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2021 -
Long-Term Fellowships - LTF

The role of metabolism in regulating pathological osteoclastogenesis in arthritis

HASEGAWA Tetsuo (JAPAN)

MRC Laboratory of Molecular Biology - University of Cambridge - Cambridge - UK

PEARCE Edward (Host supervisor)
CLATWORTHY Menna (Host supervisor)
Osteoclasts are macrophage lineage cells with unique bone-destroying capacity, playing key roles in steady-state bone remodelling inside the bone marrow (BM) and pathological joint destruction in patients with rheumatoid arthritis (RA), in which the hypertrophied synovium (called "pannus") invades the outer surface of the articular bone. While osteoclastogenesis is known to involve dynamic metabolic changes, little is known about the metabolic pathways involved in the pathological osteoclastogenesis in the inflammatory synovial tissue setting. I therefore aim to elucidate the extracellular energy microenvironment and metabolic regulation of pathological osteoclasts causing bone destruction in arthritis. I will perform imaging mass spectrometry of the knee joints of arthritic mice to elucidate the metabolic states of osteoclasts in the pannus microenvironment and compare it with physiological BM settings. I will also extract the pannus directly into the metabolite extraction buffer, and analyze the extract through mass spectrometry by comparing healthy and diseased joints. Based on these in situ information, I will elucidate how the energy microenvironment and metabolites enriched in the arthritic joints govern the function and metabolism of osteoclasts through analysing metabolic profiling assays, such as extracellular acidification rate, oxygen consumption rate, and carbon isotype (13C) tracing. Overall, these proposed studies will provide novel insights into the metabolic regulation of pathological osteoclastogenesis, potentially leading to the development of a new strategy for treating bone destruction in RA without interfering with physiological bone remodelling.
2021 -
Long-Term Fellowships - LTF

Revealing autolytic mechanisms of sieve element differentiation by improved phloem induction system

SUGIYAMA Yuki (JAPAN)

Sainsbury Laboratory - University of Cambridge - Cambridge - UK

HELARIUTTA Yrjö (Host supervisor)
In order to gain something, sometimes you have to lose something. This is true in our lives, and it is also true for cells to gain special functions. The individual component of the phloem sieve tube, the sieve element (SE), loses the cytoplasm, organelles, and even part of the cell wall to be able to transport photosynthetic products and signaling molecules over long distance. However, the molecular mechanisms behind these dramatic changes have been shrouded in mystery for more than 60 years since the discovery. The main reason for this is due to the small size of SEs and their deep location in the tissue, which makes them extremely difficult to observe with confocal live imaging. In this proposal, I aim to reveal the autolytic mechanisms of SEs by establishing a novel SE induction system that overcomes such technical hurdles. This research provides new insights into the coupling between autolysis and cell specialization. It also has the potential to expand the field of plant science, as plant development have rarely been studied in terms of degradation control.
2020 -
Long-Term Fellowships - LTF

Spatially resolved single cell profiling of plant transcriptome and microbiota

NOBORI Tatsuya (JAPAN)

Plant Molecular and Cellular Biology Laboratory - The Salk Institute for Biological Studies - La Jolla - USA

ECKER Joseph R. (Host supervisor)
In host-microbe interaction studies, genomic technologies have significantly advanced our understanding of host responses and structure of host-associated microbial communities (microbiota). However, spatial and physiological heterogeneity in host-microbe interactions has been more challenging to study. Plants are comprised of various cell types, each of which may interact with a distinct set of microbes and, at the same time, accomplish other tasks such as growth and nutrient acquisition/utilization. To date, interactions between individual plant cell types and the microbiota are poorly understood due to our lack of knowledge about cell type-specific responses of plants and the composition of associated microbes at the cellular level. Here, I employ high-throughput single cell trasncriptomics to analyze plant cell type-specific responses to the microbiota under nutrient (phosphate) replete and starvation conditions. I will mainly investigate immune response and phosphate starvation response and crosstalks between them in individual cell types. I will also develop a method for in situ microbiota community profiling by imaging-based sequencing of bacterial 16S rRNA, which provides a spatial map of the plant microbiota at the strain-level of resolution. By linking plant gene expression and microbiota distribution at the single cell resolution, I will investigate the roles of plant genes and microbiota members that show plant cell-type specificity. This project attempts to reveal plant cell type-specific responses to biotic and abiotic environmental signals and microbiota assembly that are significant and biologically meaningful.
2020 -
Long-Term Fellowships - LTF

Investigating archaeal horizontal gene transfer systems towards universal delivery tools

SAITO Makoto (JAPAN)

- Broad Institute of MIT and Harvard - Cambridge - USA

ZHANG Feng (Host supervisor)
A number of transformative molecular tools for biomedical research and therapeutics have been developed. However, a lack of safe, efficient delivery methods has precluded the widespread therapeutic application of these tools. One promising route to overcoming this limitation is to investigate the diverse biomolecule exchange systems found in nature and develop delivery tools based on these systems. Recently, a novel membrane vesicle (MV)-mediated horizontal gene transfer system was found in archaea. In this system, a relatively large 50 kbp plasmid is enclosed in a MV and delivered to other archaea. These plasmid vesicles (PVs) use a viral-like plasmid, pR1SE, that encodes the proteins required to assemble PVs and transfer the plasmid to other cells. As intercellular communication by MV is a universal process among the three domains of life, these systems may be exploited to deliver tools in human cells or the human microbiome. However, the mechanisms of archaeal MV and PV formation are largely unknown. I propose to identify the key proteins in the PV system. I will narrow down the candidates by remote homology detection among pR1SE and related viruses, and validate them by biochemical experiments. Furthermore, I will use genome-wide CRISPR screening to identify key proteins of host archaeal cells. Ultimately, I aim to reconstruct the PV system towards programmable synthetic vesicles production. This proposal will elucidate basic archaeal biology and provide a hint to the origin of viruses with a long-term objective of harnessing archaeal vesicle systems for delivery tools. Finally, this approach will serve as a paradigm for engineering archaea for molecular technologies.
2020 -
Long-Term Fellowships - LTF

Dissection of the relationship between folding stability and biological lifetime of proteins

TSUBOYAMA Kotaro (JAPAN)

Department of Pharmacology and Center for Synthetic Biology - Northwestern University - Chicago - USA

ROCKLIN Gabriel (Host supervisor)
Protein degradation systems maintain protein homeostasis. A failure of these systems causes various diseases, such as neurodegenerative diseases and cancers. In eukaryotic cells, ubiquitin is a general marker for selective degradation and determines protein lifetime in vivo. In selective degradation, E3 ubiquitin ligases determine target proteins. Although there are ~800 E3 ligases in human, only a handful of them have already shown to recognize specific short peptide motifs called “degrons”. Moreover, the feature(s) for unstable or misfolded structures recognized by E3 ligases remain unclear. In part, this is because we lack a comprehensive approach to investigate the global relationship between protein structural stability, ubiquitination status, and lifetime of the protein. To reveal the effect of protein folding stability (and other features) on ubiquitination and biological lifetime in vivo, I propose to measure these parameters for thousands of designed mini-proteins, whose folding stability has been previously characterized in detail. First, I will measure biological lifetime for these mini-proteins by flow cytometry, and monitor their ubiquitination status by using top-down proteomics approach. Then, I will analyze these data by using in silico analysis and decipher what factor(s) determine ubiquitination states and biological lifetime. This highly innovative and comprehensive approach using thousands of designed proteins will allow me to uncover the fundamental principle for protein lifetime in vivo and provide a mechanistic basis for designing better tools to manipulate protein lifetime.
2019 -
Long-Term Fellowships - LTF

Investigating the role of muscles in morphological plasticity of sea anemone Nematostella vectensis

ANZO Marie (JAPAN)

Developmental Biology Unit - EMBL, Heidelberg - Heidelberg - GERMANY

IKMI Aissam (Host supervisor)

Morphological plasticity is a key adaptive process allowing organisms to cope with changes in their environment. In animals, this process has been described in ephemeral organisms (e.g. flies, worms and mice) that typically experience a specific period in which development will respond to environmental signals producing long-lasting changes in animal body. In contrast, animals with extreme longevity are constantly patterning such that they must continuously adjust their developmental and physiological behaviours with the unpredictable fluctuations of food supply. To decipher the logic of such enhanced morphological plasticity, I am using the sea anemone Nematostella vectensis, a cnidarian laboratory model that exhibits a striking ability to adjust developmental patterns to the nutritional status of the environment while having a long lifespan. Based on preliminary data from the host lab, muscle is emerging as a central tissue that mediates the nutrient-dependent development of Nematostella tentacles. I will define this novel property of muscle cells in response to feeding using transgenic reporter lines. In parallel, I will also establish novel genetic tools to manipulate different muscle types and determine their specific contribution to post-embryonic development. I will also perform single-cell sequencing experiments to characterize the potential metabolic and developmental properties of muscles. Altogether, this proposed work will provide new insight into how morphological plasticity is encoded at the cellular and molecular levels in a long-lived animal.

2019 -
Long-Term Fellowships - LTF

Reconstituting and deciphering the TCR signaling apparatus by DNA nanotechnology

MASUBUCHI Takeya (JAPAN)

Section of Cell and Developmental Biology - UC San Diego - San Diego - USA

HUI Enfu (Host supervisor)

T cells are a type of lymphocyte that are able to recognize and destroy pathogens and tumor cells. The initial step in T cell activation is the binding of the major histocompatibility complex (MHC) presented antigen peptide to the T cell antigen receptor (TCR), which causes Lck mediated phosphorylation of its associated CD3 chains, including a CD3z/CD3z homodimer, a CD3g/CD3e heterodimer, and a CD3d/CD3e heterodimer. It is well established that CD3z/CD3z homodimer recruits ZAP70, a key cytosolic kinase, upon tyrosine phosphorylation. While much research has focused on CD3z, the roles of other CD3 chains are poorly understood. It is not clear why they are needed, what cytosolic proteins they recruit, and why they exist in a well-defined stoichiometry and geometry. To determine the role of the TCR architecture, I propose to assemble CD3 subunits on synthetic lipid bilayers with precisely controlled stoichiometry and inter-subunit distance using DNA nanotechnology. Using our recently developed fluorescence and microscopy readouts, I will dissect how the TCR architecture affects the recruitment of ZAP70 and other T cell signaling proteins. I will begin with a simple membrane reconstitution system to recapitulate each of the three types of CD3 dimerization, and determine their respective effect on signaling. I will then assemble the whole CD3 complex by precisely controlling the organization of CD3 dimers using a DNA scaffold. By exploring the potential geometry reported previously, this highly innovative approach may allow me to uncover the fundamental design principle of the TCR-CD3 complex and provide a mechanistic basis for designing better T cell-based immunotherapies.

2019 -
Long-Term Fellowships - LTF

Survival strategy of anaerobes in human microbiome using radical enzyme-assisted peptide metabolites

SUGIYAMA Ryosuke (JAPAN)

Department of Pharmacy - National University of Singapore - Singapore - SINGAPORE

MORINAKA Brandon I. (Host supervisor)

With the increasing plethora of microbial genomes, the importance of complex microbial communities in nature has been recognized. The human microbiome has particularly attracted researchers because of its direct link to health and disease. Despite their great diversity, which is apparent from the available genomic information, little is known about the bioactive small molecules secreted by the human microbiome. Few studies have investigated the presence and ecological roles of these metabolites in the human microbiome at physiological conditions, because of the lack of appropriate methodologies.
This project attempts to address uncharacterized peptide metabolites encoded in the genomes of human-associated anaerobic bacteria, and elucidate how they contribute increasing the fitness of the host microbes. These peptides are likely to have a novel scaffold made by a unique class of oxygen-sensitive enzymes. I intend to perform multidisciplinary research composed of two approaches: A) Production of the peptide metabolites in a heterologous microbe; B) A metagenome-based assay to evaluate the population changes in the cultured microbial community upon peptide treatment. The former approach has the potential to generate natural products independent of the strain; the latter enables us to decipher the biological activities of small molecules in a complex microbial community. Innovation in both concepts will increase our understanding and the availability of microbiome-derived compounds that could improve human health. Furthermore, given the broad distribution of target peptides in anaerobes, this research will also elucidate the breadth of anaerobic chemistry inside humans.

2018 -
Long-Term Fellowships - LTF

Integration of multiple diverse microbiota-derived signals by dendritic cells

NAGASHIMA Kazuki (JAPAN)

Department of Bioengineering - Stanford University - Stanford - USA

FISCHBACH Michael (Host supervisor)
The human microbiota produces a wide range of diffusible and cell-associated molecules, many of which are crucial for immune and metabolic homeostasis. The human immune system contains dendritic cells (DC), which sense bacteria-derived molecules through innate immune receptors and initiate an immune reaction that can be immunogenic or tolerogenic in tone. Previous studies have examined the effect of just a single or a few bacterial molecule(s) on the immune system. However, it is not yet understood how the immune system integrates a complex combination of numerous and diverse microbiota-derived molecules and 'decides' what kind of an immune response to mount. In this research, I will systematically elucidate the combinatorial effects of multiple microbiota-derived factors on DCs and identify genes in DCs underlying the integration of multiple signals. In Aim1, a large library of molecules from the microbiota and microenvironment will be generated. The library includes novel unpublished microbiota-derived molecules, in addition to commercially available microbial molecules and microenvironmental factors (i.e., tissue-specific cytokines). In Aim2, DCs will be cultured with combinatorial sets of molecules, and their transcriptional profiles and cytokine expression will be determined by multiplexed RNA-seq analysis. In Aim3, sets of genes required for DC responses will be identified by genome-wide CRISPRi/a screening and comprehensively analyzed using computational techniques. In Aim4, the results of the in vitro analysis will be tested in vivo using mouse experiments in which germ-free mice are colonized by genetically modified bacteria expressing multiple molecules.
2018 -
Long-Term Fellowships - LTF

Inheritance and transduction of environmental information in asexually reproducing plants

SATO Hikaru (JAPAN)

Department of Plant Biology and Forest Genetics - Swedish University of Agricultural Sciences - Uppsala - SWEDEN

KOHLER Claudia (Host supervisor)
It is estimated that about 40% of plant species can reproduce asexually, suggesting that this mode of reproduction is beneficial to allow plant survival under certain environmental conditions. Despite its prevalence, we know very little about how asexual reproduction contributes to the environmental stress response in plants. The central hypothesis of this proposal is that environmental stress induces epigenetic changes that can be stably propagated to the asexual progeny and potentially contribute to plant adaptation. To test this hypothesis, I will analyze the phenotypic, transcriptional and epigenetic changes in response to stress using Arabidopsis lyrata (A. lyrata), a plant species that asexually produces novel ramets from its root. In particular, I will investigate stress memory in novel ramets produced from parental plants that have been exposed to environmental stress (heat, salt, and cold stress). I will generate and analyze epigenetic profiles of ramets propagated over five generations under stress or non-stress conditions to identify stress-induced epimutations. I will furthermore investigate the signal transduction between maternal plants and ramets attached to each other through roots. Transcriptomic and phenotypic analyses will reveal how stress treatment of parental plants affects the attached ramets. Finally, I will test whether induced epimutations have adaptive value by investigating whether clones derived from stress-treated maternal plants are better adapted to stress. The proposed study will not only reveal a potential novel mechanism to adapt to environmental conditions, but also provide the basis for the generation of stress-adapted crops and trees.
2018 -
Long-Term Fellowships - LTF

How mechanical stress determines the direction of cell division plane in plants

TAKATANI Shogo (JAPAN)

The Plant Reproduction and Development Laboratory - ENS Lyon - Lyon - FRANCE

HAMANT Olivier (Host supervisor)
Cell geometry, hormones and mechanical stress have all been involved in Cell division plane orientation (CDPO), yet the corresponding molecular mechanisms remain unknown notably because 1- CDPOs are more variable than initially anticipated, 2- the relation between microtubule dynamics and CDPO robustness has not been analyzed, 3- the mechanotransduction pathways controlling CDPO are unknown. Using 4D live imaging, quantitative image analysis and biomechanics, I will analyze how microtubule dynamics is used, or filtered out, to generate robust cell division planes, and in turn how tissue tension feed back on microtubule dynamics to control CDPO. Using the Arabidopsis shoot apical meristem as an experimental system, I will draw a correlation map between microtubule dynamics, cell shape and growth, cell cycle, and CDPO and identify the spatio-temporal scales at which these correlations exist. I will then test the corresponding causalities using mutants affected in microtubule functions and CDPO robustness (TTP complex), mechanosensing (e.g. FER, DEK1), and micromechanics (compression, ablation, inducible lines and mosaics with cell wall defects). I will thus challenge the robustness of cell divisions and characterize the mechanotransduction pathways controlling CDPO.
2017 -
Long-Term Fellowships - LTF

In vivo imaging of synaptic plasticity through learning and sleep

MIYAMOTO Daisuke (JAPAN)

Department of Psychiatry - University of Wisconsin - Madison - USA

CIRELLI Chiara (Host supervisor)

Sleep has roles in memory consolidation. Memory consolidation is thought to include systems consolidation which reflects communication among different brain regions, as well as synaptic consolidation at the local circuitry level. I found systems consolidation during sleep among separated regions in cerebral cortex (Miyamoto et al., Science, 2016), however, synaptic consolidation pattern is still unknown. Net cortical synaptic strength changes homeostatically through wake/sleep cycle: average synaptic strength increases during wakefulness and decreases during sleep (Tononi & Cirelli, Neuron, 2014). While skill learning is known to potentiate subsets of synapses in motor cortex for encoding skill memory (Hayashi-Takagi et al., Nature, 2015), plasticity of those synapses through sleep is unknown. If sleep depresses memory-encoding potentiated synapses, it may result in impairing memory consolidation. If sleep depresses other non-potentiated synapses relatively, it may enhance signal/noise ratio and memory consolidation. Here I will use complex-wheel task for studying synaptic mechanisms of sleep-dependent skill memory consolidation in mice. Through skill learning and sleep, I will perform in vivo two-photon imaging in motor cortex to visualize fluorescence tagged AMPA receptors, since the expression of these excitatory glutamate receptors is an established index of synaptic strength. To visualize plasticity in synaptic population, I will analyze time-course of fluorescence intensity change at single-synapse resolution. This study will help clarifying how sleep favors both memory consolidation and synaptic homeostasis.

2017 -
Long-Term Fellowships - LTF

Elucidation of the cohesin mediated spatial organization of chromatin structure

NAGASAKA Kota (JAPAN)

Dept. of Molecular and Cellular Biology - IMP Vienna - Vienna - AUSTRIA

PETERS Jan-Michael (Host supervisor)

The formation of higher order chromatin structure is crucial for DNA replication, transcription, recombination, repair and chromosome segregation. The cohesin-complex is thought to contribute to formation of chromatin loops and of topologically associating domains (TADs) by entrapping two different regions of the same chromatids. Its binding along genomic DNA is thought to be controlled by two of cohesin’s cofactors, CTCF and Wapl. Cohesin has been proposed to regulate the long-range chromosomal cis-interaction by a “loop extrusion” mechanism, in which cohesin can regulate chromatin loop formation by translocating along genomic DNA. However, how cohesin controls chromatin structure remains unclear. To address this question, I will first establish assays based on FISH, Cas9-FISH in living cells, oligopaint strategy and super resolution microscopy (SRM) to visualize chromatin loops and TADs. Second, I will employ these assays and Hi-C to analyze chromatin structure in cells in which the genomic distribution of cohesin is experimentally altered. For this, I will use knowledge from my host lab which recently discovered that the distribution of cohesin in mammalian genomes depends on the DNA binding protein CTCF, the cohesin release factor Wapl and transcription. Third, I will monitor the transition of cohesin re-location and of conformational change of chromatin induced by rapid degradation of Wapl and CTCF in real time. I will then test prediction of the loop extrusion model by comparing my experimental data with the simulations recently described in the literature.

2017 -
Long-Term Fellowships - LTF

Integrative analysis of the role of mammalian Hox genes in tissue patterning

NAKAKI Fumio (JAPAN)

- EMBL - Barcelona - SPAIN

SHARPE James (Host supervisor)

How genetic programs regulate mammalian morphogenesis is one of the central problems in developmental biology. Digit patterning, a representative model to study mechanisms of morphogenesis, is regulated by multiple factors including cytokines and Homeobox-containing genes, or Hox genes, particularly Hoxa13 and Hoxd13. Recently, it has been illustrated that a reaction-diffusion model can explain this patterning in which the interactions of Sox9, a transcription factor, and WNT and BMP, cytokines, generate the periodic digit pattern. Interestingly, this periodic wave is modulated by FGF signaling and Hox genes. However, little is known about the molecular basis how Hox genes contribute to the proper digit patterning by regulating these interactions. In this project, to elucidate the role of Hox genes in mammalian digit patterning and autopod tissue identity, I will conduct integrative analyses including genetic, biochemical, and computational approaches. Firstly, single-cell RNA sequencing will be performed on both of wild type and Hoxa13 and/or Hoxd13 knockout mice to discover a gene network consisting the periodic wave pattern and analyze dosage effects of Hox genes on it. Secondly, HOXD11/D12/D13 will be epitope-tagged with genome editing tools. Thirdly, they will be immunoprecipitated to comprehensively analyze their binding specificity to the genome and other proteins. Lastly, these data are integrated with the reaction-diffusion model and investigate mechanisms of the modulation of BMP and WNT signaling by Hox genes to generate the periodic digit pattern, and consequently, establish the identity in autopod regions.

2017 -
Long-Term Fellowships - LTF

Developmental strategies to cope with nutritional stress

OHNO Hayao (JAPAN)

Developmental Biology Program - Sloan Kettering Institute - New York - USA

BAO Zhirong (Host supervisor)

Previous studies have suggested that embryogenesis is robust to environmental, genetic, or stochastic perturbations. However, little is understood about the mechanisms that enable multicellular organisms to develop robustly, and about the extent to which – and the way in which – they changes their developmental patterns in response to harmful perturbations. Especially, how organisms adapt to environmental stresses during embryogenesis is not well defined, partly because in most cases experimental organisms have been cultured under favorable conditions. Considering that organisms usually live in harsh conditions in their wild habitat, it is highly likely that they have evolved the undiscovered ability to deal with environmental stresses.
In this project, I will employ a high-performance cell tracing system and sophisticated computational techniques to accurately track the positions of ALL cell nuclei in developing embryos of the nematode Caenorhabditis elegans. By this approach, I will comprehensively examine the effects of nutrition deficiency on cell division and differentiation during embryogenesis. Additionally, I will elucidate the developmental basis of transgenerational starvation response. After the detection of the developmental differences caused by nutritional stress, I will clarify how the differences are induced (or suppressed) at the molecular level using powerful molecular genetics and mathematical analyses.
The success of this project will provide fundamental insights into embryogenesis and might lead to understanding of the mechanisms underlying teratogenesis, spontaneous abortion, and neonatal death in nutritionally challenged humans.

2016 -
Long-Term Fellowships - LTF

The role of oxytocin and vasopressin in the neural circuit of aggressive behaviors

KARIGO Tomomi (JAPAN)

Division of Biology and Biological Engineering - Caltech - Pasadena - USA

ANDERSON David J. (Host supervisor)

Innate behaviors such as aggression and reproduction are essential for the survival of animals. Neuropeptides are implicated in the regulation of various innate behaviors by changing their basal arousal state and motivation level via modulating neural activity. Oxytocin (OXT) and vasopressin (AVP) are involved in various social behaviors such as aggression, mating, pair-bonding and social recognition. However, how these peptides exert their functions at the neural circuit level is not well understood.
I plan to study the role of OXT/AVP on the neural circuit of aggressive behaviors by addressing the following questions: (1) Whether if OXT/AVP is actually released onto the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl), which was previously shown to control both the appetitive and consummatory phases of fighting and mating? (2) What is the role of the neural activity patterns responding to OXT/AVP release during aggression? (3) How is the activity of OXT / AVP neurons controlled during aggression and from where do the OXT/AVP neurons receive their input? (4) Whether if OXT/AVP control other social behaviors in VMHvl in addition to aggressive behaviors?
In contrast to my previous research experience in neuroendocrinology, the proposed research will focus on how neuropeptides modulate innate behaviors at the circuit level. Therefore, this will be a good opportunity to extend my training from endocrinological to behavioral neuroscience.

2016 -
Long-Term Fellowships - LTF

Programming of intestinal neural networks by the microbiota-immune system axis

OBATA Yuuki (JAPAN)

Mill Hill Laboratory - The Francis Crick Institute - London - UK

PACHNIS Vassilis (Host supervisor)
STOCKINGER Brigitta (Host supervisor)

The gastrointestinal tract is essential for the absorption of water and nutrients, the induction of mucosal immune responses and the maintenance of a healthy gut microbiota. Virtually all aspects of gastrointestinal physiology are controlled by the enteric nervous system (ENS), an extensive network of neurons and glial cells that is intrinsic to the gut wall. ENS interacts with components of its ‘outer’ microenvironment (gut microbiota, metabolites and nutrients) and ‘inner’ microenvironment (immune cells and stromal cells). A number of reports have demonstrated that the gut microbiota and immune system contribute to the development and homeostasis of the central and peripheral nervous system, but the mechanisms by which these cellular systems regulate the assembly and maturation of neural circuits are currently unknown. Recent studies have raised the possibility that maternal and/or early postnatal microbiota influence the organization and function of the ENS. In this proposal, we will take advantage of established expertise and reagents (including transgenic and germ-free mice) to analyse the role of the microbiota and associated products (including microbial metabolites) and the adaptive immune system on the development and functional maturation of the ENS. Our aim is to identify molecular and cellular cascades that are regulated by gut microflora and directly or indirectly control the assembly and function of intestinal neuroglial networks. This proposal will provide insight into the molecular pathways that underpin the effects of environmental factors (such as microbes) on the development and functional maturation of the central and peripheral nervous systems.

2016 -
Long-Term Fellowships - LTF

Molecular mechanism for plant developmental control by CRINKLY-type membrane receptors

OKUDA Satohiro (JAPAN)

Department of Botany and Plant Biology - University of Geneva - Geneva - SWITZERLAND

HOTHORN Michael (Host supervisor)

CRINKLY-type receptor kinases are unique plant membrane receptors with key roles in plant growth and development, in different tissues and throughout the plant's life cycle. Their cellular functions are poorly characterized and it is unknown what ligands they respond to.

I propose to combine protein biochemistry and X-ray crystallography with experiments in the model plant Arabidopsis to define what ligands CRINKLY receptors sense and how these ligands act in concert with the receptors to control very different developmental processes in different parts of the plant.

Specifically, my project aims to employ in vitro and in vivo biochemical screens to identify ligand candidates for CRINKLY receptors and to validate these interactions in genetic and structural detail. Next, I want to combine structural biology and kinetic kinase assays in vitro with reverse genetic experiments in Arabidopsis to understand ligand-induced receptor activation of CRINKLY receptors. Finally, I would like to define down-stream signaling targets of activate CRINKLY complexes in different plant tissues, by performing in vivo interaction screens.

This project sets the stage to define in molecular terms the functions of a new class of membrane receptors in plants. Identification of bona fide CRINKLY ligands will enable us to better understand plant membrane receptor activation mechanisms, the connection to downstream signaling events, and the cellular roles of CRINKLY receptors in plant development.

2016 -
Long-Term Fellowships - LTF

Synthetic morphogenesis: exploring multicellular self-organization using engineered cell signaling

TODA Satoshi (JAPAN)

Department of Cellular and Molecular Pharmacology - UC San Francisco - San Francisco - USA

LIM Wendell (Host supervisor)

During tissue formation, interacting cells generate spatiotemporal patterns of gene expression to create multicellular structures. Traditional approaches are increasing our knowledge of molecular players and their interactions for tissue formation. Nonetheless, the principles of how such molecular players work together to achieve complex multi-cellular structures remains unclear. Synthetic “bottom-up” approaches can help reveal the logic of how self-organizing multicellular systems are constructed. Here, I will build artificial communication circuits between cells to investigate their potential to build complex structures. I focus on two types of communications that we postulate to be most important for pattern formation; direct contact communication mediated by transmembrane proteins like Notch-Delta and short-range paracrine signaling by lipid-modified secreted proteins like Wnt. In preliminary unpublished work, we have designed synthetic Notch receptors that recognize artificial ligands on membrane to induce specific gene expression. Synthetic receptor/ligand pairs avoid crosstalk with native Notch/Delta and can build multiple sets of orthogonal Notch-like communication channels. I will design an analogous synthetic Wnt ligand which keeps its spatial diffusion property and is recognized by a synthetic Notch receptor. I will explore how circuits with multiple synthetic receptor-ligand combinations can generate structures by screening a library of synthetic Notch and Wnt combinations. The comprehensive elucidation of relationships between simple intercellular circuits and self-organized patterns could be useful in guiding tissue engineering and regenerative medicine.

2016 -
Long-Term Fellowships - LTF

Structural visualization of the activating full-length single transmembrane receptor complexes

TSUTSUMI Naotaka (JAPAN)

Department of Molecular and Cellular Physiology - Stanford University - Stanford - USA

GARCIA K. Christopher (Host supervisor)

Serum cytokines induce dimerization of receptor extracellular domains (ECDs) to enable the central orchestrators of cytokine signaling, Janus family tyrosine kinases (JAKs), nexus inside the cell. This event is responsible for initiating signaling cascades associated with an extensive range of functional outcomes, and therefore dysregulation of the signaling causes many types of cancers. However, the structural link between JAKs and cytokine receptors remains poorly understood. The proposed work will advance our knowledge of this essential process, combining multifaceted structural and functional approaches to visualize the entire interleukin (IL)-6 signalosome as a model for cytokine-induced JAK signaling. In the first aim, we will determine an X-ray crystal structure of a receptor intracellular domain (ICD) bound to JAK1 N-terminal FERM domain. Given that the JAK domains are highly conserved, the binding mode elucidated from our structure should be widely applicable to all JAK-receptor pairs. In the second aim, we will explore JAK activation from an ultrastructural perspective. Cryo-electron microscopy (cryo-EM) single-particle reconstructions of a transmembrane holocomplex will yield an unprecedented 3-dimensional view of the orientation of signaling molecules in the working state. Taken together, our work will provide a thorough mechanistic view of JAK activation and inform design of novel molecular therapeutics for cancer.