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Awardees

2022 -
Cross Disciplinary Fellowships - CDF

An extreme approach to biomineralization: biomineral selection by extremophiles

KNOLL Pamela (.)

. - University of Edinburgh - Edinburgh - United Kingdom

COCKELL Charles (Host supervisor)
CARTWRIGHT Julyan (Host supervisor)
In biomineralization, organisms actively build structures (e.g., bones) or initiate a reaction from the secretion of precipitation-inducing chemicals (e.g., stromatolites). Even within extreme temperature and pH environments, bacteria-induced mineral precipitation uses enzyme-mediated reactions and/or controls the surrounding metal redox state to create dense mats of mineral filaments. While inorganic reactions are capable of mimicking these morphologies, such as laboratory grown chemical gardens, biominerals are often composed of mineral phases that are seldom selected in inorganic precipitation. Complicating the understanding of mineral selection in extremophiles versus similar inorganic counterparts is the large variation between their environmental conditions during precipitation. Using temperature/pressure vessels, I will investigate biominerals formed by thermoacidophiles and thermoalkaliphiles and compare their crystal products to those of chemical gardens formed under the same conditions. The goal is to understand the distinction between precipitation in living versus nonliving systems including the modification of crystal structure and habit within the silica-rich environments where both structures are found as well as the effect of metabolic processes on existing inorganic and biomineral structures. Tackling knowledge gaps in extremophiles will provide insights into the mechanisms for biomineralization and survival of these organisms in mineral-rich environments (at toxic levels for most organisms). This work can also pave the way to understanding how life could withstand the harsh conditions of early Earth and current conditions on other planetary bodies.
2022 -
Long-Term Fellowships - LTF

Systematic characterization of SNPs in CREs associated with congenital heart disease

LINDENHOFER Dominik (.)

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

STEINMETZ Lars (Host supervisor)
Genome wide association studies (GWAS) produced a wealth of information on associations between distinct genetic loci and human diseases. Over 90 percent of predicted variants for common diseases are located in the non-coding genome mainly affecting cis-regulatory elements (CREs) like enhancers. Single nucleotide polymorphisms (SNPs) in enhancers can affect gene expression and contribute to disease mechanisms, but for the majority of SNPs their functional contribution to disease is not understood. Current high-throughput methods to study enhancers use CRISPRi to perturb them entirely. This neglects the differential influence of individual SNPs at CREs on disease-relevant gene expression. I aim to establish a method to modify SNPs in CREs in high-throughput using precision genome editing with a targeted scDNA-scRNAseq readout to link variable genomic editing outcomes with disease-relevant gene expression. Screening at this scale will require to advance methods for precision genome editing in mammalian cells, while the single-cell multiomic readout is crucial to precisely link genotype with gene expression profiles due to differential editing efficiencies at distinct loci. I will use this method to systematically test SNPs in CREs associated with congenital heart disease (CHD), a disorder resulting in structural heart abnormalities at birth. CHD is highly heritable and GWAS identified hundreds of CHD-associated SNPs at risk loci in non-coding regions with their function remaining elusive. Screening for these SNPs in human heart organoids will allow us to determine associated gene expression changes systematically and how they contribute to the disease mechanism of CHD.
2022 -
Grant Awardees - Program

Regulation of neuronal physiology by the electromechanical effects of the action potential

LOIS Carlos (SPAIN)

biology and biological engineering - california institute of technology - Pasadena - USA

ROYLE Stephen (UK)

Centre for Mechanochemical Cell Biology - Warwick Medical School - Coventry - UK

SEZGIN Erdinc (TURKEY)

SciLifeLab - Karolinska Institute - Stockholm - SWEDEN

Cell signaling has traditionally been thought to exclusively depend on biochemical processes, but in recent years we have begun to appreciate how physical forces can control cell-cell communication. Mechanical forces generated by cells regulate many physiological processes, including selection of optimal antibodies by lymphocytes, sorting of chromosomes and organelles within the cell, and blood clotting in response to turbulence. Among all cells, neurons and muscle cells have special features because they have excitable membranes capable of generating large voltage fluctuations (~100 mV) known as action potentials. Over the past decades, researchers have accumulated evidence that action potentials generate membrane movements up to 100 angstroms. However, it remains unknown what are the biological consequences of this neuronal electromechanical force. The central hypothesis of this proposal is that the membrane motions associated with action potentials generate mechanical forces that regulate the intercellular communication between neurons and intracellular signaling events within individual neurons. To investigate how the mechanical force of action potentials regulates intercellular communication we will focus on the activation of the mechanoreceptor Notch, a key molecule involved in cell-cell interactions. To explore the effects of the neuronal electromechanical forces on intracellular signalling, we will focus on the regulation of membrane trafficking processes, including endocytosis and sorting of intracellular vesicles. To investigate these issues we have assembled a team with expertise on different aspects of cellular signalling including membrane biophysics, single-particle imaging in living cells, and neurophysiology. A central goal of the proposal is to take advantage of new technologies that can be used to investigate the regulation of cell physiology at several scales of biological complexity, spanning individual molecules, living cells, and transgenic animals. We anticipate that this research will provide unanticipated insights about how mechanical forces control intercellular and intracellular signaling events in neurons.
2022 -
Grant Awardees - Program

Super-resolution multifunctional scanning ion conductance microscopy: tapping the cell's energy grid

MACHESKY Laura (USA)

Migration, invasion and metastasis lab - CRUK Beatson Institute - Glasgow - UK

SASAKI Atsuo (JAPAN)

Department of Internal Medicine / Cancer Energy Metabolism Lab. - University of Cincinnati College of Medicine - Cincinnati - USA

TAKAHASHI Yasufumi (JAPAN)

Graduate School of Engineering Electronics - Nagoya university - Kanazawa - JAPAN

Cells show a remarkable resiliency when energy-starved, maintaining their ability to migrate to a new energy-rich environment, where they can re-purpose their cytoskeleton to take up nutrients via macropinocytosis. ATP and GTP are the major energy currencies of the cell, driving motor activity, cytoskeletal polymer dynamics and signaling. While the cell generally maintains robust global levels of ATP and GTP in nutrient-starved conditions, it is the local concentrations and ratios of ATP/ADP and GTP/GDP that drive motility and signaling. Multiple parameters control ATP/GTP flux, including: -Diffusion -Biosynthesis vs consumption -Shuttling and compartmentation However, how the cell meets energy demand is largely unclear, because we lack tools to measure multiple parameters simultaneously at sub-cellular resolution over time in live cells. Our multidisciplinary team, with expertise in motility (Machesky), metabolism (Sasaki), and Scanning Ion Conductance Microscopy (SICM) (Takahashi) will address this unmet need. SICM offers advantages of measurements without perturbation, correlation with high-resolution confocal imaging and simultaneous measurement of multiple parameters. Our team will thus engineer the first live single-cell multiscale platform, using cellular feedback to guide sampling and stimuli, providing the means to acquire a holistic view of cellular energy flux. We have two main objectives, each with new technology driven by our biological questions. Objective 1. Develop metabolic and protein mapping SICM to discover how cells use membraneless compartmentation to control metabolite supply and demand at the leading edge. We will establish how diffusion, biosynthesis and consumption generate and maintain intracellular nucleotide gradients using subcellular metabolic sampling SICM. We also probe subcellular localization and organization of metabolic enzymes, using femtoliter scale subcellular protein proximity labelling. Objective 2. Develop cell-to-machine feedback loop controlled SICM to determine how a cell decides to walk or to eat Using cell feedback guided SICM, we discover how mechanosensing via plasma membrane tension, integrins and the cytoskeleton, as well as nutrient sensing, allowing the cell to either locomote or use its cytoskeleton to perform nutrient uptake via macropinocytosis.
2022 -
Long-Term Fellowships - LTF

Understanding ancient woolly mammoth gene function through multiplex gene editing

MAJEWSKI Dorothy (.)

. - President and Fellows of Harvard College, Harvard Medical School - Boston - United States

CHURCH George (Host supervisor)
The ability to sequence ancient DNA from extinct species is a relatively recent development in evolutionary biology with the potential to provide direct information on evolutionary pressures spanning tens of thousands of years. However, our ability to functionally characterize these genes is limited to predictions based on orthologues in living ancestors. Using multiplex gene editing techniques currently being developed in the Church lab at Harvard University, I will modify Asian elephant cell lines with multiple candidate mutations simultaneously based on the related woolly mammoth genome. Functional characterization of mammoth genes in the context of elephant tissues can provide insight into cold-resistance adaptations selected for by harsh environmental conditions. Several loci of interest from the mammoth genome will be targeted based on sequence comparison with the elephant genome, specifically aiming to find orthologous proteins altered from a common ancestor over the course of speciation. Multiplex gene editing methods will be optimized for the elephant cell lines, both in immortalized somatic cells and eventually induced pluripotent stem cells. Functional analysis will be performed by a combination of cell-based and biochemical approaches. The technology to modify many genes simultaneously will create a foundation for the long term goal of generating Asian elephant – woolly mammoth hybrid embryos, a part of de-extinction efforts to fill lost ecological niches that can be applied to several species in the future.
2022 -
Long-Term Fellowships - LTF

Feeding or folding? Untangling the ecology of spatial patterning in microbial consortia

MEACOCK Oliver (.)

. - University of Lausanne - Lausanne - SWITZERLAND

MITRI Sara (Host supervisor)
The ecology of microbial communities is characterised by a rich interplay between metabolic and mechanical processes. In sessile communities, these processes typically play out through changes in the spatial structure of the system, such as the strong intermixing of strains that rely on each other’s secretions, or the buckling of a biofilm due to growth-induced forces. However, as species in naturally-occurring communities can differ widely in both their metabolic and physical properties, it has thus far proven difficult to disentangle the contributions of these two types of process during the emergence of spatial structure in multi-species consortia. To address this, I propose a three-pronged course of research, using the Mitri group’s recently characterised four species consortium as a testbed. In stream one, I will develop microfluidic techniques to physically separate different species while allowing chemical exchange between them, isolating those spatial patterns driven purely by metabolic mechanisms. In stream two, I will adapt pre-existing ecological theory to uncover the underlying metabolic interactions necessary to explain these patterns, as well as investigate patterning’s importance in enabling the community to perform a user-defined function such as toxin degradation. Finally, in stream three, I will reintroduce the previously excluded mechanical interactions in a controlled manner, engineering the emergent patterns to improve the community’s ability to fulfil its target function. This project will combine my proven analytical capabilities with the ecological insights of my host group to bring a fresh, more predictive perspective to microbial ecology.
2022 -
Long-Term Fellowships - LTF

Neural basis of relative aversive value coding in mice

MIRANDA Magdalena (.)

. - Institute for Functional Genomics (Institut de Génomique Fonctionnelle, IGF-CNRS) - Montpellier - FRANCE

TROUCHE Stéphanie (Host supervisor)
Absolute (good or bad), but also relative (better or worse), values can be assigned to an experience to guide appropriate actions (approach or avoidance). While previous work mainly focused on appetitive relative value coding, a detailed understanding of the neural mechanisms allowing animals to compare relative aversive value is at its infancy. Additionally, the extent to which value computed during learning is affected by specific internal states to alter value-based decisions remains elusive. The paraventricular thalamus (PVT) is modulated by hypothalamic inputs and also integrates positive and negative emotional states. Moreover, through its glutamatergic projections to the Nucleus Accumbens (NAc), the PVT is therefore ideally positioned to impact value coding and appropriate behavior selection. In this project I will use a novel behavioral task combined with optogenetics and in vivo physiology to examine the neural mechanisms for absolute versus relative value assignment during learning in mice and how internal state affect value signals. By monitoring and manipulating the neural activity simultaneously with dopamine kinetics from genetically defined neuronal populations in behaving mice, this project will investigate 1) the role of the PVT-NAc pathway, 2) the physiological changes of the DA release in the NAc and 3) the effect of acute changes in internal state (e.g. thirst) during both relative and absolute aversive learning and choice. This project therefore aims to build the first functional circuit model in mice for relative aversive value coding and highlights how internal states integrated in the PVT modulate value coding and value-based decisions.
2022 -
Long-Term Fellowships - LTF

Delineating control of translation upon iron starvation

MOLENAARS Marte (.)

. - New York University Grossman School of Medicine - New York - United States

POSSEMATO Richard (Host supervisor)
Iron is the most abundant transition metal in biological systems. Iron and iron-containing cofactors support essential processes like energy metabolism or DNA replication. As with many essential nutrients, cells have mechanisms to sense shortages and alter metabolically intensive processes like translation. My over-arching goal is to gain mechanistic understanding of the translational program dedicated to cope with the iron deprivation. Known mechanisms for iron sensing involve Iron regulatory proteins (IRP) 1 and 2, which have RNA binding domains that bind iron-responsive elements (IRE) upon iron starvation. Known IRP targets are involved in iron metabolism: IRPs bind the 3`UTR of mRNA related to iron uptake or the 5`UTR of mRNAs related to iron storage to stabilize or inhibit translation, respectively. Interestingly, I have found that (1) iron starvation rapidly inhibits mammalian translation globally and (2) novel IRP-bound mRNA species are selectively affected. Here, I will determine how iron sensing pathways regulate translation globally, and delineate which mRNAs are directly regulated by IRPs. Using polysome profiling I will define how mRNA translation rates are affected by iron starvation and determine whether these effects are dependent upon IRPs or other common translational control mechanisms such as mTORC1. I will isolate polysomal RNA and perform RIP-seq of IRP-bound mRNAs upon induction of the iron starvation response pharmacologically or genetically to identify novel transcripts regulated by the IRP system. These findings will have an impact on diseases where iron metabolism is dysregulated, such as Friedrich’s Ataxia, iron storage diseases, or cancer.
2022 -
Grant Awardees - Program

Mental 3D space-time travel in fission-fusion animal societies

MOSS Cynthia (USA)

Dept. of Psychological and Brain Sciences - Johns Hopkins University - Baltimore - USA

PEREMANS Herbert (BELGIUM)

Dept. of Engineering Management - University of Antwerp - Antwerp - BELGIUM

VON BAYERN Auguste (GERMANY)

Dept. of Behavioural Ecology and Evolutionary Genetics - Max-Planck-Institute for Ornithology - Seewiesen - GERMANY

WAHLBERG Magnus (SWEDEN)

Marine Biological Research Center - Dept. of Biology - Kerteminde - DENMARK

Flying and swimming animals can move freely in three-dimensional space, but their ability to use past information to inform future decisions remains largely unknown. Time flows in one direction, and no living creatures can physically move backwards into the past or forwards into the future. Animals can, however, use memories to adapt their behavior in future events. For humans, it is even possible to mentally travel backwards and forwards in time; we can ‘see’ ourselves in past situations and foresee ourselves in future ones. Mental time-travel is regarded as a fundamental property of human success, enabling us to organize societies and travel to the moon. Here we study to what degree other animals possess mental time travel abilities. We study dolphins, bats and parrots, which all travel and forage in 3-D space and live in large groups where they are constantly engage in social interactions, both cooperation and competition. Any degree of mental time travel abilities would be advantageous for them to anticipate the best solutions in the execution of future tasks. We investigate how our model species perceive time, and any evidence for them making use of episodic memory and perform future planning. By using similar experimental approaches for all model species, we compare mental time travel in animals living in vastly different environments and exposed to a large variety of challenges. By comparing the performance of our model species, we can decipher the underlying evolutionary mechanisms and requirements for mental time travel. We will use the results of our investigations to construct robots that exhibit different degrees of mental time travel functions. By studying the success rate with which robots solve problems, and by manipulating the robots’ abilities to mentally travel in time and collaborate, we will aim to pinpoint which mental time travel traits are most pertinent for solving different types of problems. The robot studies not only inform us about how our model species function, but may also inspire the development of robots that successfully solve difficult problems requiring complex decision-making, such as responding appropriately in emergency situation and executing complex construction operations.
2022 -
Long-Term Fellowships - LTF

Neuronal changes following pathogen infection: mechanisms underpinning sickness-related behaviors

MURIA Aurélie (.)

. - University of Bonn (Rheinische Friedrich-Wilhelms-Universität Bonn) - Bonn - GERMANY

GRUNWALD KADOW Ilona (Host supervisor)
When sick, animals undergo adaptive changes related to both infection control and recovery. The molecular and neuronal changes induced by infection, in particular central brain adaptations following pathogen ingestion or injury (peripheral infection), are still poorly understood. The immune system has been proposed to play a crucial role in behavioral adaptations. Characterizing the neuronal changes following activation of peripheral immune signaling would thus permit to better understand how infections impact, in the short and long term, the physiology and function of the brain. The innate immune system of Drosophila is a useful model to assess this question, thanks to its neurogenetic tools. The host lab showed that pathogen ingestion leads to adaptive feeding through immune signaling in neurons and changes in the mushroom bodies. Using in vivo ROS sensing and calcium/voltage whole brain imaging, I will first i) establish a map of neuronal activity and plasticity in flies infected with pathogens (i.e. Ecc15 and Pseudomonas) of various virulence, by oral ingestion or injury (i.e. before, during sickness, after recovery). Through 2-photon imaging and RNAi approaches, I will ii) define the role of the innate immune pathway in the affected brain regions and iii) characterize the immediate and long-lasting impacts of infection on specific neurons and synapses, such as serotoninergic and octopaminergic, in brain regions associated with sickness-related behavioral adaptations, such as feeding. I anticipate that the results of this project will significantly widen our understanding of how immune signaling and infection change the nervous system, in the short- and long-term.
2022 -
Long-Term Fellowships - LTF

Exploring the combinatorial space of plant immune receptors and pathogen signals

OFIR Gal (.)

. - Max Planck Institute for Biology Tübingen - Tübingen - GERMANY

WEIGEL Detlef (Host supervisor)
To defend themselves against pathogens, plants employ an amazing diversity of immune receptors responding to pathogen signatures or to pathogenic manipulation of plant cells. While several major families of immune receptors are known, most of their members respond to unknown signals from unknown pathogens. Better understanding of the immune repertoire against a diverse array of pathogens can shed light on the ecological and evolutionary forces shaping plants’ immune arsenal. I will establish a high-throughput screening pipeline for immune responses to an array of plant viruses. I will first focus on the well-studied NLR immune receptors. NLR genes of unknown specificities will be bioinformatically identified in fully assembled genomes, and will be co-expressed in plant cells with a library of plant viruses. Immune response activation will be monitored using multiple approaches - transcriptome changes, production of reactive oxygen species and signaling molecules, and programmed cell death. In a second step, the proposed pipeline will be used for discovery of novel immune-related genes. Candidate genes will be identified using genomic hallmarks of immune genes – excessive diversity and copy number variation within species, sharing of alleles between species, presence/absence between closely related strains, diversification by unequal crossing over and immune-related protein domain signatures. Candidate genes will then be tested experimentally for immune responses using co-expression with a collection of plant viral infectious clones to discover new antiviral mechanisms in plants.
2022 -
Long-Term Fellowships - LTF

Spatial and temporal scales of serotonin neuromodulation

ÖZÇETE Özge demet (.)

. - Harvard Medical School - Boston - United States

KAESER Pascal (Host supervisor)
Serotonin controls brain functions including mood, affection and reward, and modulating serotonergic activity is used to treat anxiety, depression and other disorders. While serotonin is thought to act as a volume transmitter, the spatial and temporal scales of serotonin transmission are not well understood. At classical synapses, position, timing and extent of neurotransmitter release are controlled by the active zone, a specialized protein complex. I here propose to dissect the protein machinery for serotonin release. I will use superresolution microscopy to identify secretory protein clusters that embody release hotspots in serotonin axons. To assess the spatiotemporal dynamics of serotonin release, I will employ the recently developed fluorescent serotonin sensors in mouse striatum. I will record serotonin release in striatal brain slices and in vivo using imaging and photometry in combination with chemo- or optogenetic control of serotonergic activity. I will next determine whether proteins that mediate spatial precision and rapid release dynamics at synapses, for example active zone scaffolds, fast calcium sensors, and voltage-gated calcium channels, are needed for serotonin release. My host laboratory has developed a series of conditional mouse mutants for this purpose. In summary, I expect to uncover the key features of serotonin release. Building on my experience with synaptic signalling, I will be able to generate new models on the spatiotemporal organization of serotonin transmission. This work will provide insights into the principles of neuromodulation and may offer new targets for modulating serotonergic activity in disease.
2022 -
Grant Awardees - Program

New ways to generate color: light manipulation by crystal-forming pigments

PALMER Benjamin (UK)

Department of Chemistry - Ben-Gurion University of the Negev - Rehovot - ISRAEL

STUART-FOX Devi (AUSTRALIA)

School of Biosciences - The University of Melbourne - Melbourne - AUSTRALIA

TZIKA Athanasia (GREECE)

Department of Genetics and Evolution - University of Geneva - Geneva - SWITZERLAND

Recently discovered organic crystals in the eyes of shrimp exhibit remarkable optical properties, never observed in synthetic materials. Intriguingly, the same crystal-forming pteridine pigments are abundant in the skin of many vertebrates. Pteridines are the dominant class of pigment within the xanthophore pigment cells of reptiles, amphibians and fish; yet the optical properties of crystalline pteridines remain completely unexplored. How do these pigments contribute to vivid skin colors and what is the mechanism of molecular self-assembly? We seek to answer these questions by integrating comparative biology, materials chemistry, optics, molecular evo-devo and transcriptomics, and exploiting opportunities provided by reptilian models. In Aim 1, we will characterize the ultrastructure of pigment cells and the chemical, material and optical properties of crystalline pteridine pigments for multiple dragon lizard species and color forms of the corn snake. We will relate the optical properties of pteridine crystals within xanthophore pigment cells to those of guanine crystals within iridophore pigment cells. Aim 1 will enable us to integrate pigment-structure interactions at different scales of structural organization (cellular ultrastructure and subcellular optics) to produce a comprehensive model of skin color generation in reptiles. By discovering the special optical properties that arise from the crystallinity of certain pigments and their ordered assembly, we will reveal new ways that vertebrates generate vivid colors. In Aim 2, we will elucidate the formation and molecular control of crystalline materials within pigment cells throughout ontogeny in bearded dragons and corn snakes. Currently, we do not know what proteins control crystal nucleation, growth or assembly or which proteins are involved in transport of molecular precursors. Understanding the link between gene and crystal ‘phenotype’ would reveal how organisms exquisitely regulate the formation and properties of molecular crystals. This new and exciting connection between molecular biology and materials science could pave the way for the development of genetic tools to design new synthetic molecular crystals.
2022 -
Long-Term Fellowships - LTF

Genome-wide profiling and targeted editing of chromatin state at double-strand breaks in cancer

PARNANDI Nishita (.)

. - The Francis Crick Institute Ltd - London - United Kingdom

BOULTON Simon (Host supervisor)
DNA double-strand breaks (DSBs) are toxic lesions that drive cancer, aging and neurological disorders. DSBs are predominantly repaired by two competing repair pathways, classical non-homologous end joining (c-NHEJ) and homologous recombination (HR), and the choice between these two pathways is extensively regulated. Exploiting pathway choice forms the basis for several cancer therapies. Yet, there are no genome-wide tools to accurately predict pathway choice at individual DSBs, which limits the efficacy of chemotherapies. Induction of DSBs causes dynamic changes in nearby chromatin that influences repair. Key changes that occur in chromatin state are DSB-induced histone post-translational modifications (PTMs), which then recruit specific DSB repair factors to constrain pathway choice. Identifying histone PTMs near DSB sites could serve as a critical “marker” for pathway choice. However, limitations of current tools hamper our understanding of the correlation between chromatin state and pathway choice. To overcome these limitations, I will develop the first genome-wide high-throughput method for joint profiling of histone modifications and DSBs in the same cell population. I will validate the method by comparing measured chromatin state changes against the expected effects of well-characterized processes such as the cell cycle. Based on the improved understanding of the link between chromatin state and DSB repair pathway choice, I will use dCas9-fusion to engineer chromatin state and direct pathway choice to optimize sensitivity to chemotherapeutic drugs, including PARP and POLQ inhibitors. Ultimately, these results can be harnessed to advance chemotherapies in patients.
2022 -
Long-Term Fellowships - LTF

Molecular mechanism of ERAD-M by in vitro reconstitution

PFITZNER Anna-katharina (.)

. - President and Fellows of Harvard College, Harvard Medical School - Boston - United States

RAPOPORT Tom (Host supervisor)
ER-associated degradation (ERAD) is a quality control pathway clearing misfolded proteins from the ER by retrotranslocation across the ER membrane and subsequent proteasomal degradation. It is linked to many diseases, e.g., cystic fibrosis. Membrane destabilization by a dedicated channel is thought to facilitate translocation of luminal substrates (ERAD-L). Although in vivo studies have evidenced Dfm1 as channel for misfolded transmembrane (TM) proteins (ERAD-M), the molecular details remain elusive. This project aims to study the mechanism of TM protein extraction from membranes and to explore lipid bilayer destabilization as a general mechanism for retrotranslocation. To this purpose, we will reconstitute ERAD-M translocation in vitro and quantify destabilization of lipid bilayers in ERAD-pathways with diverse substrates. First, we will analyze the Dfm1 complex in vivo, before coreconstituting it with substrate (NBD-labeled) into liposomes (Rhodamine-PE) and follow extraction activity by release of Rhodamine quenching on NBD fluorescence. Contribution of functional domains, known interaction partners and substrate ubiquitination will be tested. Secondly, bilayer destabilization of ERAD-L and ERAD-M channels, reconstituted in liposomes, will be quantified using a fluorescent reporter probe for lipid order (FliptR). Influence of lipid composition (lipid shape, bending rigidity) on bilayer destabilization and translocation activity will be evaluated. Our work will not only provide experimental verification of the role of Dfm1 in ERAD-M, but also help to understand how proteins with diverse energetical requirements overcome the thermodynamical barrier to cross membranes.
2022 -
Long-Term Fellowships - LTF

The cellular and developmental genetic mechanisms underlying germline response to climate change

RAJAKUMAR Arjuna (.)

. - Whitehead Institute for Biomedical Research - Cambridge - United States

LEHMANN Ruth (Host supervisor)
Fertility is declining globally for a range of animals including insects, fish, and mammals. Rising temperatures due to climate change are expected to worsen this problem because temperature is known to influence key biological processes including immunity and cellular physiology, and abnormal temperature levels can elicit a maladaptive response. While organisms have evolved the capacity to respond to fluctuating environments in a constant and predictable way, a capacity known as canalization, previous work indicates that the germline is temperature sensitive, where high temperatures induce sterility. We propose to use Drosophila melanogaster as a model to understand the mechanistic basis of temperature-induced sterility through the following aims: 1) Combining developmental genetics with super-resolution microscopy, we will test how temperature affects germplasm assembly and whether intrinsic disordered domains, characteristic of germ granule proteins, are involved in temperature sensing. 2) Using hybrid dysgenesis, a system that generates sterile progeny and is exacerbated by high temperatures, we will test whether piRNA pathway control and transposable element activity change across a temperature gradient. 3) Using Drosophilid populations adapted to high temperatures, we will test if mitochondria purifying selection within the germline is temperature-optimized and whether mitochondria transplantation from adapted to non-adapted individuals can modulate temperature-induced sterility. Altogether this work will further our understanding of the cellular and molecular mechanisms underlying the germline response to temperature and how this may be impacted by climate change.
2022 -
Cross Disciplinary Fellowships - CDF

Understanding and controlling the sub-motors of bacterial rotary nanomachines

RIEU Martin (.)

. - University of Oxford - Oxford - United Kingdom

BERRY Richard (Host supervisor)
The bacterial flagellar motor (BFM) consists of hundreds of proteins that assemble into a transmembrane rotary nanomachine. It propels the flagella that drive bacterial swimming. New structural studies suggest that the motor is itself powered by smaller rotary motors. The latter, called stator complexes, are now thought to be autonomous rotary machines, powered by transmembrane ion flux, that drive the rest of the BFM. They share the structural motif of a pentamer surrounding a dimer (5:2), but it is not yet known whether these complexes really are rotary machines, and if so how rotation drives their functions. Here, we will deliver the stators into Droplets on Hydrogel Bilayers, which will allow single-molecule fluorescence imaging and membrane energization. Coupled to FRET and precise sodiometry based on organic sensors, this will allow us to determine the relation between the ion flux, the conformation and the rotation of the units. Finally, we will decipher their interaction with the whole BFM by fast measurements of their discrete dynamics: attaching gold nanorods to the BFM and following rotation in vivo by polarization microscopy, with angular resolution of a few degrees at sub-microsecond timescales. This project will be the first to characterize the molecular mechanisms behind the activation and the dynamics of the sub-motors of the BFM, using advanced techniques from bilayer biochemistry and single-molecule fluorescence, and will bring a new understanding of large rotary machines. It will also open avenues for synthetic biology, making important steps towards the synthesis of artificial cells endowed with the ability to self-propel.
2022 -
Long-Term Fellowships - LTF

Revealing the control of epithelial mechanics during wound healing using in vivo force manipulation

ROGALLA Svana (.)

. - Instituto Biofisika, Basque Centre for Biophysics (UPV/EHU, CSIC) - Leioa - SPAIN

SOLON Jerome (Host supervisor)
Wound healing is a dynamic process in which a living organism replaces lost or damaged tissue based on molecular, hormonal and cellular responses. Understanding the mechanics behind the cellular responses during wound healing processes is a key question in biology and biomedicine, however, the contribution of tissue mechanics (setting tissue deformation upon forces) to wound healing in live animals remains poorly understood. In this project, I will use live Drosophila melanogaster embryos as a model system to reveal how changes in epithelial mechanics are controlled during wound repair. Combining Drosophila genetics with laser dissection, and a novel procedure allowing the application of controlled forces on a single magnetic particle embedded within the tissue in vivo, I will measure the changes in epithelial mechanics within the wounded tissue and link these to specific wound healing regulatory signals. The project will comprise three consecutive stages: 1) I will measure changes in mechanical forces during the different phases of wound healing and correlate them with activation of biochemical signals, such as Hippo and JnK pathways. 2) Based on these measurements and in collaboration with theoretical physicists, I will create a biophysical model of wound healing processes. 3) I will investigate the effect of ectopic forces on the kinetics of wound healing and on cell response, particularly focusing on potential modulation in gene activity by mechanical perturbation. Altogether, these experiments will allow to reveal the control of wound healing in vivo encompassing mechanical and biochemical signals and the resulting changes in epithelial mechanics and cellular forces.
2022 -
Long-Term Fellowships - LTF

Deciphering the nature of genomic conflict using locus-specific chromatin perturbation and capture

RUDNIZKY Sergei (.)

. - Johns Hopkins University School of Medicine - Baltimore - United States

HA Taekjip (Host supervisor)
Chromatin modulates DNA accessibility and hence serves as a fundamental layer in the complex regulation of gene expression and DNA integrity. However, the role of chromatin organization in resolving a genomic conflict between two seemingly competing processes – transcription and DNA damage response (DDR) is not clear. This stems from the dynamic nature of chromatin that is transcribed and repaired in an asynchronous and position-dependent manner. Moreover, locus-specific purification of mammalian chromatin, usually found with only two copies per cell, presents a major challenge regarding the necessary assay specificity and sensitivity. To overcome these limitations, I will develop a single-molecule (SM) pulldown approach based on multi-step enrichment with CRISPR Cas9 and Cas12a, followed by Oligopaint to capture and dissect any chromatin locus of interest. Using light-activated vfCRISPR, I will introduce lesions at transcriptionally active or silent regions to induce DDR with high spatiotemporal control. Captured chromatin will be characterized by SM and super-resolution imaging regarding its composition and organization and will be used as a native substrate to access the dynamics of transcription and DNA repair factors in real-time. Coordination between transcription, DDR, and local chromatin will be examined by guiding vfCRISPR to the promoter and gene body of the heat-shock induced Hsp70 model gene. The developed method can be expanded to mouse models and can be broadly applied to genomic regions of high relevance to health and disease, providing an exciting opportunity for understanding the fundamentals of transcription and DDR in vivo at unprecedented resolution.
2022 -
Long-Term Fellowships - LTF

Are endothelial cells regulated differently during limb regeneration than during development?

SAVAGE Aaron (.)

. - President & Fellows of Harvard College - Cambridge - United States

WHITED Jessica (Host supervisor)
Biological question My question is: how is vascular regrowth regulated during limb regeneration? I aim to understand— at a precise molecular, genetic, and cellular level— how vascular regeneration is regulated in axolotls; whether by angiogenesis or novel mechanisms remains unclear. Scientific content This multi-disciplinary project spans developmental biology, genomics, and regenerative medicine, combining cutting-edge transcriptomic, imaging, and molecular biology practices analyze vascular regeneration in axolotl limbs. Specific aims: 1) Generate the first axolotl vasculature map, using CRISPR knockin-generated transgenics, contrasting native vascular limb patterning to post-regeneration limb patterning; 2) Analyze regenerating vascular cells transcriptionally to elucidate key genes/pathways, using single cell RNA sequencing; 3) Perform in vitro and in vivo assays (morpholino/CRISPRi knockdown; mutagenesis, etc.) to characterize how interplay between key genes/pathways regulates how blood vessels and other tissues, such as fibroblasts interact, informing limb regeneration. Novel transgenics will express both vascular-specific fluorescent proteins and the TVA receptor, enabling visualization of expression and spatiotemporal delivery of molecular modulators, for precise control of gene expression. Significance To understand limb regrowth, it is essential to understand complex interactions between different cell types during regeneration. This project will pioneer characterization of regenerating vessels, informing both wider understanding of limb regeneration and how vessels can be reactivated, generally, to expand in response to injury and disease.