-like propagation of strigolactone perception dynamics within the seeds (Fig. 2). We are studying how this pattern is created and how this dynamics is linked to germination.

Fig. 1. Striga is a parasitic plant of maize with pink flowers.

By identifying of strigolactone receptors along with the development of convenient tool to monitor their functions, it became possible to rationally develop synthetic agonists of strigolactone receptors. We succeeded in developing Sphinolactone-7, a hybrid molecule of synthetic molecule and natural strigolactones, which is extremely potent germination stimulant for Striga. This molecule led Striga seeds to germinate in the absence of host plants and let them die. We are currently testing the suicide germination method using sphinolactone-7 in Kenya.

Fig. 2. Strigolactone recognition patterns occurring in waves in striga seeds (from left to right: before treatment, after 4 hours, after 11 hours, and after 27 hours).

To elucidate the molecular mechanisms of the circadian clock, we have been applying chemical biology approaches, which use chemical compounds to study the mechanisms of biological processes. Among tens of thousands of compounds with diverse structures, we identified several compounds that alter the circadian rhythm of human cells. From functional analysis of these new compounds, we discovered the first compounds that selectively act on each of the clock proteins CRY1 and CRY2, which constitute the circadian clock (Fig. 1). Furthermore, by analyzing the crystal structures of these compounds in complex with CRY proteins, we revealed the unique mechanism underlying the isoform selectivity. We are using our original compounds to uncover the mysteries of the circadian clock.

Figure 1. Compounds selectively target each of the clock proteins CRY1 and CRY2.

In modern society, disturbances of the circadian clock and resulting various diseases are becoming increasingly serious problems. Compounds that regulate the circadian clock are expected to provide the starting point for drugs to treat these diseases. We have found that compounds targeting CRY inhibit hepatocyte glycogenesis, promote brown adipocyte differentiation, and inhibit glioblastoma stem cell proliferation. Furthermore, we found compounds that specifically inhibit CK2, a kinase associated with the circadian clock, and inhibit the proliferation of acute myeloid leukemia cells (Fig. 2). By analyzing the effects of these compounds on animal models of metabolic syndrome and cancer, we are also conducting basic research for the treatment of diseases.

Figure 2. Compound specifically inhibits clock-related kinase CK2.

Understanding cellular functions in plant developmental processes and responses to the environment requires real-time quantification of intracellular biochemical and biophysical processes with high-resolution spatial information. Our research group systemically engineers biosensors utilizing fluorescent proteins to facilitate the quantitative analysis of biomolecules such as sugars and plant hormones. We are also working with a group of Chemistry at ITbM to develop imaging tools using fluorescent small molecules (Figure 1). Moreover, we are also developing tools employing a synthetic approach to regulate plant cellular processes precisely, thereby unraveling the intricacies of metabolic signaling networks.

Figure 1. Arabidopsis root with fluorescence dye（Cyan：Microtubule）and Fluorescent proteins（Blue：Nuclei, Pink：Cell Membrane）

Transporters and enzymes are known to recognize diverse chemical structures, generally without absolute specificity. For example, transporters, such as the SWEET sugar transporter and the NRT/PTR family of transporters known as nitrate ion and peptide transporters, have been reported to possess phytohormone transport functions. Through collaboration with physics groups at ITbM, our research group aims to elucidate the molecular mechanisms of substrate selectivity in membrane transporters and investigate the physiological relevance of each transport substrate. Simultaneously, collaboration with the ITbM chemical library center and the Molecular structure center facilitates the identification of transporter inhibitors and the development of their applicability in agricultural research.

Plants alter their shapes as part of their responses to developmental processes and environmental cues. The pattern of cortical microtubules, located just beneath the plasma membrane, is crucial in cellular morphogenesis (Figure 2). These cortical microtubules dynamically respond to diverse stimuli, such as gravity, mechanical stress, light, and phytohormones. Our research group aims to elucidate the spatiotemporal regulatory mechanisms governing the microtubule organization that underlie plant morphologies by employing chemical genetics, biochemical approaches, and live cell imaging.

Figure 2. Cortical microtubules in Arabidopsis leaf epidermal cells

We study the response of plant cells when encountering obstacles. In nature, plant cells may encounter spaces smaller than their own size. For instance, pollens adhering to the stigma extends pollen tubes through intricate and narrow spaces within the female tissues in order to produce seeds for the next generation. In the soil, root hairs extend through the narrow gaps between soil particles to absorb water and nutrients. In both cases, the pathways for pollen tubes or toot hairs are not pre-existing. The question is how these cells behave when encountering spaces narrower than their own size. We have discovered that pollen tubes and root hairs have the ability to deform themselves and pass through gaps as small as 1 micrometer using microfabrication techniques (Fig. 1). Indeed, plant cells possess a turgor pressure comparable to that of a car tire, serving as the force for elongation. When pollen tubes and root hairs elongate within tissues or soil, it is believed that they constantly regulate the rigidity of the cell walls and turgor pressure effectively.　We are conducting research to discover and unravel the mechanisms behind the potential resilience and strength of plant cells.

Fig 1. Pollen tubes passing through narrow gaps within microdevices.

We are conducting research to elucidate the regulatory mechanisms controlling the number and distribution of organelles, especially in chloroplasts (Fig. 2). In cells constituting plant body, there are organelles responsible for cellular functions, and their number and distribution are properly regulated to be in the right place, at the right time, and in the right quantity. For instance, the luminous moss, which inhabits dark rock crevices, is known to arrange chloroplasts around the nucleus within specialized rounded form cells, also known as lens-shaped cells. However, the mechanism behind the formation of the lens-shaped cells and the positioning of chloroplasts remains unknown. We are focusing on the microtubule- and actin-associated proteins involved in the regulation of the number and distribution of organelles.

Fig. 2. Chloroplasts and microtubules in the moss Physcomitrium patens.

We are also conducting research to unravel the mystery behind the remarkable regeneration capacity in plants. Indeed, plants exhibit a high regeneration capacity, as evidenced by the sprouting of shoots from stumps and propagating by cuttings. These abilities of plants depend on high capacity of differentiated cells to transform into pluripotent stem cells (reprogramming ability). It is known that plant hormones such as auxin and cytokinin are involved in the reprogramming of plants, but the process is still largely unknown. We are employing single-cell analysis devices, AI robotics technology, plant hormone imaging techniques, and other molecular tools to investigate the mechanisms underlying the regeneration capacity of plants.

Plants have a remarkable ability to communicate with the signals from their surroundings, and effectively employ these signals to protect themselves and to control their own developmental strategies. Our study delves into the intricate processes through which plants detect environmental changes using a range of signaling pathways, leading to various physiological reactions. Through this exploration, we aim to reveal fresh perspectives on how plants ensure their survival, which are instrumental in increasing crop productivity and their resilience to changing environmental dynamics.

Fig.1 Leaf epidermal tissue (left) and root apical meristem (right) of Arabidopsis thaliana

Plant stem cells play a critical role in the growth and vitality of plants, especially in adapting to environmental changes. These cells exhibit an extraordinary capacity to regenerate new tissues. Precise regulation of stem cell activities is essential to maintain and optimize plant development. Our current research focuses on identifying key regulators crucial for initiating and maintaining plant stem cell activities. Furthermore, we aim to investigate the intricate interactions of these factors and how they are influenced by environmental stimuli. This study seeks to not only elucidate the fundamental mechanisms governing plant stem cell regulation but also offers potential for the creation of novel plant varieties with improved environmental resilience.

Fig.2 WOX5 expression in the quiescent centre (left) and false color representation of gene expression patterns related to root stem cell activity (right)

Cell motility is a fundamental activity of life, and the elucidation of the operating principles of the apparatus responsible for motility is a quite important question of life science. Bacteria, which consist of only one cell, use motility apparatus as a means of escaping from toxic substances and moving toward nutrient in order to survive in the environment. Many bacteria use the flagellum as motility apparatus, with spiral flagellar filaments rotated by a motor embedded in the cell surface layer at its base to generate the driving force for motility.

The flagellar motor (Figure 1) harnesses electrochemical gradient of ions across the cell membrane (ion driving force) to generate rotational force. The flagellar motor is ~ 50 nm size of supramolecular membrane complex. What makes unique about this motor is that it can convert, not ATP, but transmembrane ion motive force to rotational force. Such an ion-driven rotary motor does not exist in eukaryotic organisms. Furthermore, this motor is a high-performance molecular machine that can rotate as fast as 1000 revolutions per second in both directions, change direction instantly, and adjust its energy conversion capacity by sensing the load. Although the flagellar motor has attracted attention across the fields of science, engineering, and medicine, much of the energy conversion mechanism is still in a mystery. We are attempting to clarify the rotational mechanism by focusing on both the stator, which is the energy conversion unit, and the rotor ring, which is involved in controlling the direction of rotation, and elucidating the relationship between structure and function.

Figure 1: The cryo-electron tomographic image of the flagellar motor of marine Vibrio.

Our bodies are shaped by functional molecules which are under appropriate spatiotemporal control during the process of development. In addition, in order to maintain tissues and organs normally, those molecules must work in appropriate amounts at each site. If this mechanism fails, it may cause disease. Thus, placement of biomolecules in appropriate amounts at functional sites is a fundamental function of all living organisms. In bacteria, the number and position of flagella are strictly determined to function optimally, so that each cell can move appropriately in the environment in its habitat. Using marine Vibrio as a model organism, which form a single flagellum at the pole of the cell, we are trying to find out why “only one” flagellum can be formed at the special position called the “pole of the cell” (Fig. 2). Instead of using complex living organisms, we use simple and fast-growing bacteria to conduct research aimed at elucidating the mechanism of the proper arrangement of biological supramolecules.

Figure 2: The electron micrographic image of wild-type Vibrio cell with only one polar flagellum at cell pole (left) and a mutant with multi-polar flagella at a cell pole (right).

The major challenges to studying the aging of neuronal functions are the long aging time and the complexity of the nervous system. Therefore, we use the nematode C. elegans, which has a short lifespan and a simple nervous system, as a model. C. elegans becomes an adult within three days after fertilization, undergoes a reproductive period of a few days, and completes its ephemeral life span in about two weeks (Fig. 1). During this time, it exhibits various behaviors using only 302 neurons. Many of these behaviors diminish around the time they finish reproducing. The existence of a genetic program for aging is suspected to cause behavioral changes because age-dependent behavioral decline happens in only one week in C. elegans, whereas in humans, it spans several decades. We, therefore, conduct novel genetic screening for mutants that do not exhibit age-dependent behavioral decline and analyze these mutants. Since molecular mechanisms of critical biological processes are often shared between C. elegans and mammals, we hope our research will elucidate the fundamental principles of neuronal aging.

Figure 1. Aged C. elegans (10-day-old adult).

Not only genetic programs but also environmental factors such as diet affect the aging of neuronal function. We found that bacteria, the food of C. elegans (Figure 2, left), influence behavioral aging. To elucidate the neural basis of these diet- and aging-induced behavioral changes, we measure neuronal activity manifested as changes in intracellular calcium concentration. Because C. elegans is translucent (Figure 2, right), we can readily monitor the neuronal activity of a live animal using a protein whose fluorescence intensity reflects calcium concentration.

Figure 2. Bacteria in the C. elegans intestine (green, left) and neuronal nuclei in the C. elegans head (red, right).

The behavioral decline in C. elegans is mainly observed in behaviors that navigate animals toward bacterial food. Perhaps the alteration of neural function due to aging and the accompanying behavioral changes may be a mechanism by which aged individuals do not compete with younger individuals for food, maximizing the group’s benefits at the individual’s expense. C. elegans is as tiny as one mm long, making it a powerful tool for population analyses. In the future, we would like to explore the significance of aging in populations using mutants less prone to behavioral aging obtained through our genetic screening.

Triple negative breast cancer (TNBC) is a type of breast cancer that is resistant to common hormone therapy and molecular targeted drugs (Herceptin). TNBC accounts for 15 to 20% of breast cancers with poor prognosis. It has been reported that the Wnt signaling pathway is over-activated in TNBC, so that we have started to search for inhibitors of Dvl that may block Wnt signaling, using the molecular structure of Dvl as a clue. During this project, we succeeded in elucidating the structure of the new inhibitor named NPL-3009 bound to the Dvl-PDZ. This achievement is expected to contribute to the design and development of new anti-cancer drug molecules (Fig. 1).

Figure 1. two conformations in the crystal of the anticancer drug candidate, “NPL-3009” (left) and the complex structure with the Dvl1-PDZ domain (right)

The Nobel Prize in Physiology or Medicine 2023 was awarded to Drs. Catalin Carrico and Drew Weissman, for their “discovery of nucleic acid modifications that enabled the development of an effective mRNA vaccine against COVID-19”. Natural RNA and DNA molecules are known to be easily and rapidly degraded in vivo, making it difficult for them as pharmacologically active therapeutics. We have designed and synthesized many nucleosides with the designed sugar moieties. These modified nucleosides will be incorporated into oligonucleic acid molecules, and further analyzed their conformation when bound to the target mRNA.

Stomatin (Band 7.2 protein) is a membrane protein that is highly abundant in erythrocyte membranes. The SPFH domain is a domain of unknown function in the central part of the stomatin. We eventually discovered that the domain can form linear fibrils (Figure 2). It is possible that changes in physiological conditions cause stomatin to assemble and act as a cytoskeleton just beneath the erythrocyte membrane to reguralte membrane stiffness.

Figure 2: Electron microscopic image of fibers formed by the SPFH domain of the human erythrocyte protein Stomatin.

Gap junction channels are known to be direct communication channels between adjacent cells, but the structural basis for the gating mechanisms remains unclear. We have reported the cryo-EM structures of the Caenorhabditis elegans innexin-6 gap junction channels. By comparing the structures of the solubilized and lipid nanodisc-reconstituted states, we have proposed a gating model in which lipid diffusion occludes the channel passage (Fig. 1).

Figure 1 Cryo-EM image and 3D reconstruction of a gap junction channel

We are studying the mechanism of the transporters driven by ATP hydrolysis, including lipid flippases that transport lipids (phosphatidylserine) that act as “Eat me” signals, using biochemistry and structural analysis by cryo-EM (Fig. 2). We are also designing drugs based on the protein structure using artificial intelligence.

Figure 2 Gastric proton pump

G-protein coupled receptors are membrane proteins that capture extracellular ligands and activate G proteins at the cytoplasmic side. We have reported the cryo-EM structures of the MrgD-Gi complex, which are involved in itch and pain. We evaluated the stability of the modeled ligand, β-alanine, using molecular dynamics simulations and confirmed that the orientation was correct (Fig. 3).

Figure 3 MD simulation of GPCR and ligand distribution