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2018 -
Grant Awardees - Program Grants

Integrating mechanotransduction in development: how does cell shape dictate chromatin remodeling?


Institut de biologie moléculaire des plantes - CNRS UPR 2357 - Strasbourg - FRANCE


Laboratoire Matière et Systèmes Complexes - Unité Mixte de Recherche 7057 - Paris Cedex 13 - FRANCE


Department of Environmental and Life Sciences - School of Food and Nutritional Sciences - Shizuoka - JAPAN


Sainsbury Laboratory - University of Cambridge - Cambridge - UK

Plant development and growth are linked to cellular shape changes, which are controlled by genetic programs but also by perception of environmental signals, including mechanical cues. While both genetic regulation and mechanical control of morphogenesis were studied independently, there is a need to explore how cellular shape-associated strain and stress can mechanically regulate gene expression during differentiation. In animals, mechanical stimuli are known effectors of differentiation. They involve propagation of mechanical forces through the cytoskeleton to the nucleus, leading to chromatin remodeling and modification of gene expression. Thus, nuclear envelope proteins that control nuclear shape and transmit forces to chromatin play a key role in rapid triggering of gene expression. In plants, less is known about mechanotransduction from cell surface to the nucleus.
Using a systems biology approach and an interdisciplinary network, we propose to investigate how mechanical cues affecting cellular shaping are sensed at the nuclear envelope to drive chromatin remodeling in Arabidopsis. We will sudy a unique cellular model, the single root hair in an epidermal tissue context, with well-defined morphogenetic programs linked to cytoskeleton and nuclear dynamics. We will analyze root hair formation and growth in WT and mutants affected in either root hair development or nuclear shape. Combining in vivo live imaging and micro-mechanical measurements (rheometry), we will evaluate mechanical properties of cells and nuclei during root hair development and their dependence on cytoskeleton and nuclear dynamics in relation to gene expression. We will also determine how these mechanical, structural and biological properties are modified when a controlled mechanical stress is applied to the root hair cell during development. Our data will highlight proteins involved in mechanosensing, and we will evaluate their interaction with the nuclear envelope network. Live imaging and rheometry data will be correlated to finite element modeling to estimate strain and stress in the system for predicting chromatin remodeling following cellular and nuclear shape changes.
Altogether, this will highlight the molecular networks involved in mechanosensing at the nucleo-cytoplamic interface and reveal how gene expression is robustly regulated during cellular morphogenesis in higher plants.

2018 -
Cross Disciplinary Fellowships - CDF

Non-invasive deep brain stimulation and imaging using near-infrared upconversion nanocrystals


Helen Wills Neuroscience Institute - University of California, Berkeley - Berkeley - USA

FOSTER David (Host supervisor)
JI Na (Host supervisor)

Non-invasive transcranial stimulation and imaging of targeted neurons deep in brain are a central goal of neurobiology and related engineering science. Optical techniques, such as imaging and optogenetics, have revolutionized experimental neuroscience but are limited by the inability of visible light to penetrate deep into brain tissue. Here I propose an approach for transcranial near-infrared (NIR) deep brain stimulation and imaging by taking advantage of upconversion nanoparticles (UCNPs). UCNPs are lanthanide-doped nanocrystals unique in their ability to absorb tissue-penetrating NIR light and convert into emission in both visible and NIR spectral ranges. First, I will demonstrate UCNP-mediated optogenetics for deep brain stimulation, where UCNPs will be employed to convert transcranial NIR irradiation into visible emission to stimulate rhodopsin-expressing neurons in mouse brain. Second, I will develop blood-brain barrier-crossing UCNPs for targeted imaging of molecular markers in deep brain. For proof of concept, UCNPs will be conjugated to curcumin, a specific binder to beta amyloid - a molecular marker of Alzheimer’s disease, therefore allowing transcranial imaging of amyloids by UCNPs’ NIR emission. Finally, I will integrate the dual functions of UCNPs in deep brain imaging and stimulation, by taking advantage of their simultaneous NIR and visible emission, to achieve transcranial diagnostic imaging and optogenetic treatment of neurological diseases in model mice. The new techniques developed here will allow for non-invasive brain stimulation and imaging without the need for implants, and hold potential for next-generation remote neural therapeutics.

2018 -
Long-Term Fellowships - LTF

The role of bacterial symbionts in development and homeostasis of the immune system


Microenvironment and Immunity - Institut Pasteur - Paris - FRANCE

EBERL Gerard (Host supervisor)

Pattern recognition receptors (PRRs) of the innate immune system recognize structures that are shared by a large group of microbes. Originally posited as the initiators of immunity recognizing pathogen-associated molecular patterns (PAMPs), it was realized that PRRs also detect structures on symbionts which were therefore termed microbe-associated molecular patterns (MAMPs). In addition to initiating inflammation, MAMPs are also involved in the maintenance of symbiosis and in the development and regulation of the immune system. Given the very large microbiota resident in the intestine, a significant amount of intestinal MAMPs penetrates the circulation and modulate diverse aspects of immunity and physiology. For this project, we have developed a transgenic mouse system to control the expression and delivery of a MAMP into the host and assess its impact on immunity and physiology. We will assess the molecular mechanisms governing MAMP detection in the host and impact on development of immunity both basally to maintain homeostasis and during disease. As early life exposure of MAMPs likely modulates effects later in life, we will determine if fetal and neonatal exposure to MAMPs imprints the host. Moreover, as the penetration of MAMPs into the host is normally controlled by the innate immune system, we will use transgenic mice lacking this compartment to assess how innate immunity modulates physiology through the control of MAMPs. We thereby aim to determine the impact of MAMPs on the host, and the threshold levels of MAMPs beyond which immunity and physiology cross into pathology. This study will open the way for the use of MAMPs as modifiers of human physiology.

2018 -
Long-Term Fellowships - LTF

Unraveling co-evolution of multiple pathogens in humans using genomics and “big data”

DATTA Manoshi (USA)

Department of Biology - Technion, Israel Institute of Technology - Haifa - ISRAEL

KISHONY Roy (Host supervisor)

While microbial pathogens evolve during infection to adapt to the human environment and overcome antibiotic treatments, little is known about how adaptation is affected by other co-infecting species. Recent advances in whole-genome sequencing and phenotyping of microbial pathogens derived from human hosts during infection reveal that bacterial pathogens can diversify quite rapidly within a single patient, accumulating mutations that allow them to adapt to challenges presented by the human environment, as well as to overcome antimicrobial treatments. Yet, pathogens within the human body often do not evolve alone. Indeed, many complex infections, especially long-term chronic infections, involve multiple co-infecting pathogenic species within the same patient. In such polymicrobial infections, interactions between pathogens can determine virulence and alter the nature of evolutionary adaptation for each species. As an HFSP Fellow, I will investigate how interspecies interactions influence pathogen evolution in patients. First, I will focus on the cystic fibrosis lung, a canonical site of polymicrobial infections. Second, I will expand to a wide range of infections, leveraging clinical data from >2 million patients. This research will improve our ability to predict how pathogens will evolve in the human body and to design new strategies to thwart their evolution.

2018 -
Cross Disciplinary Fellowships - CDF

Protein liquid-liquid phase separation as a mechanism of peroxisome matrix membrane transport


Dept. of Biochemistry - Université de Montréal - Montreal - CANADA

MICHNICK Stephen W. (Host supervisor)

Proteins in the cell can undergo liquid-liquid phase separation (LLPS), forming liquid droplets - the so-called “non-membranous organelles”. The Michnick group has shown that protein liquid droplets generate work to drive membrane invagination in clathrin-mediated endocytosis. I propose an entirely new role for protein liquid droplets in membrane biology: protein transmembrane transport. My studies will focus on exploring a molecular-level mechanism of protein import from the cytosol to the peroxisome lumen, a mysterious process that occurs without the need of proteins being unfolded. Although this process is crucial to peroxisome biogenesis, its mechanisms remain unknown, due to lack of evidence of detectible transmembrane channels in these organelles. The Michnick group has evidence that two of the proteins involved in peroxisome protein translocation (Pex5 and Pex13) have both transmembrane domains and sequences that favor LLPS. My hypothesis is that these proteins form liquid droplets inside the peroxisome membrane (i.e. between the lipid bilayer leaflets), which would act as a conduit for transmembrane protein transport. After being recognized by a C-terminal peroxisome-targeting sequence, the cargo proteins could then dissolve in the droplet and emerge on the other side of the membrane. My studies will employ the budding yeast S. cerevisiae and will mainly use genetic manipulation with super-resolution and electron micrographic methods to characterize liquid droplets. Further spectroscopic and biophysical methods (e.g. fluorescence recovery after photobleaching, FRAP) will be used to assess protein dynamics inside the droplets and transmembrane protein transport.

2018 -
Grant Awardees - Program Grants

Protein nanocages as single molecular reactors to understand biocatalysis in crowded environments


Física de la Materia Condensada C-III - Universidad Autónoma de Madrid - Madrid - SPAIN


Australian Institute for Bioengineering and Nanotechnology - The University of Queensland - St Lucia, Brisbane - AUSTRALIA


Dept. of Chemistry - Indiana University - Bloomington - USA

Enzymes catalyze molecular transformations in living systems while being pushed and squeezed by other biomacromolecules. This molecular crowding generates excluded volume effects that result in large quantitative consequences on the rates and the equilibria of enzymatic activity by molecular mechanisms that are not yet fully elucidated. To recreate these features, we propose packing tens to hundreds of enzymes within bacteriophage capsids to create nanoreactors (NRs). Our nanoreactors allow control over packing density, protein environment, and boundaries at the nanoscale. Regulation of these conditions is key for understanding in vivo enzyme behaviour, and facilitating metabolic engineering efforts. We will focus on the mevalonate (MVA) isoprenoid pathway, which, despite demanding enzymatic cascade reactions, is widely used for production of industrially useful biochemicals. Limitations may be related to the lack of proximity between partner enzymes, unfavourable interactions under molecular crowding conditions, and/or unfavourable molecular ratios between cascade enzymes. Here we pursue an integrated study to produce materials where MVA enzyme packing and stoichiometry are controlled to mimic the complexity of the intracellular environment. The kinetics of enzymes within NRs will be measured using both ensemble and single-molecule techniques. The enzymatic activity of single nanoreactors will be monitored using a combination of atomic force and fluorescence microscopies, which will allow the in situ study and manipulation of single NRs in real time to provide individualized details not accessible with averaging ensemble approaches. These studies will be supported by multiscale modelling of the crowded enzyme environment. Our experiments will reveal the optimum biophysical conditions for maximizing metabolic flux in the MVA pathway. Further, designed NRs will be assembled and assessed within living cells. Our project has both fundamental and applied aims. First, to understand how crowding affects enzymes at the nanoscale, including metabolite diffusion and protein conformational changes. Second, to improve the biosynthesis of isoprenoids in biological and industrial systems.

2018 -
Long-Term Fellowships - LTF

Defining the key regulators of cell fate decisions by single cell co-assays


Department of Genome Sciences - University of Washington - Seattle - USA

SHENDURE Jay (Host supervisor)

In mammalian development, a zygote gives rise to three germ layers and ultimately hundreds of cell types. How a single genome drives the cell fate decisions underlying development remains one of the most intriguing questions in biology. Extensive work over recent decades has identified some 'master regulator' transcription factors (TFs) that specify certain lineages upon expression, e.g. MYOD for muscles. However, for many lineages the TFs and regulatory regions governing cell type specification remain in the dark. In addition, the sequence of events that leads to open and TF-bound chromatin at developmental enhancers is unknown. These cell fate decisions inherently occur in heterogeneous cell populations, making them difficult to study with bulk techniques. The ability to simultaneously measure changes in chromatin accessibility and gene expression in each of many single cells, coupled to cell lineage information, would provide unprecedented insights into mammalian development. Here I propose to develop and apply such single-cell ‘co-assays’ using mouse embryoid bodies (EBs) as a model system. This will allow me to identify the TFs and regulatory regions involved in early mammalian lineage specification. I will then genetically perturb candidate genes and characterize the effects on lineage establishment. In addition I propose a systematic screen to identify lineage-specifying TFs that serve as ‘pioneer factors’, capable of accessing binding sites in previously closed chromatin. The approach developed in this easily accessible and perturbable system will provide unique insights into intricate biological processes in the future, from in vivo mouse development to disease.

2018 -
Career Development Awards

A new epitranscriptome mark - deciphering function and mechanism of N1-methyladenosine (m1A) in mRNA


Cancer Research Center - Sheba Medical Center / Tel-Aviv University - Tel-Aviv - ISRAEL

The primary sequence of mRNA transcripts contains essential regulatory information in the form of internal chemical adducts, coined the epitranscriptome. Groundbreaking studies of N6-methyladenosine (m6A), by us and others, revealed that this abundant mRNA modification participates in many aspects of the mRNA life cycle to affect cellular, developmental and disease processes. It does so primarily through recruitment of specific binding proteins that channel modified transcripts to various processing pathways and fates. I recently identified a new mRNA modification, N1-methyladenosine (m1A), which occurs on thousands of human and mouse transcripts. It is strongly enriched at the 5’UTR and positively correlates with translation. These distinct features, together with its evolutionary conservation and dynamic nature, suggest a fundamental role in gene expression. The objective of this proposal is to establish the function of m1A in mRNA metabolism, focusing on 5’UTR and translation, and delineate the mechanisms through which it is executed. Aim 1 will systematically identify m1A-binding proteins that will be characterized to expose pathways controlling methylated transcripts. Aim 2 will functionally interrogate the effect of individual 5’UTR m1A sites on translational output in vitro and in vivo (now made possible by method advancement in my lab allowing robust mapping of m1A with single-base precision). Aim 3 will combine results of Aim 1 and 2 to mechanistically dissect the combination of factors and sequence features required for translation initiation. This study is expected to expose a new and central player in translation regulation affecting thousands of genes.

2018 -
Grant Awardees - Program Grants

Probing persistence paradigms: synthetically, immunologically and ecologically


Institute of Virology - Charité – Universitätsmedizin Berlin - Berlin - GERMANY


Center for Vaccine Research - University of Pittsburgh School of Medicine - Pittsburgh - USA


MRC-University of Glasgow Centre for Virus Research - University of Glasgow - Glasgow - UK

As intracellular, obligate parasites viruses rely on a diversity of strategies to survive. Some, like most RNA viruses, cause acute “hit-and-run” infections and require large susceptible host populations. Others, mostly DNA viruses, persistently infect their hosts. Morbilliviruses, like measles virus (MV) and canine distemper virus are examples of RNA viruses that can cause persistent infections. The exceptional circumstances which converge to drive RNA virus persistence remain unclear, since these viruses need to replicate continually in vivo. Bats are evolutionary and ecologically unique mammals standing out in their ability to tolerate viral infections. We recently showed that bats host major mammalian paramyxoviruses and described an ancient morbillivirus in vampire bats. This discovery provides a unique opportunity to explore RNA virus persistence in an important reservoir host that has high rates of contact with humans and domestic animals through its blood feeding habits. The primary objective of the proposed research is to unravel fundamental mechanisms that govern morbillivirus persistence and understand the consequences for immunity and infection. We hypothesize that within-host persistence in canonical morbilliviruses is a vestigial relic of a more common strategy that evolved in bats. Research activities comprise a multidisciplinary program involving investigations of natural and experimental infection of bats, reverse genetics to resurrect viruses from infectious clones, comparative studies of canonical morbilliviruses and hosts together with investigation of pathobiology and immunity underpinned by mathematical modeling. Existing materials, a unique field network for long-term monitoring vampire bats, novel approaches and critical insights from systematic field and laboratory studies make this project both exciting and feasible. This study represents an unusual integration of approaches and ideas from disease ecology, evolutionary biology and synthetic biology. We expect this fusion to resolve fundamental knowledge gaps in our understanding of how RNA viruses persist in hosts; a critical component for the understanding of rare disease manifestations, the consequences of viral persistence on the host immune system and for identifying patterns in risk of virus transmission between species.

2018 -
Long-Term Fellowships - LTF

Mechanisms of radiation resistance in neural stem cells


Physiology and Metabolism Laboratory - The Francis Crick Institute - London - UK

GOULD Alex (Host supervisor)

Protecting genome integrity from environmental stresses is essential for the stable propagation of genetic information from stem cells to their daughters. The most important significance of this work is to add to our basic knowledge of how some cell types, especially neural stem cells, are able to cope with ionising radiation damage better than other cells. The two main aims of this project are to identify the cell-autonomous mechanism of radiation resistance in neuroblasts and to determine the non-cell autonomous role of the stem cell niche in neuroblast resistance. To pursue both aims, I will use a combination of confocal microscopy with molecular markers, cell-type specific genetic manipulations, genetic screens and in vitro stem cell cultures. Using the power of fruit fly genetics to get at some of the underlying mechanisms for neural stem cell radiation resistance may also help to provide us with tools for manipulating the resistance process in many different cell types. The findings of this project will have long-term implications for improving our understanding of how stem cells react to challenging environments and the inputs they receive from their niche.

2018 -
Grant Awardees - Program Grants

Handling OXPHOS structural heterogeneity and metabolic plasticity


Myocardial Pathophysiology area - Centro Nacional de Investigaciones Cardiovasculares Carlos III (FSP) - Madrid - SPAIN


Systems Pharmacology and Experimental Therapeutics - University of Pennsylvania - Philadelphia - USA


Dept. of Biology - Westfälische-Wilhelms-Universität Münster - Münster - GERMANY


Institute of Anatomy - University of Bern - Bern - SWITZERLAND

The OXPHOS system is the only process in animal cells with components encoded by two genomes, maternally transmitted mitochondrial DNA (mtDNA) and biparentally transmitted nuclear DNA (nDNA). The protein products of both genomes have to physically assemble with their counterparts to build functional respiratory complexes. Therefore, variability in the OXPHOS encoded genes is limited by a physical match constraint. This imposes a close-fitting co-evolution of both genomes challenged by the very different mechanism to generate variability for nDNA (by sexual reproduction, mutation and co-existence of two alleles) and mtDNA encoded OXPHOS genes (by mutation, polyploidy and segregation). Since the simultaneous co-existence of alternative mtDNA encoded alleles for OXPHOS proteins has been shown to be detrimental for the organism, we postulate that the co-expression of nuclear encoded alternative alleles may have similar adverse consequences, and that specific regulatory mechanism prevent them. Indeed, random mono-allelic expression is not rare and was suggested for about 30 OXPHOS genes in mice. We postulate that is part of a sophisticated and multi-level system of quality control and functional testing to select for the best combination for providing metabolic plasticity.
Potential regulatory mechanisms are: selective transcription of one allele per cell or selection of the expressed alleles at the import or assembly of the OXPHOS complexes. For testing this hypothesis, we integrate different skills for the analysis of mitochondrial functional and genetics profiling (Enriquez), single cell transcriptomic (Eberwine) and functional and dynamic analysis (Busch) in mouse (Enriquez) and zebrafish (Mercader). If confirmed, we will set the ground for a novel theory of genetic interaction for OXPHOS function. If discarded, the existence of mtDNA and its particular way of inheritance would be necessary to group those genes for which allelic variability is detrimental.

2018 -
Grant Awardees - Program Grants

Sleep, the clock, and the brain: a neuromathematical approach


Dept. of Mathematics - University of Michigan - Ann Arbor - USA

UEDA Hiroki R. (JAPAN)

Dept. of Systems Pharmacology - Graduate School of Medicine - Tokyo - JAPAN

BROWN Steven A. (USA)

Chronobiology and Sleep Research Group - University of Zurich - Zurich - SWITZERLAND

One of the most basic aspects of sleep is that it happens at a particular time of day. Neuroscientists have known for almost half a century that this consolidation requires the suprachiasmatic nuclei of the hypothalamus (SCN); beyond this point, the circuit remains untraced. Equally mysterious, healthy young humans sleep in a single consolidated bout, while infants and older individuals, as well as laboratory mice, can have highly fragmented sleep. Informed by a combined cellular and circuit-based model for slow-wave or “deep” sleep (Forger Group), we propose to trace the signals leading from SCN to cortical slow waves at both physical and molecular levels, and then manipulate them to artificially consolidate sleep. Starting from cortex, aided by a novel “triple-CRISPR” approach to generate knockout mice efficiently in a single generation, we shall examine roles of individual cortical ion channels and the signaling pathways they regulate in creating these slow waves both in vivo and in newly developed whole-brain culture (Ueda Group). This information will be incorporated into a cellular and then a thalamocortical model of slow-wave sleep (Forger Group). In parallel, synaptic tracing in cleared brain, as well as calcium imaging correlation analysis using miniature skull-mounted microscopes, will establish physical and functional connectivity from SCN to cortex (Brown Group), which can also be tested in whole-brain culture (Ueda group). Modeling these data comprehensively and then optimizing this whole-brain model (Forger group), we can predict and test molecular sources of homeostatic and circadian influence upon sleep, as well as combined transgenic and optogenetic strategies to create consolidated bouts of sleep from the normally fragmented sleep of the mouse (Brown and Ueda groups). In this way, by using the power of large-scale quantitative modeling to explore synergies in sleep-dependent signaling, we hope to provide a starting point for novel multimodal therapies with the capacity to fundamentally alter the sleep-wake landscape.

2018 -
Grant Awardees - Young Investigator Grants

Evolutionary puzzles: Do microbes in the Atacama Desert harvest UV as an energy source?


Dept. of Bioengineering - University of California-Riverside - Riverside - USA


Dept. of Planetology and Habitability - Centro de Astrobiología, CSIC-INTA - Madrid - SPAIN

Melanin is a unique molecule synthesized by all domains of life (bacteria, archaea, and eukarya), but it is most commonly known as a UV screening pigment in humans and other animals. However, surprisingly little is known about the secondary or tertiary roles of the molecule. The recent discovery that melanin-containing fungi located at the ruptured Chernobyl reactor display enhanced growth in the presence of gamma radiation, which is usually thought to be harmful, has hinted at the diverse functions of the molecule. A defining feature of melanin is the chemical and structural variability found across and within microbial species. This feature along with the ability to broadly absorb over the UV and visible spectrum has led to the hypothesis that microbial cells could have evolved to capture and utilize this energy. Low resource environments such as those found in deserts are the most probable habitats in which these adaptations could exist. The main objective of this proposal is to assemble a diverse team of scientists to apply new single cell and single molecule technology to studying divergent biology in one of the most extreme environments on the planet, the Atacama Desert.
Here we test the hypothesis that melanin is a key player in producing biochemical energy in microorganisms that are exposed to high amounts of radiation in the UV range. As part of the project, our international team, consisting of engineers, biochemists and microbiologists, will carry out a comprehensive analysis of the microbes living in the Atacama Desert, one of earth’s most UV-irradiated regions. We will identify alternate functions of melanin using state-of-art technologies to 1) sort out and identify melanin producers; 2) sequence their DNA for melanin pathway analysis; 3) characterize how melanin is being synthesized; and 4) link melanin to the photochemical conversion of UV light. Since microbial communities and melanin represent mixed populations, technology is needed which can probe individual cells and molecules. Only once this is accomplished, can sub-populations within each group be thoroughly examined. The impact of understanding melanin not only extends the fundamental knowledge of a commonplace pigment, but also can also prove useful for searching for life on other UV-irradiated environments such those on other planets.

2018 -
Long-Term Fellowships - LTF

How are genes born de novo and integrated into cellular systems?


Department of Biology - Massachusetts Institute of Technology - Cambridge - USA

LAUB Michael T. (Host supervisor)

Organisms must innovate to survive. While adaptation often occurs via mutations in existing proteins or through protein domain shuffling, evolution sometimes must invent functional proteins de novo. Such proteins can arise when organisms acquire the ability to express a short open reading frame encoding a peptide of benefit. Although nearly a third of the genes in many genomes are predicted to have emerged de novo, the mechanisms and constraints governing gene birth remain mostly elusive. During my post-doctoral studies, I will use the molecular, biochemical, and next-generation sequencing-based approaches that are fully accessible in the Laub lab to study how genes emerge during evolution and become integrated into cellular processes. I will develop an approach that explores the link between environmental challenges, existing cellular systems, and de novo gene birth – a largely uncharted area. Specifically, I will (i) screen for beneficial and deleterious randomized protein sequences under various conditions, (ii) characterize those sequences to reveal the molecular mechanisms behind their cellular effects, and (iii) generate models that identify and predict the potential functions of natural de novo genes. This project will provide insight into how cellular innovation arises and how the existing complexities of the cell shape such innovation.

2018 -
Cross Disciplinary Fellowships - CDF

In vivo voltage imaging of cortical circuits


Chemistry and Chemical Biology - Harvard University - Cambridge - USA

COHEN Adam E. (Host supervisor)

Neuronal activity consists of electrical transients on the millisecond timescale. Recently developed genetically encoded voltage indicators (GEVIs) have potential to record optically those transients in thousands of neurons simultaneously. Despite great efforts and improvements on the GEVIs, many technical challenges must be overcome for GEVIs to being used in large-scale recordings in live animals.
Here, we propose to develop and implement new imaging modalities to enable recordings of voltage dynamics from neuronal networks in layer 2/3 of the mouse cerebral cortex in vivo. As a voltage indicator, we will use a recently developed Archaerhodopsin mutant, called 'NovArch', offering optical sectioning by nonlinear activation. We will use protein engineering to further improve trafficking of NovArch to minimize background fluorescence, and we will co-express it with an actuator to allow for simultaneous optical stimulation.
We also plan to develop a microscope specifically optimized for voltage imaging in vivo. It will contain adaptive optical elements to direct optical excitation to the two-dimensional cell membranes where the GEVIs reside, thereby minimizing illumination of non-signal-bearing regions of the sample. This approach will provide high signal levels, while minimizing light scattering and photodamage. We will use this novel microscope to map neural activity in cortical sensory circuits under conditions of sensory stimulation in vivo. The recordings will probe the precise activity levels, sub-threshold voltage dynamics, and intercellular correlations in activity of genetically defined neurons. These data will aid in constraining models of cortical function.

2018 -
Cross Disciplinary Fellowships - CDF

Quantitative in situ liquid cell transmission electron microscopy of microtubules


Department of Chemistry - Northwestern University - EVANSTON - USA

GIANNESCHI Nathan (Host supervisor)

Microtubules form the basic structure, and transport systems in cells, responsible for fundamental processes including cell division, and hence are important drug targets for diseases including cancer, with common taxane-based drugs having them as their biological target of action. Broadly, one of the mysteries of cell biology is the basic set of properties involved in the seed and growth of microtubules and the processes underlying how these systems are initiated. New avenues for controlling cell development and division, possibly related to anticancer treatments will come with a fundamental understanding of the basic mechanisms that lead to unusual switching behavior – dynamic instability. After decades of studies, both in cells and in reconstituted systems there are many open questions about this fascinating behavior. The major bottleneck in exploring microtubules is that the process of seeding is short-lived and the molecular species are structurally very small making standard microscopy observations very difficult, especially for capturing dynamics. In situ liquid cell transmission electron microscopy (LCTEM) has the potential to solve this crucial problem. To enable these kinds of experiments, the microtubule growth, and concentration need to be optimized for confinement within the liquid-cell. Imaging conditions need to be optimized to maximize contrast (as biological systems produce very low contrast under electron beam). We contend that LCTEM experiments replicating naturally occurring nucleation, growth, shrinkage, and re-growth process of microtubules and quantitative image analysis would offer us unique insights into their formation and reorganization.

2018 -
Grant Awardees - Program Grants

Quantitative dissection of molecular determinants of enhancer function


Dept. of Evolutionary Ecology - Ludwig-Maximilians-Universität München - Planegg-Martinsried - GERMANY


Preibisch Lab - Berlin Institute for Medical Systems Biology - Berlin - GERMANY


Depts. of Biological Sciences, Chemistry, Physics, and Computer Science - University of Southern California - Los Angeles - USA

Enhancers are relatively short DNA sequence elements (<1 kb) that determine the timing, location and levels of gene transcription. They harbor specific binding sites for transcription factors (TFs) that control the enhancer. While the necessity of particular TF binding sites (TFBSs) can be assayed with mutations, it is not yet understood which DNA features in an enhancer sequence collectively give rise to the regulatory activity. To identify the molecular determinants that impart a regulatory activity to a DNA sequence element, we have devised a quantitative experimental paradigm and propose to apply statistical analysis and machine learning approaches to the molecular dissection of a model enhancer.
Specifically, we will use a model enhancer driving patterned spatial expression in the wings of fruit flies (Drosophila). First, we will create tens of variants of this enhancer, introducing mutations along its sequence, and describe their regulatory effect. To this end, we will build an automated imaging pipeline to measure the levels and spatial distribution of reporter gene in the wings. The resulting quantitative expression data in a flat tissue will define a morphospace, i.e., a mathematical multidimensional space representing the possible variation of enhancer activity. Second, we will extract DNA feature sets to quantitatively describe molecular variation along the sequence of our mutant enhancers. This will capture structural changes (DNA shape readout), changes in nucleotide sequence per se (DNA base readout), as well as other features (e.g., TFBSs). Third, leaning on dimensionality reduction techniques and machine learning, we will develop predictive models that describe the relationship between DNA features and morphospace. Finally, we will test our predictions of enhancer functionality using synthetic enhancers in transgenic flies. Because enhancers are complex biological objects, we aim with this proposal at developing appropriate mathematical tools to capture the essence of this complexity.
Since this proposal aims at developing a comprehensive mathematical approach to modeling of enhancer functionality, it has the potential to unravel complex molecular mechanisms underlying transcriptional regulation.

2018 -
Long-Term Fellowships - LTF

Quantitative analysis of single-cell regeneration dynamics in living planarians


Morgridge Institute for Research - University of Wisconsin - Madison - USA

HUISKEN Jan (Host supervisor)

Unlike humans, some multicellular organisms are able to regenerate their organs after severe injuries. Understanding how regeneration properties emerge from fundamental biological processes such as gene expression and stem cell differentiation is of utmost importance for basic science and regenerative medicine. Nevertheless, little is known on the mechanisms underlying the initiation, maintenance and control of regeneration. Therefore, many fundamental biological questions remain unanswered, i.e.: (i) how do stem cells migrate, divide and differentiate? and (ii) how do regenerating organs control their size and orientation?
Thanks to their astonishing regenerative properties and simple body plan, planarians form an ideal model system for the study of regeneration. However, due to their opaque tissues, time-lapse microscopy of living planarians has so far been impossible and a comprehensive understanding of the mechanisms underlying their regeneration is still lacking. In the proposed research, I aim to use an interdisciplinary approach to characterize, for the first time, the single-cell dynamics of planarian regeneration. I will use light sheet microscopy, an ideal technique for live imaging of large specimens, and design microfluidic devices to keep the organism at physiological conditions. Fluorescently labelled animals will be used to follow the regeneration dynamics at the single-cell level. Together with complementary genetic and molecular biology techniques, this approach will give us new insights on single stem cells and gene expression dynamics during regeneration.

2018 -
Cross Disciplinary Fellowships - CDF

In vitro control of cell collective flows and tissue folding by means of surface patterns


Department of Biochemistry - University of Geneva - Geneva - SWITZERLAND

ROUX Aurélien (Host supervisor)

In nature, cellular tissues efficiently remodel to attain crucial biological processes, both in health (organogenesis and development) and disease (cancer invasion and metastasis). Such reshaping events require collective cell migration, by means of which tissues can generate forces at large length scales. Although studies in vivo have evidenced the essential role of collective migration modes and flows within reshaping tissues, in vitro studies are necessary to explore these processes in simpler and better controlled microenvironments. In particular, this project aims at inducing reshaping events within confluent monolayers of cells by controlling cell flows through patterns of differential adhesiveness. First, we will explore the effects of geometrical confinement in the steady state positioning of topological defects in cellular nematics as a tool for localizing stress singularities of different strengths within cell tissues. Second, we will gain control over cellular active flows in order to induce unidirectional collective migration patterns. To this purpose, we will use gradients of surface-bound cues to control cell migration (haptotaxis) that, together with geometrical constraints, will provide with the tools for controlling flow and force fields within tissues. Finally, we will design patterns combining geometrical confinement and adhesion gradients to locally frustrate active flows and induce the creation of folds in preassigned regions within proliferative cell monolayers. Our work not only will provide experimental verification of the link between tissue mechanodynamics and reshaping, but also envisions the development of living 3D tissue structures in vitro.

2018 -
Long-Term Fellowships - LTF

Block-Seq, an innovative strategy to decipher cell-cell signaling in root meristems


Department of Biology - New York University - New York - USA

BIRNBAUM Kenneth (Host supervisor)

The indeterminate growth of plant meristems is mediated by the long-term maintenance of stem cells. While patterns like the placement of stem cells are highly dependent on cell-cell communication in plants, there is incomplete understanding of the sources of signals that pattern the meristem. In addition, there is even less knowledge about how stem cells form during organogenesis and regeneration. In plant roots and other organs, patterning processes have been shown to be highly dependent on symplastic signaling through plasmodesmata -- membrane lined channels that connect every plant cells and allow trans-cellular movements of proteins (including transcription factors), miRNAs, RNAs, sugars and ions.
To study cell-cell signaling on a broad scale, the host institute has developed a novel system to monitor complex communication from one cell to another during development. The system relies on recently developed tools to inducibly block plasmodesmata combined with single-cell RNA-seq profiling in plants. Here I propose to use this new set of techniques to sequentially block different cell identities and assess the consequent transcriptional changes using single-cell RNA-seq – a strategy we call “Block-Seq.” In particular, single-cell resolution offers the advantage of detecting responses in a subset of cells rather than expecting a tissue to respond uniformly. For the first time, these approaches will allow me to map communication from one cell type to another in the root meristem.