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Awardees

2023 -
Grant Awardees - Program

Evolution of protein multifunctionality

CHANG Belinda (.)

University of Toronto, Department of Cell & Systems Biology - . - .

FEUDA Roberto (.)

University of Leicester - Leicester - United Kingdom

GOEPFERT Martin (.)

University of Göttingen (Georg-August-Universität Gottingen) - . - .

MENON Anant (.)

Weill Medical College of Cornell University - . - .

Like moonlighting people, proteins can have several seemingly unrelated functions. How multiple functions can coexist within the sequence of a single polypeptide chain remains an open question, especially if the individual functions are not separated as in a Swiss army knife but intermingled such that selection for one function might compromise others. Merging expertise in evolutionary genomics, biochemistry, biophysics, and physiology, implemented via in silico, in vitro and in vivo approaches, we now propose to elucidate fundamental principles underlying protein moonlighting using the Drosophila melanogaster opsin protein Rh1 as a prototype. Rh1 (i) initiates visual transduction in photoreceptor cells, (ii) organizes cilia in mechanoreceptor cells, (iii) acts as a chemoreceptor protein in taste receptor cells, (iv) functions in the sensory discrimination of environmental temperature, and (v) acts as a phospholipid scramblase, contributing to membrane lipid homeostasis. We will use two approaches to scan for variants that may be important for Rh1 multifunctionality, which will then be investigated experimentally. First, we will investigate natural variation of the Rh1 sequence across populations/species and identify targets of selection. Second, we will conduct deep scanning mutagenesis experiments of Rh1 to identify all allowable variants which permit proper protein maturation and visual signaling. Allowable mutations not found in nature may be important for non-visual functions. Natural and allowable sequence variants will be quantitatively phenotyped using in vitro and in vivo analyses to determine their effects on the photo-, mechano-, chemo-, and thermosensory functions of Rh1, its scramblase activity, spectral tuning, and downstream signaling. These genetic and functional data will then be used to (i) link multiple aspects of Rh1 functions to specific amino-acid residues, (ii) determine to what extent the diverse functions make use of common residues, (iii) reveal whether the respective residues are conserved or vary in nature, (iv) elucidate the functional consequences of this variation and finally, (v) illuminate the evolutionary constraints associated with moonlighting by opsins. Thus, we will obtain a molecular understanding of how hyper-diverse functions are realized and maintained in super-multifunctional proteins.
2023 -
Long-Term Fellowships - LTF

Functional exploration and machine learning of the solute binding domain transcription factors

CHEN John (.)

. - The Australian National University - Canberra - AUSTRALIA

COLIN Jackson (Host supervisor)
The LacI superfamily of Transcriptional Regulators (TRs) are common in bacteria and allow for the sensing of molecules to regulate gene expression. TRs have a DNA binding domain and a solute binding protein (SBP) domain, which binds specifically to a variety of small metabolites. Ligand binding results in conformational change and allosteric control of the TR’s DNA binding affinity, coupling small molecule binding to gene regulation. The recent explosion in genomic and metagenomics sequencing has unveiled a wealth of sequence diversity. However, only a handful of TR sequences have been characterized, leaving the vast majority of the metabolite binding sequence space unexplored; we estimate that >95% of sequences clusters are ‘orphans’, with no closely related (>35% sequence identity) sequences of known function. Thus, there is great opportunity to expand our understanding of TRs and molecular recognition in general, as there is a plethora of undiscovered ligand binding solutions with unknown mechanisms. In this project, I propose to survey the unexplored sequence space, by characterizing and functionally mapping orphan TR sequences, followed by a multifaceted machine learning approach to predict ligand binding throughout the rest of the family. We will first generate a sequence similarity network (SSN) of the ~1 million TRs collected from the NCBI database, which clusters sequences by sequence identity. Using the SSN, we will identify approximately 200 diverse ‘orphan’ TR clusters, then synthesize and clone approximately 200 of these sequences into expression vectors. To characterize each TR in terms of their function as an activator or repressor, each gene will be co-expressed into a library of reporter plasmids expressing an On/Off selection system that contains a binding site with randomized DNA promoter sequence; this system selects (by survival) for transcriptional activation or repression. The TRs will then be screened using a small molecule library (~1,000s of compounds), where each gene/DNA library is screened with and without the small molecule, and co-selected for active or repressed expression of the reporter. Finally, each condition in which activation/repression is observed will be identified with a molecular barcode, pooled and sent for deep sequencing, allowing mapping of all DNA recognition sequences and all responsive ligands for every screened TR by highlighting the surviving DNA/ligand combinations under On/Off selection. We will analyze novel TRs using protein crystallography, structure prediction (alphafold) and phylogenetic analysis. Most importantly, this data will be used to train machine learning algorithms to allow us to accurately predict both DNA binding specificity and ligand binding throughout the entire superfamily. We expect this project to greatly enhance our knowledge of gene regulation and molecular recognition, allowing for further expansion of the fundamental study of prokaryotic gene regulation.
2023 -
Cross Disciplinary Fellowships - CDF

Highly parallel determination of protein identity and function with single-molecule resolution

CHOI Hansol (.)

. - Boston Children's Hospital - Boston - United States

WESLEY Wong (Host supervisor)
While advances in single cell RNA sequencing (scRNA-seq) technology have revolutionized biomedical research (Papalexi et al., Nat. Rev. Immunol., 2017), equivalent approaches for proteomic analysis remains a challenge. Two significant reasons why analyzing proteins is more challenging than analyzing DNA or RNA 1. The high complexity of the proteome: proteins show higher complexity than genes not only due to the increased diversity of their building blocks, but also due to diversification during translation. 2. Lack of technology that can efficiently identify and characterize proteins in trace samples: unlike DNA which can be easily amplified with polymerase chain reaction (PCR), proteins cannot be easily replicated. To meet these challenges, the development of high-throughput single-molecule approaches is a promising avenue, which should enable the deciphering of complex proteomes, including the high heterogeneity between cells that can arise in cancer. Furthermore, proteins can provide additional information to scRNA-seq technology since they contain information that arose during translation. In addition to measuring posttranslational modifications, proteomics methods can also characterize protein conformation, which cannot be inferred from scRNA-seq data and is closely related to its functionality, with misfolded proteins potentially leading to various diseases (Cohen et al., Nat., 2003). Because misfolded proteins cannot easily be purified or labeled, protein conformation would ideally be analyzed using single-molecule approaches. However, conventional approaches for studying protein conformation are often lacking or limited in terms of throughput or their ability to analyze trace samples (Serebryany et al., bioRxiv, 2022; Aebersold et al., Nat., 2016). The overall goal of this proposal is to develop a high-throughput, single-molecule proteomics technology with the potential to revolutionize cancer proteomics. The highly parallel protein profiling and structural analysis technology that we develop will complement more traditional single-cell profiling approaches such as scRNA-seq. Compared to mass spectrometer (MS)-based and ELISA-based single cell proteomics, our single-molecule approach should have advantages in sensitivity and multiplexity, respectively (Labib et al., Nat. Rev. Chem., 2020).
2023 -
Grant Awardees - Program

Deciphering the role of dynamics in vascular network remodeling and determination

CORNELISSEN Annemiek (.)

Laboratoire Matière et Systemes Complexes, CNRS, UMR 7057 - . - .

JONES Elizabeth (.)

Catholic University of Leuven (Katholieke Universiteit Leuven, KU Leuven) - . - .

KATIFORI Eleni (.)

University of Pennsylvania, Philadelphia, PA - Philadelphia - United States

Vascular networks in animals are constantly in flux, remodeling their topology and architecture during development and adulthood, to respond and adapt to different stimuli and environmental conditions. The resulting topologies are typically complex, involving several levels of hierarchy, and display features that can be inconsistent with steady flow adaptation models, like suppression of vascular shunting. In this project, we will explore how these biological transport networks are built and maintained. In particular, we will combine our expertise in theoretical and computational fluid dynamics, soft matter physics, and experimental developmental biology of two very different animal classes, mammals and jellyfish, to understand how pulsatility and other dynamics influences the long-term adaptation and remodeling of the vascular system. Pulsatility is present in all living organisms and yet our understanding of the role this stimulus plays is minimal. We propose that pulsatility is vital to the organism, playing an essential role in preserving vascular loops and preventing shunting. To do this, we will first build computation models that calculate the flow dynamics in complex elastic networks. We will test these models with real in vivo measurements of whole mouse embryos and jellyfish. We will then use experiment and computation to study how short-term dynamics in the flow and variations in the pumping force that propels the fluid are propagated by fluid structure interactions of the elastic vascular walls. We will investigate mechanisms, both on the physiological and genetic level, including gain-of-function and loss-function models. The earlier is obtained by increasing vessel stiffness, both in jellyfish and mouse, thereby generating faster and larger pulse waves. The latter takes advantage of recently developed mechanical assist devices for the heart that create continuous flow in the vasculature. Our final aim is to to understand the role of wave reflection and additive/subtractive effects of the pulse wave forms and perform a comparative analysis of our findings in jellyfish and mouse, to eventually uncover the fundamental biological rules that shape vascular network architectures.
2023 -
Grant Awardees - Program

Electrogenetic control of bacterial metabolism, communication, and biofilm formation

CRAIG Lisa (.)

Simon Fraser University - . - .

FRANCETIC Olivera (.)

Institut Pasteur, Paris - . - .

MALVANKAR Nikhil (.)

Yale University - New Haven - United States

SALGUEIRO Carlos (.)

Faculdade Ciências e Tecnologia, Universidade Nova de Lisboa - . - .

Electrogenetics is an emerging biotechnology platform that uses electric fields to control gene expression. Current electrogenetic methods rely on diffusive ions which become ineffective due to high fluid flow present in most environments. To achieve fast and directional electron transport, soil microbes including Geobacter have evolved long thin proteinaceous “nanowires” capable of ultra-fast delivery of excess metabolic electrons to external mineral acceptors or to other microbes in a process called Direct Interspecies Electron Transfer (DIET). DIET is crucial to prevent toxic electron build-up in the anaerobic habitats where Geobacter lives in biofilm communities with other microbe species. Crucially, many archaea capture these electrons via DIET and use them to produce methane (methanogens) or to degrade methane (methanotrophs). Nanowires and DIET thus have a major impact on the global levels of methane, a greenhouse gas 30 times more potent than CO2 in trapping heat. Despite their global importance, the conditions and mechanisms driving nanowire biogenesis and function in DIET are poorly understood. Recently, PI Malvankar showed that nanowires are polymers of cytochromes that are secreted to the Geobacter cell surface via an unusual type IV pilus machine. Electric fields induce nanowire production by upregulating expression of genes involved in their biogenesis. Thus, the Geobacter system is an ideal model to study electrogenetics. To elucidate how the electric fields control nanowire biogenesis and signalling, we will identify Geobacter genes upregulated during DIET in co-cultures of Geobacter donor and acceptor species. We will deploy solid-state physics, spectroelectrochemistry, biochemistry, structural biology and genetics to characterize nanowire secretion and assembly, electron wiring and transport in DIET, and its effects on gene expression. By reconstituting nanowire secretion and assembly in Escherichia coli we can characterize their biogenesis and function. This information can be used to design nanowire-based electrogenetics systems that form conductive biomaterials, generate energy (biofuels) and produce biological products. Importantly the Geobacter system can be paired with methane-utilizing microbes to investigate electrogenetics in these species, with the long term goal of controlling DIET to reduce atmospheric carbon levels.
2023 -
Long-Term Fellowships - LTF

Structure of the Synaptic Vesicle

DAS Poulomi (.)

. - The Regents of the University of California, San Francisco - San Francisco - United States

ROBERT Edwards (Host supervisor)
ROBERT Stroud (Host supervisor)
Synaptic transmission is crucial for brain function and defects are associated with many neuropsychiatric disorders. Presynaptic mechanisms regulating neurotransmitter release decode the information stored in neural firing, and functions have been assigned to several of the key proteins involved in synaptic vesicle docking, Ca++ sensing and membrane fusion. However, the mechanisms, regulation and coordination are not well understood. The function of many other synaptic vesicle proteins also remains unknown. Indeed, previous work has focused on the function of individual proteins without considering the larger context, leading to the assumption that membrane proteins are randomly distributed on the synaptic vesicle. However, interactions between several of them suggest organization which would have profound implications for neurotransmission, behavior and disease. The long-term objective of this project is to develop a holistic understanding of the diverse mechanisms involved in neurotransmitter release and their role in information processing. The strategy is to determine the structure and organization of proteins embedded in the synaptic vesicle membrane at near-atomic resolution and test the functional implications of the most salient findings. I hypothesize that the organization of synaptic vesicle membrane proteins serves to coordinate their function in neurotransmitter release. Previous work on individual proteins involved in synaptic vesicle docking and fusion has suggested interactions between several of them. For instance, membrane fusion requires multiple SNARE complexes, and the synaptic vesicle contains many copies of the principal v-SNARE VAMP2. The Ca2+ sensor synaptotagmin 1 has been suggested to link these complexes into a ring, which may confer cooperativity. The synaptic vesicle also contains t-SNAREs that may act in cis to regulate interaction in trans with t-SNAREs at the plasma membrane. Determining the organization of these proteins will help to understand their role in release. This work will also provide a foundation to understand how synaptic vesicles interact with other elements of the release machinery such as plasma membrane docking sites and presynaptic Ca++ channels. Synaptic vesicles isolated by differential centrifugation and gradient fractionation will be used for structure determination by Cryo-electron tomography, in collaboration with the Stroud lab. The issue of heterogeneity will be addressed by selective immunoisolation and/or class averaging. Imaging and electrophysiology in primary neuronal cultures will be used to test the predictions for synaptic vesicle exocytosis and neurotransmitter release. The results will provide a framework to understand the function, regulation, and interactions among synaptic vesicle proteins, providing a broad context to understand neurotransmitter release, its role in information processing, behavior, and dysfunction in disease.
2023 -
Long-Term Fellowships - LTF

Investigation of uropathogenic E. coli virulence mechanisms in a human bladder microtissue model

DE BENTO FLORES Carlos Eduardo (.)

. - Biozentrum University of Basel - Basel - SWITZERLAND

CHRISTOPH Dehio (Host supervisor)
Urinary tract infections (UTIs) are among the most common infections in the world and the primary cause for the prescription of antibiotics. WHO has recently highlighted UTI treatment as a critical exacerbating factor in the global antimicrobial resistance (AMR) crisis. However, the pathophysiological mechanisms underlying UTI and its frequent recurrence remain largely understudied, hindering the development of effective therapeutics. The vast majority of UTI research has been performed in mouse models or cancer cell lines using uropathogenic E. coli (UPEC) as the predominant agent. However, the obtained results often cannot be fully translated to humans due to differences in bladder ultrastructure, physiology/function and immunity. Therefore, the main goal of this project is to unveil the main strategies and virulence factors underlying UPEC colonization and persistence using a physiologically relevant human microtissue bladder model. The following hypotheses will be addressed: i) Does bacterial adhesion, invasion and the formation of intracellular bacterial communities occur in the human model as previously described for the mouse model?; ii) Which virulence factors (e.g. flagella, type I- or P-pili) are critical for the establishment and persistence of UTI?; iii) What is the role of the innate immune response (e.g. diapedesis and phagocytosis) for the outcome of the infection (persistence vs. clearance); iv) Which of these strategies and molecular mechanisms are shared between different UPEC strains? A multidisciplinary approach will be taken by the in-depth characterization of a perfused, urine-tolerant, fully stratified 3D-human bladder microtissue model, that is under development in the host lab at Biozentrum, Univ. of Basel. Bladder biopsies and urine from UTI patients obtained from the University Hospital Basel will be used for validation purposes. The initial assessment of pathophysiological mechanisms will be performed using UPEC strain CFT073 (broadly used in mice infection models), together with mutants in virulence factors identified by a genome-wide CRISPRi screen in the host lab. To depict the general infection strategies (adhesion, intracellular communities, etc.), I will also address mechanistic aspects of the underlying virulence processes, targeting host compartments and bacterial factors (e.g. flagella, pili, etc.) at single cell resolution using 4D spinning disk live cell imaging. Selected clinical UPEC isolates will be used as comparison and immune cells (i.e., neutrophils) will be added to the model to assess UPEC clearance. Overall, this project will allow the study of basic pathways underlying UTIs and mimic recurrence in a powerful platform, serving as a crucial complement (or even alternative) to the current mice models that dominate the field. We will be able to dissect with high-resolution the specific mechanisms and players involved in the UTI establishment and its persistence, which is a current demand.
2023 -
Grant Awardees - Program

From disorder to order: mechanism of specialised assemblies formation essential for muscle function

DJINOVIC-CARUGO Kristina (.)

European Molecular Biology Laboratory (EMBL-Grenoble) - Grenoble - FRANCE

HINSON John (.)

University of Connecticut, Farmington - . - .

ODA Toshiyuki (.)

University of Yamanashi - . - .

RIES Jonas (.)

European Molecular Biology Laboratory (EMBL-Heidelberg) - . - .

Muscle force production and mechanotransduction rely on the ordered assembly of functionally and molecularly specialised actin cytoskeletal complexes. The sarcomere - the basic contractile unit of myocytes - assembles from nascent actin-rich Z-bodies into paracrystalline multiprotein structures called Z-discs, which contain numerous proteins frequently mutated in inheritable muscle disorders such as familial myopathies and cardiomyopathies. Yet, the assembly rules and protein components regulating these complex structures remain largely unknown. The project aims to elucidate the assembly processes of multiprotein actin complexes essential for cardiomyocyte contractility and mechanotransduction. We will examine the hierarchy and interdependence of interactions of cytoskeletal complexes in intercalated and Z-discs (Objective 1), resolve the structure of cytoskeletal complexes in vitro in and in cellula at different stages of sarcomere biogenesis and assess the dynamics of cytoskeletal components in different stages of maturation (Objective 2). Finally, we will combine the data in a 4D multiscale molecular model and validate the model with structure-informed in cellula and in vitro assays (Objective 3). Capitalising on the team’s complementary and synergic expertise in cardiovascular development and disease, advanced imaging, structural biology and modelling, we will determine the biophysical and structural rules underlying complex assembly, molecular components and regulators, and combine the data in a 4D multiscale integrative molecular model. Of special interest is, how highly ordered cytoskeletal structures arise from disorder through maturation and specialisation of the actin cytoskeleton.
2023 -
Grant Awardees - Program

Nuclei as mechanical sensors and actuators in epithelial folding

ERZBERGER Anna (.)

European Molecular Biology Laboratory (EMBL-Heidelberg) - Heidelberg - GERMANY

WANG Yu-Chiun (.)

- . - .

The nucleus is the largest and stiffest organelle in a cell. It functions not merely in cellular information processing, but also in the spatial organization of cells and tissues, as well as mechanotransduction impacting gene expression. Prior experimental investigations in nuclear mechanics are primarily in vitro or ex vivo, while most physical theories on tissue mechanics neglect the presence of nuclei. Thus, how large-scale in vivo tissue architecture is shaped by — and feeds back onto — nuclear properties is unclear. During epithelial folding (EF), nuclei characteristically relocalize along the apico-basal axis, raising the question if nuclear movements mechanically contribute to tissue deformation, and/or if the positioning or deformation of the nucleus senses and encodes tissue curvature information to subsequently modulate tissue architecture and cell state. We propose to investigate the coupled dynamics between cell shape, nuclear position and deformation, and address how nuclear distribution and mechanics orchestrates the transitions from the simple-columnar to the pseudostratified, or from the flat to the curved tissue architectures. Combining physics and biology, we will employ innovative, multi-scale theoretical approaches to account for parameters ranging from the subcellular to the tissue and embryo scales, and use the gastrulating Drosophila embryo to experimentally inform and verify our theoretical framework. Exploiting the prowess of Drosophila in quantitative imaging and controlled perturbation, and in particular, optogenetics, we will take a comparative approach to accentuate common mechanical principles of cell-nucleus coupling from the mechanistically distinct EFs that form during Drosophila gastrulation. Furthermore, we will propose lineage-specific feedback parameters to predict the way in which deformed or displaced nuclei influence long-timescale EF morphodynamics, and experimentally explore mechanosensitive changes of nuclear properties, chromatin states and transcription in search of corresponding mechanisms. We expect to uncover fundamental and scale-crossing principles of tissue self-organization driven by nucleo-cellular coupling, thereby testing the hypothesis that nuclei function as sensors and actuators for tissue deformation and differentiation.
2023 -
Grant Awardees - Program

The role of lipid physical properties for the multifunctionality of insect cuticular hydrocarbons

FEDERLE Walter (.)

University of Cambridge - Cambridge - United Kingdom

KANEKO Fumitoshi (.)

Graduate School of Science, Osaka University - . - .

MENZEL Florian (.)

Johannes Gutenberg University of Mainz - . - .

Insects wear a multifunctional suit on their body surface - a thin film of cuticular lipids that fulfils many different biological tasks. This outermost layer has a complex chemical composition that varies greatly between species. It not only serves as a vital barrier against desiccation, but the lipids are also essential for chemical communication. Besides, cuticular lipids also have various biomechanical functions in surface adhesion, joint lubrication, and self-healing. How can this lipid layer perform so many functions at the same time? Why is the chemical composition of insect lipid films so complex, with up to 100 different compounds? What selection pressures explain the complex variation in the chemical composition of lipid films? All the biological functions of cuticular lipids and possible trade-offs between them crucially depend on the physical properties of the films, which are still poorly understood. Current models assume that waterproofing is based on relatively solid lipids, while the other functions require lipids in a fluid state. We hypothesize that cuticular lipid layers can meet these conflicting requirements due to their complex phase behaviour and other physical properties, and that insects can tune these to different demands by varying lipid composition. We will investigate four main questions: 1) What are the physical properties of cuticular lipid layers, and how are they influenced by their chemical composition? 2) How do cuticular lipid properties depend on temperature, and how do insects change their lipid layers when acclimating to different environmental conditions? 3) How do the physical properties of cuticular lipids affect their biological functions? 4) How do insect lipid properties correlate with their ecological niche? We will study the multifunctionality of insect cuticular lipid films by integrating physical chemistry, physiology and evolutionary ecology. By combining modern techniques for measuring physical properties of lipid layers with novel biological experiments on insects, we will determine how lipid composition, phase state and physical properties affect each function. With this we will gain novel insights into the functions of cuticular lipids, and better understand the adaptive value of the complex composition of cuticular lipids and how this multifunctional trait evolved.
2023 -
Grant Awardees - Program

Social immunity in honeybee - SoBee

FIEHN Oliver (.)

The Regents of the University of California (University of California Davis) - . - .

GALIZIA C Giovanni (.)

University of Konstanz (Universität Konstanz) - Konstanz - GERMANY

JENSEN Michael (.)

Technical University of Denmark - . - .

Insects play crucial roles in pollination of the world’s crops. Yet insect populations are on decline, partially due to pesticide-intensive agriculture, climate change-induced disruptions, and diseases. With respect to disease, timely recognition of and response to, eminent contagions has proven essential for eusocial insects like honey bees (Apis mellifera). Without an adaptive immune system, honey bees rather rely on social immunity as part of maintaining hive hygiene, involving both removal of corpse from hives and altered social interactions. However, while there is a growing body of evidence linking disease-related odors to honey bee physiology and behavior, the causal molecular underpinnings of olfaction, or the sense of smell, driving behavioral disease defenses, like social immunity, in response to hive infestation, remains elusive. One key piece in decoding the sense of smell is to map the specificity and affinity of honey bee olfactory receptors (ORs). The honey bee genome encodes approx 170 ORs, yet odors consist of hundreds of small molecules. Adding to the complexity, the OR:odorant map is bow-tied, with many ORs detecting the same odorant, and some ORs detecting multiple odorants. Here we join expertise in entomology, synthetic biology, and chemistry to test the working hypothesis that the evolution of social immunity in honey bees is controlled by olfaction, or more specifically that certain ORs in worker bees are under strong selection, and even causal to olfaction-induced social immunity leading to survival and next-generation courtship. To address this hypothesis, the consortium on social immunity in honeybees (SoBee) will search bacterial and honey bee cues causal to recognizing infected sisters, by i) testing volatile organic compounds (VOCs) produced from metagenomic libraries of infectious bacteria against honey bee ORs expressed in a microbial olfaction platform, ii) performing metabolite profiling of honey bee glands and clonal isolates from metagenomic library screens to search for chemical cues correlating with social immunity, and, iii) testing glands in bioassays, bee brain activity, and conduct hygienic behavioral studies on honey bees. Thus, the project seeks to identify causal factors underlying social immunity with the ultimate goal to support sustainable honey bee disease mitigation and crop pollination.
2023 -
Grant Awardees - Early Career

Exploration of the structure-function space of prebiotic to biological proteins – RENEWAL APP

FREELAND Stephen (.)

University of Maryland Baltimore County - . - .

FRIED Stephen (.)

Johns Hopkins University JHURA - . - .

FUJISHIMA Kosuke (.)

National University Corporation Tokyo Institute of Technology - Meguro-ku Tokyo - JAPAN

HLOUCHOVA Klara (.)

Charles University (Univerzita Karlova) - . - .

All life on Earth depends on proteins, now composed of a quasi-universal alphabet of 20 amino acids. The alphabet has been fixed in the Central Dogma for eons of evolutionary history, creating the impression that it is somehow “optimal” for folding sequences into complex structures and performing biochemical functions. The possibility however that the canonical amino acid alphabet is a “frozen accident” (to quote Francis Crick) has yet to be critically examined because whether alternative amino acids would lead to “bad” alphabets has not been empirically tested. Here, we propose to design “xeno-alphabets” composed primarily of non-canonical amino acids, and then to synthesize peptide libraries “written” in these new chemical languages. Our goal is to test whether alternative life (or evolutionary paths) could have produced successful proteins from entirely different building blocks and to learn about the chemical logic of the set found within life’s genetic code. As the number of possible xeno-alphabets is infinite, chemoinformatic approaches will be used to propose representative xeno-alphabets that vary in their chemical distance from canonical and capture different regions of physicochemical property space. After designing such alphabets in silico, we will synthesize peptides composed of these letters using solid phase peptide synthesis (which will allow us to focus on evaluating the properties of the peptides, such as the ability to fold or bind with other biomolecules) or using wet-dry cycling (which will focus on the capacity of the building blocks to form peptides autonomously under prebiotically-relevant conditions). In characterizing these peptide libraries, our principal aim will be to compare the structure-forming and functional potential of the alphabets (using a range of biophysical and biological methods) with the canonical set, to answer: How large is the chemical space that could support life as we know it (or don’t know it)? We, therefore, propose a research program by which unique computational tools will be adapted to design xeno-alphabets, whose peptides will be tested with laboratory techniques – some of which draw from our previous experience but most require significant methodological innovation, including next-generation peptide sequencing.
2023 -
Grant Awardees - Early Career

Switchable immunomodulation of mRNA transport and local translation in microglia by bioactive RNAs

FU Meng-Meng (.)

- . - .

L. J. BROERE Daniël (.)

Universiteit Utrecht - . - .

LEPPEK Kathrin (.)

University Hospital Bonn (Universitätsklinikum Bonn) - . - .

MILOVANOVIC Dragomir (.)

German Center for Neurodegenerative Diseases (Deutsches Zentrum für Neurodegenerative Erkrankungen - DZNE) - Bonn - GERMANY

Defects in microglia, the resident immune cells of the brain, have been implicated in neurological diseases, including Alzheimer’s, Parkinson’s, and multiple sclerosis. Microglia perform diverse functions, such as developmental synapse elimination, immune surveillance, and phagocytosis of pathogens and cellular debris. As highly ramified cells, microglia spatially regulate locally branched domains. In response to local signals, microglia can rapidly switch their shape from highly ramified to amoeboid. How this dramatic morphological change is achieved remains unclear. Recent transcriptome profiling of microglial processes uncovered a subset of local mRNAs encoding cytoskeletal protein. This suggests that mRNA transport and local translation may regulate microglia morphology. Though both of these mechanisms are well understood in neurons, they have not been studied extensively in microglia. In addition, few tools are available to manipulate mRNA transport and local translation with spatial precision. We will capitalize on our expertise in glial cell biology (Fu GLIA Lab), regulatory RNA structure, translation and therapeutics (Leppek RNA Lab), protein biochemistry and liquid condensates (Milovanovic CONDENSATE Lab), and synthetic organic chemistry (Broere SYNTHESIS Lab). We hypothesize that manipulating mRNA granules can lead to local changes in translation and morphology. We aim to: (i) identify key local mRNAs that affect microglial function using primary microglia cultures; (ii) characterize the mesoscale assembly of mRNAs and RNA-binding proteins (RBPs) inside condensates; (iii) leverage mRNA structure-function to design and develop a photoswitchable bio-active RNA tool (SMARTswitch); (iv) validate that SMARTswitch can manipulate local translation in microglia. Collectively, we will provide innovative synthetic RNA-based approaches to modulate local cellular functions and reprogram microglial responses.
2023 -
Grant Awardees - Early Career

Experimentally evolving budding yeast cell size to test scaling laws in cell biology

FUMASONI Marco (.)

Fundacao Calouste Gulbenkian - Lisboa - PORTUGAL

GIOMETTO Andrea (.)

Cornell University - . - .

Genome and organelle sizes vary with cell volume following scaling laws that are thought to reflect physical and molecular constraints on life. Despite cell size spans over several orders of magnitude across the tree of life, individual species are able to maintain a characteristic, narrow range of volumes, following a handful of homeostatic principles. To cover a large range of sizes, scaling patterns were derived by comparing distantly related species, highlighting their conservation but limiting causal inference. On the other hand, much of the work on the mechanisms that maintain size homeostasis was done in single species, which typically cover cell size ranges that are too narrow to investigate scaling patterns. The molecular and physical links that govern size scaling patterns and homeostasis principles, and how they evolve to produce the variability we observe in nature have remained elusive. To overcome previous limitations, we will evolve and engineer a panel of much larger/smaller Saccharomyces cerevisiae cells, aiming at finding the physical or evolutionary limits for this species. We will track genomic and cellular changes associated with the altered size, identify causative mutations, and dissect the mechanisms they act on. Single cell measurements in a genetically amenable species over an unprecedented range of sizes will allow us to test the robustness of homeostasis principles and scaling laws in cell biology, and observe how evolution can alter physical constraints on cell, genome and organelle size.
2023 -
Grant Awardees - Early Career

Unraveling the multi-layer relationship between archaeal symbionts and their viruses

GHOSAL Debnath (.)

University of Melbourne (Melbourne University) - . - .

GOOD Benjamin (.)

The Board of Trustees of the Leland Stanford Junior University - . - .

QUAX Tessa (.)

Rijksuniversiteit Groningen - Groningen - NETHERLANDS

SAKAI Hiroyuki (.)

RIKEN BioResource Research Center (BRC) - . - .

Associations between organisms and viruses are complex and encompass everything from beneficial to parasitic relationships. Here we will map the molecular mechanisms that underlie the interactions between crenarchaea, their DPANN archaeal symbionts and their viruses. Archaea are ubiquitous microorganisms in the environment, but the number of isolated species is modest and their cell biology remains underexplored. They are regarded direct ancestors of eukaryotes and important for understanding the origin and evolution of life. Current knowledge of microorganism–virus interactions relies primarily on laboratory studies of one virus and one cell. Yet, based on culture independent approaches, the fraction of microbial species that is involved in a symbiotic relationship is estimated to be considerable, although most are unculturable. Symbiotic microorganisms sustain relationships with another organism (called the host) over a considerable fraction of the life of the host. In addition, this relationship is beneficial for at least one of the organisms. Since symbionts often lack anti-viral defence systems, it has been hypothesized that symbionts can protect their host-cells from viral infection, by serving as decoy. However, empirical relevance of this hypothesis has not yet been tested. Recently multiple fast-growing symbiotic archaea belonging to the phylum DPANN were isolated by HS, which now offers the exciting possibility to unravel molecular mechanisms underlying viral infection in a symbiotic system. The aerobic fast-growth allows us to apply to this system recently developed imaging techniques. We will combine virology, microbiology, microscopy and structural biology with mathematical modelling to connect the microscopic properties of the individual cells with the emergent behavior at the population level. This will allow us to assess if symbiotic DPANN serve as viral decoys. Viruses of DPANN archaea are not studied, even though these archaea are widespread. With this project, we will generate unprecedented detail of their infection cycles, structure and the biology of their DPANN hosts, which will lay foundations for a novel field of research. This project will be one of the first to study viral infection in a symbiotic system, and thus provide essential insights into the impact of viruses on symbiotic organisms, which are so widespread in nature.
2023 -
Long-Term Fellowships - LTF

Unraveling limb size determinants using mouse-jerboa chimera

GOEL Isha (.)

. - University of Cambridge - Cambridge - United Kingdom

ALBERTO Rosello Diez (Host supervisor)
Background: From a tiny insect to a giant blue whale the diversity in the animal kingdom is truly remarkable. An understanding of this poorly explored variation in size of organisms can provide a more elaborate map of evolution and will be useful in commercial applications such as increasing meat production from specific body parts. Given the complexity of the animal body, it is more practical to start with one organ that can offer a high range of diversity among different species such as the limb. The differences in size and proportions demonstrating their adaptations during evolution open a door to the factors controlling organ size. Two main steps can be distinguished during limb growth: the patterning phase, in which the different limb segments are specified, and the post-patterning phase, where most of the elongation and differentiation take place. Aim: We aim to generate jerboa iPSCs for the first time to study how they form limbs in host mouse embryos. We plan to compare the development of chimeric jerboa-mouse limbs with that of normal mouse limbs so that their intrinsic differences can be found. Central hypothesis: Based on classic and recent studies, we hypothesize that final limb size is controlled by the signal balance during the patterning phase, whereas internal limb proportions are tuned by intrinsic differences in the tempo of the post patterning phase. This predicts that if early limb bud cells are placed within the signal environment of a different species, the limb bud cells will adapt the duration of their patterning phase, leading to a change in the limb size, while the internal proportions will be preserved. This decoupling between intrinsic and extrinsic processes will allow us to identify the key genes and networks involved in the control of limb size. Experiments: The experiments will take place in three stages: 1) Preparation of jerboa iPSCs under the expert supervision of Dr. Rio Tsutsumi, Kyoto University. The jiPSCs will be chemically transformed in four stages and then characterized by RNA-seq and teratome assays for pluripotency. They will be modified for GFP expression 2) Preparation of hindlimb-less mouse model (Hoxb6-Cre; Ctnnb1(flox/flox)) and complementation of its blastocyst with jiPSCs. Litters will be collected to characterize the expression of segment markers Meis1/2, Hoxa11, Hoxa13 by in situ hybridisation, characterize bone length by micro-CT. Morphological and size analysis will be done according to protocols from the Cooper Lab 3) Relative RNA-seq and ATAC-seq analysis of transcriptome and accessible chromatin of limbs. Conclusion: These experiments are designed to generate the first iPSC line from jerboa providing new resources for research. jiPSCs are expected to form most of the hindlimb in chimeric animals. The size and genetic analysis of the developed hindlimb will provide an insight into what aspects of limb growth are controlled by intrinsic mechanisms, and which aspects by extrinsic ones.
2023 -
Grant Awardees - Program

Cellular and molecular basis of bilaterian symmetry

GUIGNARD Léo (.)

Aix-Marseille University (Université d'Aix-Marseille) - . - .

PAVLOPOULOS Anastasios (.)

FORTH Institute of Molecular Biology and Biotechnology (IMBB-FORTH) - Heraklion - GREECE

XIE Liangqi (Frank) (.)

Cleveland Clinic Foundation - . - .

One of the most pervasive, yet poorly understood, hallmarks of most metazoans is bilateral symmetry. The external left and right sides of bilaterians develop separately but somehow manage to produce symmetric matching halves, presumably via deterministic developmental programs, homeostatic buffering mechanisms or combinations thereof. We will probe these mechanisms in a crustacean embryonic epithelial monolayer that acquires a stereotypic, geometrically ordered and bilaterally symmetric tissue architecture composed of less than a thousand cells, while maintaining the capacity to restore left/right symmetry after unilateral cell ablations. Through live imaging with multi-view light-sheet fluorescence microscopy and image analysis of intact and ablated embryos, we will first decompose the shaping of each side into the individual cell contributions to quantify their levels of stereotypy and variability across sides, embryos and conditions during the normal emergence of bilateral symmetry and during its restoration after cell ablation. Guided by these cellular maps, we will then screen for spatially and temporally structured gene networks operating at the beginning, middle and end of the restoration process, using a genome-wide time-course profiling of gene expression and chromatin accessibility with established spatially resolved methods. To probe the regulatory rules of symmetry, we will also develop a new method for dual spatial transcriptomics and epigenomics by converting and amplifying the open chromatin DNA regions into polyadenylated RNA molecules that will be assayed together with cellular mRNA on existing platforms for spatial transcriptomics. We will devise novel computational frameworks to register the cellular atlases from the microscopy datasets with the molecular atlases from the multi-omics datasets in time and space, and to generate average, statistical representations from multiple input embryos. Data integration will enable us to measure at cellular resolution the intraindividual and interindividual variability between sides and across embryos, respectively, and to probe the qualitative and quantitative differences in the corrective molecular and cellular mechanisms that bilaterians have evolved to promote symmetry by buffering the effects of inherent developmental noise (developmental stability) and the effects of perturbation (canalization).
2023 -
Long-Term Fellowships - LTF

Utilizing spatial transcriptomics for uncovering the hypoxic transcriptional landscape of tumors

GUSTAFSSON Johan (.)

. - Broad Institute (Eli and Edythe L. Broad Institute of MIT and Harvard ) - Cambridge - United States

GAD Getz (Host supervisor)
The tumor microenvironment is characterized by a dense extracellular matrix in combination with leaky and irregular vasculature, creating acidic hypoxic tumor regions with small influx of oxygen and nutrients. Most therapies, such as radiation therapy, immune therapies, and chemotherapy are ineffective in these regions due to lack of oxygen, limited drug influx, immune exclusion, and low proliferation rate. Effective ways to target the cancer cells in hypoxic regions are lacking and means to address this problem could potentially improve the clinical outcome for many groups of patients. An important step towards finding effective treatments targeting hypoxic regions is to understand their biology. The arrival of spatial transcriptomics has made possible high-resolution studies of the transcriptional landscape in tumors. In spatial transcriptomics, spots containing 1-10 cells are sequenced and associated with a physical coordinate from a thin tissue section, forming a spatial network of gene expression. Hypoxic regions can be identified using immunofluorescence staining for hypoxic markers, enabling identification of biological traits per level of hypoxia. In this project, we will collaborate with the Human Tumor Atlas Network (HTAN) to obtain spatial transcriptomics data overlayed with hypoxia staining from lung and breast cancer patient samples. The first aim is to establish a method to determine the level of hypoxia per spot based on staining for hypoxic markers, but also from the transcriptomic profiles for use with datasets without staining information. The second aim is to identify regions within the hypoxia range with similar cellular behavior. The third aim is to explore and understand the biology and identify potential drug targets in each hypoxic region. To this end, I intend to use methods such as differential expression analysis, gene set enrichment analysis, genome-scale metabolic modeling, and utilize gene essentiality information from the DepMap database. The fourth aim is to collaborate with the PRISM lab at the Broad Institute, which performs high-throughput screening for drug targets in hundreds of cell lines. To validate our biological theories and provide potential drug targets for hypoxic regions, we will develop a hypoxic setup to test previously identified drug targets as well as perform screening of existing drug libraries for the hypoxic regions previously identified. In this project, I will bring my metabolic systems biology knowledge into the Getz lab and learn about spatial transcriptomics and cancer biology. The results are envisioned to be an atlas over the transcriptional landscape in tumors defined per region of hypoxia and a list of potential drug targets for these regions. These could serve to find a complementary treatment to existing therapies, specifically targeting the hypoxic regions, which could lead to combination treatments with an increased survival chance for cancer patients.
2023 -
Long-Term Fellowships - LTF

Investigating the mechanistic role of the gut microbiome in modulating depressive behavior in mice

GUZZETTA Katherine (.)

. - Swiss Federal Institute of Technology in Zurich (ETH Zurich/ETH Zürich) - Zurich - SWITZERLAND

RANDALL Platt (Host supervisor)
Background: Depression is the leading cause of disability worldwide, creating immense psychological and economic burdens for individuals, families and society. Despite depression’s pervasiveness, the pathology and mechanism of action for treatments remain elusive, and roughly half of patients do not improve with antidepressant use. The high global prevalence, poor treatment efficacy and lack of mechanistic understanding has created a large knowledge gap that needs to be addressed to better understand the disease and help patients. The gut microbiota is an emerging contributor to depression onset and recovery, and can regulate brain function, behavior, and mood of its host by altering the metabolome and immune system. Intriguingly, gut microbial composition is altered in depressed individuals, post-antidepressant treatment and by stress. However, the exact pathways by which these microbes act on the brain, including in depression, remain elusive largely due to an inability to mechanistically interrogate microbe-host interactions. Overall aim: Uncover how the gut microbiota contributes to the development and recovery from stress-induced depressive behavior (initially in mice). Hypothesis: The gut microbiota contributes to stress-induced depressive behavior and amelioration through microbe-host signaling (including modulating host serotonin levels and aryl hydrocarbon receptor immune signaling through microbial tryptophan metabolism), and manipulating these pathways is sufficient to augment depressive-like phenotypes in mice. Experimental Approach: To reveal how stress and antidepressants impact the gut microbiome and physiology of mice, I will analyze behavior and physiology (gene expression, corticosterone/neurotransmitter levels, etc) and use contemporary multi-omics methods (metagenomics/transcriptomics) combined with the novel Record-seq technology recently created by the host laboratory (Schmidt, Nature, 2018; Schmidt, Science, 2022). My bioinformatics analysis will focus on understanding microbe-host pathways altered by stress and alleviated by antidepressant (fluoxetine or probiotic) consumption. I will then functionally validate key findings using CRISPR-based manipulation of top identified pathways in the gut microbiome (with host institutional knowledge) and microbiota transplantation to reveal novel mechanisms by which the gut microbiota contributes to anti-depressive or depressive-like behavior. Conclusions: This research has the potential to shift paradigms for depression research by incorporating novel microbiome-based techniques and centering the gut microbiota in the pathophysiology of depression. This project will expand on my background of the microbiota-gut-brain axis and allow me to pursue new scientific disciplines and techniques, including CRISPR, microbial engineering, bioinformatics, and Record-seq. The use of cutting-edge non-invasive methods (Record-seq) also provides an entry point for follow-up clinical studies.
2023 -
Long-Term Fellowships - LTF

Investigating the cross talk between glioma cells and oligodendrocytes in brain tumors

HAN Jichang (.)

. - Miller School of Medicine of the University of Miami - Coral Gables - United States

ANTONIO Iavarone (Host supervisor)
Glioblastoma multiforme (GBM) is the most common high-grade glioma in adults and has an exceptionally dismal prognosis. Neuronal activity has recently emerged as a critical regulator of GBM progression by regulating membrane depolarization of glioma cells via neuron-glioma synapses or secreting neurotrophic factors from neurons. Oligodendrocytes can wrap around axonal segments, enabling fast saltatory conduction of action potentials and supporting axons' energy metabolism. Whether a functional interaction between oligodendrocytes and cancer cells drives GBM remains unknown. To better understand the interactions between tumor and non-tumor cells, the host lab analyzed larger scRNAseq dataset of GBM to uncover the ligand-receptor interactions from the tumor microenvironment (TME) using a recently developed single-cell tumor-host interaction (scTHI) tool and identified that the oligodendrocytes express high levels of NLGN1 that interacts with NRXN3 expressed by neuronal tumor cells. We propose a model wherein oligodendrocytes directly interact with the tumor cells through ligand-receptor interactions and generate oligodendrocyte-glioma myelin sheaths to promote cancer development. This proposal will uncover the functional downstream consequences of the newly identified ligand-receptor interactions and how they drive glioma progression. A key innovative component of our proposal will be to interrogate the ability of glioma cells to form functional myelin sheaths with oligodendrocytes thus hijacking the normal activities of oligodendrocytes. A diverse range of cutting-edge techniques will be used to address these goals. To distinguish and verify the interactions between tumor and non-tumor cell types in GBM, I will deploy spatial transcriptomic technology and multiplexed error-robust fluorescence in situ hybridization (MERFISH). For these analyses, a panel of genes will be designed based on the host lab scRNA-seq data and cell-type/state annotations, to decipher spatial and functional organization. I will also investigate the ultrastructure of oligodendrocyte-glioma myelin sheaths in mouse models and patient tumors by enhanced focused ion beam scanning electron microscopy (FIB-SEM) technology enabling high-throughput, large volume, and high-resolution 3D imaging. Finally, to elucidate the molecular mechanisms of ligand-receptor interactions governing tumor growth or oligodendrocyte recruitment in glioma, I propose to examine the gene expression profiling and gene set enrichment analysis of ligand-null (CRISPR-KO) versus parental ligand-wild-type in cell lines or genetically engineered mouse models of gliomas. The outcome of this work will unravel the regulation and functional impact of the crosstalk between glioma cells and oligodendrocytes in GBM and provide the key knowledge for the design of innovative therapies. It will also provide the paradigm-shift discovery and functional characterization of myelination as driver of GBM progression.