ERC FUNDED PROJECTS

Synthetic biology is an emerging discipline that offers powerful tools to control and manipulate fundamental processes in living matter. We propose to develop and apply such tools to modify the genetic code of cultured mammalian cells and bacteria with the aim to study the role of lysine acetylation in the regulation of metabolism and in cancer development. Thousands of lysine acetylation sites were recently discovered on non-histone proteins, suggesting that acetylation is a widespread and evolutionarily conserved post translational modification, similar in scope to phosphorylation and ubiquitination. Specifically, it has been found that most of the enzymes of metabolic processes—including glycolysis—are acetylated, implying that acetylation is key regulator of cellular metabolism in general and in glycolysis in particular. The regulation of metabolic pathways is of particular importance to cancer research, as misregulation of metabolic pathways, especially upregulation of glycolysis, is common to most transformed cells and is now considered a new hallmark of cancer. These data raise an immediate question: what is the role of acetylation in the regulation of glycolysis and in the metabolic reprogramming of cancer cells? While current methods rely on mutational analyses, we will genetically encode the incorporation of acetylated lysine and directly measure the functional role of each acetylation site in cancerous and non-cancerous cell lines. Using this methodology, we will study the structural and functional implications of all the acetylation sites in glycolytic enzymes. We will also decipher the mechanism by which acetylation is regulated by deacetylases and answer a long standing question – how 18 deacetylases recognise their substrates among thousands of acetylated proteins? The developed methodologies can be applied to a wide range of protein families known to be acetylated, thereby making this study relevant to diverse research fields.

1 499 375 €

Start date: 2016-07-01, End date: 2021-06-30

NK cells that are well known by their ability to recognize and eliminate virus infected and tumor cells were also implicated in the defence against bacteria. However, the recognition of bacteria by NK cells is only poorly understood. we do not know how bacteria are recognized and the functional consequences of such recognition are also weakly understood. In the current proposal we aimed at determining the “NK cell receptor-bacterial interactome”. We will examine the hypothesis that NK inhibitory and activating receptors are directly involved in bacterial recognition. This ground breaking hypothesis is based on our preliminary results in which we show that several NK cell receptors directly recognize various bacterial strains as well as on a few other publications. We will generate various mice knockouts for NCR1 (a major NK killer receptor) and determine their microbiota to understand the physiological function of NCR1 and whether certain bacterial strains affects its activity. We will use different human and mouse NK killer and inhibitory receptors fused to IgG1 to pull-down bacteria from saliva and fecal samples and then use 16S rRNA analysis and next generation sequencing to determine the nature of the bacteria species isolated. We will identify the bacterial ligands that are recognized by the relevant NK cell receptors, using bacterial random transposon insertion mutagenesis approach. We will end this research with functional assays. In the wake of the emerging threat of bacterial drug resistance and the involvement of bacteria in the pathogenesis of many different chronic diseases and in shaping the immune response, the completion of this study will open a new field of research; the direct recognition of bacteria by NK cell receptors.

2 499 800 €

Start date: 2013-03-01, End date: 2018-02-28

Peptide building blocks serve as very attractive bio-inspired elements in nanotechnology owing to their controlled self-assembly, inherent biocompatibility, chemical versatility, biological recognition abilities and facile synthesis. We have demonstrated the ability of remarkably simple aromatic peptides to form well-ordered nanostructures of exceptional physical properties. By taking inspiration from the minimal recognition modules used by nature to mediate coordinated processes of self-assembly, we have developed building blocks that form well-ordered nanostructures. The compact design of the building blocks, and therefore, the unique structural organization, resulted in metallic-like Young's modulus, blue luminescence due to quantum confinement, and notable piezoelectric properties. The goal of this proposal is to develop two new fronts for bio-inspired building block repertoire along with co-assembly to provide new avenues for organic nanotechnology. This will combine our vast experience in the assembly of aromatic peptides together with additional structural modules from nature. The new entities will be developed by exploiting the design principles of small aromatic building blocks to arrive at the smallest possible module that form super helical assembly based on the coiled coil motifs and establishing peptide nucleic acids based systems to combine the worlds of peptide and DNA nanotechnologies. The proposed research will combine extensive design and synthesis effort to provide a very diverse collection of novel buildings blocks and determination of their self-assembly process, followed by broad chemical, physical, and biological characterization of the nanostructures. Furthermore, effort will be made to establish supramolecular co-polymer systems to extend the morphological control of the assembly process. The result of the project will be a large and defined collection of novel chemical entities that will help reshape the field of bioorganic nanotechnology.

3 003 125 €

Start date: 2016-06-01, End date: 2021-05-31

Clinical experience with globally-acting cannabinoid-1 receptor (CB1R) antagonists revealed the benefits of blocking CB1Rs for the treatment of obesity and diabetes. However, their use is hampered by increased CNS-mediated side effects. Recently, I have demonstrated that peripherally-restricted CB1R antagonists have the potential to treat the metabolic syndrome without eliciting these adverse effects. While these results are promising and are currently being developed into the clinic, our ability to rationally design CB1R blockers that would target a diseased organ is limited. The current proposal aims to develop and test cell- and organelle-specific CB1R antagonists. To establish this paradigm, I will focus our interest on the kidney, since chronic kidney disease (CKD) is the leading cause of increased morbidity and mortality of patients with diabetes. Our first goal will be to characterize the obligatory role of the renal proximal tubular CB1R in the pathogenesis of diabetic renal complications. Next, we will attempt to link renal proximal CB1R with diabetic mitochondrial dysfunction. Finally, we will develop proximal tubular (cell-specific) and mitochondrial (organelle-specific) CB1R blockers and test their effectiveness in treating CKD. To that end, we will encapsulate CB1R blockers into biocompatible polymeric nanoparticles that will serve as targeted drug delivery systems, via their conjugation to targeting ligands. The implications of this work are far reaching as they will (i) point to renal proximal tubule CB1R as a novel target for CKD; (ii) identify mitochondrial CB1R as a new player in the regulation of proximal tubular cell function, and (iii) eventually become the drug-of-choice in treating diabetic CKD and its comorbidities. Moreover, this work will lead to the development of a novel organ-specific drug delivery system for CB1R blockers, which could be then exploited in other tissues affected by obesity, diabetes and the metabolic syndrome.

1 500 000 €

Start date: 2016-04-01, End date: 2021-03-31

"Communication between the nervous and the immune system is pivotal for maintaining homeostasis and ensuring rapid and efficient reaction to stress and infection insults. The emergence of microRNAs (miRs) as regulators of gene expression and of acetylcholine (ACh) signalling as regulator of anxiety and inflammation provides a model for studying this interaction. My hypothesis is that 1) a specific sub-group of miRs, designated ""CholinomiRs"", may silence multiple target genes in the neuro-immune interface; 2) these miRs compete with each other on the interaction with their targets, and 3) mutations interfering with miR binding lead to inherited susceptibility to anxiety and inflammation disorders by modifying these interactions. Our preliminary findings have shown that by targeting acetylcholinesterase (AChE), CholinomiR-132 can intensify acute stress, resolve intestinal inflammation and change post-ischemic stroke responses. Further, we have identified clustered single nucleotide polymorphisms (SNPs) interfering with AChE silencing by several miRs which associate with elevated trait anxiety, blood pressure and inflammation. To further study miR regulators of ACh signalling, I plan to: (1) Identify anxiety and inflammation-induced changes in CholinomiRs and their targets in challenged brain and immune cells. (2) Establish the roles of these targets for one selected CholinomiR by tissue-specific manipulations. (3) Study primate-specific CholinomiRs by continued human DNA screens to identify SNPs and in ""humanized"" mice with knocked-in human AChE and transgenic CholinomiR-608. (4) Test if therapeutic modulation of aberrant CholinomiR expression can restore homeostasis. This research will clarify how miRs interact with each other in health and disease, introduce the dimension of complexity of multi-target competition and miR interactions and make a conceptual change in miRs research while enhancing the ability to intervene with diseases involving impaired ACh signalling."

2 375 600 €

Start date: 2013-03-01, End date: 2018-02-28

During the human lifetime 10000 trillion cell divisions take place to ensure tissue homeostasis and several vital functions in the organism. Mitosis is the process that ensures that dividing cells preserve the chromosome number of their progenitors, while deviation from this, a condition known as aneuploidy, represents the most common feature in human cancers. Here we will test two original concepts with strong implications for chromosome segregation fidelity. The first concept is based on the “tubulin code” hypothesis, which predicts that molecular motors “read” tubulin post-translational modifications on spindle microtubules. Our proof-of-concept experiments demonstrate that tubulin detyrosination works as a navigation system that guides chromosomes towards the cell equator. Thus, in addition to regulating the motors required for chromosome motion, the cell might regulate the tracks in which they move on. We will combine proteomic, super-resolution and live-cell microscopy, with in vitro reconstitutions, to perform a comprehensive survey of the tubulin code and the respective implications for motors involved in chromosome motion, mitotic spindle assembly and correction of kinetochore-microtubule attachments. The second concept is centered on the recently uncovered chromosome separation checkpoint mediated by a midzone-associated Aurora B gradient, which delays nuclear envelope reformation in response to incompletely separated chromosomes. We aim to identify Aurora B targets involved in the spatiotemporal regulation of the anaphase-telophase transition. We will establish powerful live-cell microscopy assays and a novel mammalian model system to dissect how this checkpoint allows the detection and correction of lagging/long chromosomes and DNA bridges that would otherwise contribute to genomic instability. Overall, this work will establish a paradigm shift in our understanding of how spatial information is conveyed to faithfully segregate chromosomes during mitosis.

2 323 468 €

Start date: 2016-07-01, End date: 2021-06-30

Transcription factors (TF) regulate genome function by controlling gene expression. Comprehensive characterization of the in vivo binding of TF to the DNA in relevant primary models is a critical step towards a global understanding of the human genome. Recent advances in high-throughput genomic technologies provide an extraordinary opportunity to develop and apply systematic approaches to learn the underline principles and mechanisms of mammalian transcriptional networks. The premise of this proposal is that a tractable set of rules govern how cells commit to a specific cell type or respond to the environment, and that these rules are coded in regulatory elements in the genome. Currently our understanding of the mammalian regulatory code is hampered by the difficulty of directly measuring in vivo binding of large numbers of TFs to DNA across multiple primary cell types and their natural response to physiological stimuli. Here, we overcome this bottleneck by systematically exploring the genomic binding network of 1. All relevant TFs of key hematopoietic cells in both steady state and under relevant stimuli. 2. Follow the changes in TF networks as cells differentiate 3. Use these models to engineer cell states and responses. To achieve these goals, we developed a new method for automated high throughput ChIP coupled to sequencing (HT-ChIP-Seq). We used this method to measure binding of 40 TFs in 4 time points following stimulation of dendritic cells with pathogen components. We find that TFs vary substantially in their binding dynamics, genomic localization, number of binding events, and degree of interaction with other TFs. The analysis of this data suggests that the TF network is hierarchically organized, and composed of different types of TFs, cell differentiation factors, factors that prime for gene induction, and factors that bind more specifically and dynamically. This proposal revisits and challenges the current understanding of the mammalian regulatory code.

1 500 000 €

Start date: 2012-10-01, End date: 2017-09-30

The staggering diversity of the living world is a testament to the amount of variation available to the agency of natural selection. While it has been assumed that variation is entirely uniform and unbiased, recent work has challenged this notion. Evolutionary developmental biology seeks to understand the biases on variation imposed by developmental processes and their distinction from selective constraints. Metazoan development is best described by developmental gene pathways which are composed of transcription factors, signaling molecules, and terminal differentiation genes. A systematic comparison of such pathways across species would reveal the patterns of conservation and divergence; however this has not yet been achieved. In the EvoDevoPaths project we will develop a new approach to unravel pathways using both single-cell and tissue-specific transcriptomics. Our aim is to elucidate the evolution of developmental gene pathways using intricate embryology in the nematode phylum, a single-cell transcriptomic method we have developed, and sophisticated computational approaches for pathway comparisons. We will ask how variation is distributed across the specification and differentiation modules of a pathway using the nematode endoderm pathway as a model system. We further propose that the evolutionary change in the tissue specification pathways of early cell lineages is constrained by the properties of cell specification pathways. To test this hypothesis we will, for the first time, determine early developmental cell lineages from single cell transcriptomic data. Finally, we will attempt to unify the molecular signatures of conserved stages in disparate phyla under a framework in which they can be systematically compared. This research collectively represents the first time that developmental gene pathways are examined in an unbiased manner contributing to a theory of molecular variation that explains the evolutionary processes that underlie phenotypic novelty.

1 500 000 €

Start date: 2012-10-01, End date: 2017-09-30

Plants evolved a unique molecular mechanism that spatially regulate auxin, forming finely tuned gradients and local maxima of auxin that inform and direct developmental patterning and adaptive growth processes. Recent findings call into question the uniqueness of polar auxin transport in the sense that more plant hormones seem to be actively transported. Although still lacking many mechanistic details, as well as comprehensive functional connotations, these findings warrant a more thorough investigation into the prospect of a broader scope for plants spatial regulation capacity in the context of additional hormones. Critically, we lack an effective set of tools to directly investigate and dissect the particulars of plant hormones mobility at the molecular level. My long-term goal is to provide a molecular and mechanistic understanding of plant hormones dynamics that will augment our evolving model of how they are regulated and how they convey information. Here, I hypothesize that GA mobility in plants is controlled and directed by an active transport mechanism to form distinct distribution patterns that affect signaling. I will test my hypothesis with a multi-faceted and multi-disciplinary approach, combining: fluorescent labeling of key gibberellins to map their accumulation sites in whole plants and at the sub-cellular level; chemical-biology strategies that facilitate manipulation of GA “origin point” in planta to map and quantify GA flow pathways; probe-based genetic screens and un-biased photo-affinity labeling to identify proteins affecting GA mobility; and genetic and molecular biology techniques to characterize identified proteins’ functions. I expect to offer an exceptional, detailed view into the inner workings of gibberellins dynamics in planta and into the mechanisms driving it. I further anticipate that the strategies developed here to specifically address gibberellins could be straightforwardly re-tailored to investigate additional plant hormones.

1 500 000 €

Start date: 2016-02-01, End date: 2021-01-31

DNA methylation, the covalent binding of a methyl group (CH3) to cytosine base, regulates the genome activity and plays a fundamental developmental role in eukaryotes. Its epigenetic characteristics of regulating transcription without changing the genetic code together with the ability to be transmitted through DNA replication allow organisms to memorize cellular events for many generations. DNA methylation is mostly known for its role in transcriptional silencing; however, its functional output is more complex and is influenced in part by its DNA context. Recent genomic studies, have found DNA methylation to be targeted inside sequences of actively transcribed genes, thus termed gene body methylation. Despite being an evolutionary conserved and a robust methylation pathway targeted to thousands of genes in animal and plant genomes, the function of gene body methylation is still poorly understood at both the molecular and functional level. Similar to the chicken and egg conundrum, because we do not know what gene body methylation does, therefore scientists could not apply its function to discover its regulators either. Gene body methylation is targeted to a very specific subset and subregions of genes, thus we strongly believe that specific factors exist and are missing simply because that no one has ever searched for them before. Hence, to make major breakthroughs in the field, our approach is to artificially generate gene-body-specific hypomethylated plants that together with customized genetic and biochemical systems will allow us to discover regulators and interpreters of gene body methylation. Using these unique genetic tools and novel molecular factors, we will be able to ultimately explore the particular biological roles of gene body methylation. These findings will fill the gap towards a full comprehension of the entire functional array of DNA methylation, and to its more precise exploitation in yielding better crops and in treating human diseases.

1 500 000 €

Start date: 2016-10-01, End date: 2021-09-30

Vaccination is widely used to prevent human diseases by inducing the formation of cellular and antibody-mediated immune responses for induction of long lasting immunological memory. Although most studies focus on immune responses elicited against injected immunizations, the simplest delivery of a vaccine regimen is by oral administration. The cellular and molecular components of the antibody immune response in peripheral lymph nodes in response to immunization are well described, however, much less is known about the dynamics of immune cells in gut associate lymphoid tissues (GALT) and adjust intestinal mucosal tissues. In the proposed research plan I will implicate intravital in vivo imaging for analysis of the cellular component of the antibody immune response in intestinal tissues. My goals are: 1. To track germinal center (GC) T cells for prolong time periods in peripheral lymph nodes and GALT and determine if they enter the memory compartment. For this purpose I will develop a new photoactivation method for permanently labeling immune cells and fate tracing of their daughter cells. 2. To examine T-B interactions and their regulation by intraceullar signaling pathways in GALT and to determine where and when class switch recombination to IgA takes place. For this purpose I will use intravital imaging of fluorescent reporter mice. 3. I will analyze the dynamics of plasma cell migration from Peyer’s patches to the mucosa by implementing state of the art photoactivation and imaging techniques that allow prolonged cell tracking. I will also use photoactivation approaches for sorting plasma cells from specific intestinal layers and perform gene expression analysis. 4. I will develop a new method to study dynamics and fate of B cells specific for commensal microbes in the GC, memory and plasma cell compartments. This research plan will extend our knowledge of the antibody immune response in intestinal tissues towards the future design of improved oral vaccinations.

1 375 000 €

Start date: 2016-05-01, End date: 2021-04-30

The global demand for dairy products is expected to surge by 36% over the next decade in a manner that is progressively insatiable by existing technologies. The dairy industry relies on bovine reproduction, yet cow fertility is declining and the exact causes are not fully understood. It is clear, however, that the quality of bovine oocytes is decreasing. In mammals, the ovarian reserve of oocytes stored within quiescent primordial follicles is non-renewable. Oocyte develop and mature within distinctive follicular microenvironments under tightly regulated molecular and physical conditions. Similarly, preimplantation embryo development is supported within a specialized microenvironment that is surrounded by the zona pellucida and insulated from external soluble and mechanical inputs. Characterizing and understanding these environments and how they affect reproductive processes is a key toward improving assisted reproductive technologies in bovine species. Our premise is that molecular characterization of endocrine and paracrine signalling pathways must be complemented with understanding the mechanical regulation of reproductive biology. This premise is supported by recent finding showing that physical stresses and the mechanical compliance of the extracellular surroundings serve as potent regulators of cell fates in regeneration processes, development, and disease. I propose to employ a biophysical and computational toolbox to study the mechanobiology of reproduction with application to bovine embryo-based technologies. By mimicking the mechanical properties of the ovarian cortical niche, which I will characterize using freshly derived ovaries, I will design an in vitro system for supporting follicle growth. Mechanical profiling of the entire developmental course from oocyte maturation to preimplantation embryogenesis will generate mechanistic insights into the physical regulation of reproductive processes.

1 500 000 €

Start date: 2016-06-01, End date: 2021-05-31

Signaling, genetic regulatory circuits, and tissue morphology are inherently coupled to each other during embryonic development. Although changes in cellular and tissue morphology are commonly treated as a downstream consequence of cell fate decision processes, there are multiple examples where morphological changes occur concurrently with the differentiation processes. This suggests that a feedback between cell morphology and regulatory processes can play an important role in coordinating tissue development. Currently, however, we lack the experimental, theoretical, and conceptual tools to understand this interplay between cell morphology, signaling, and regulatory circuits. In particular, we need to understand (1) how intercellular signaling depends on the cellular morphology and on the properties of the boundary between cells, and (2) how intercellular signaling, genetic circuits, and cell morphology integrate to generate robust differentiation patterns. Here, I propose to combine quantitative in-vitro and in-vivo experiments with mathematical modeling to address these questions in the context of Notch signaling and Notch mediated patterning, typically used for coordinating differentiation between neighboring cells during development. We will utilize novel reporters and micropatterning technology to analyze Notch signaling between pairs of cells. We will elucidate how the geometry and the molecular composition of the boundary between cells affect signaling. At the tissue level, we will study how the interplay between cell morphology and Notch signaling gives rise to robust patterning in the mammalian inner ear. We will use cochlear inner ear explant imaging to track the transition from disordered undifferentiated state to ordered pattern of hair and supporting cells in the cochlea. Together with a novel hybrid modeling approach, we will provide the foundation for a systems level understanding of development that interconnects morphology and regulatory circuits.

2 000 000 €

Start date: 2016-06-01, End date: 2021-05-31

The mammalian hippocampal formation contains place cells, grid cells, head-direction cells and border cells, which collectively represent the animal’s position (‘map’), distance traveled (‘odometer’) and direction (‘compass’), and are thought to underlie navigation. These neurons are typically studied in rodents running on linear tracks or in small empty boxes, ~1×1 m in size. However, real-world navigation differs dramatically from typical laboratory setups, in at least three ways – which we plan to study: (1) The world is not empty, but contains objects and goals. Almost nothing is known about how neural circuits represent goal location – which is essential for navigating towards the goal. We will record single-neuron activity in bats flying towards spatial goals, in search for cells that encode vectorial information about the direction and distance to the goal. Preliminary results support the existence of such cells in the bat hippocampal formation. This new functional cell class of vectorial goal-encoding neurons may underlie goal-directed navigation. (2) The world is not flat, but three-dimensional (3-D). We will train bats to fly in a large flight-room and examine 3-D grid cells and 3-D border cells. (3) The world is not 1-m in size, and both rodents and bats navigate over kilometer-scale distances. Nothing is known about how the brain supports such real-life navigation. We will utilize a 1-km long test facility at the Weizmann Institute of Science, and record place cells and grid cells in bats navigating over biologically relevant spatial scales. Further, we will compare neural codes for space in wild-born bats versus bats born in the lab – which have never experienced a 1-km distance – to illuminate the role of experience in mammalian spatial cognition. Taken together, this set of studies will bridge the gap – a conceptual gap and a gap in spatial scale – between hippocampal laboratory studies and real-world natural navigation.

2 000 000 €

Start date: 2016-11-01, End date: 2021-10-31

In order to survive and maintain normal function, the cell depends on a dynamic system of spatial specificity and fidelity of signaling pathways that can respond to both internal and external changes over space and time. This cell-cell communication is mediated by ligand-receptor mechanisms. In the case of highly polarized cells such as neurons trafficking mechanisms mediated by motor proteins are used to achieve precise signal targeting. Alterations in the trafficking machinery may results in incorrect signaling, that in some cases leads to neurodegeneration. An example for such phenomenon may be found in Amyotrophic Lateral Sclerosis (ALS). ALS is a motor neuron disease characterized by a non-cell autonomous neurodegeneration process, which involves neighboring cells via an unknown mechanism. This proposal focuses on the elucidation of basic cell-cell communication mechanisms by using the motor neuron degeneration process as a model. I aim to reveal critical communication mechanisms between the neuron and its environment for cell survival and synapse maintenance. My working hypothesis is that alterations in extrinsic and intrinsic signals may lead to the neurodegeneration seen in ALS. I will develop unique compartmental platforms mimicking the natural environment of the motor neuron. Then using differential “omics” approaches followed by functional assays I will reveal and characterized vital factors essential to neuron synapse integrity and neuron survival. Using state of the art live-cell imaging techniques I will reveal also the molecular mechanism for signals localization and targeting driven by the motor protein dynein. I will elucidate the molecular mechanism of neuronal communication with its diverse environment essential to its survival and proper function. The project will bring revolutionary new mechanistic insight to a truly fundamental problem in cell biology, how the cell communicates and how signals arrive at the right place at the right time?

1 499 800 €

Start date: 2013-02-01, End date: 2019-01-31

The liver is the main organ responsible for the systemic regulation of human metabolism, responding to hormonal stimulation, nutritional challenges, and circadian rhythms using fast enzymatic processes and slow transcriptional mechanisms. This regulatory complexity limits our ability to create efficient pharmaceutical interventions for metabolic diseases such as fatty liver disease and diabetes. In addition, circadian changes in drug metabolism can impact pharmacokinetics and pharmacodynamics affecting our ability to optimize drug dosage or properly assess chronic liver toxicity. The challenge in rationally designing efficient drug interventions stems from current reliance on end-point assays and animal models that provide intermittent information with limited human relevance. Therefore, there is a need to develop systems capable of tracking transcriptional and metabolic dynamics of human tissue with high-resolution preferably in real time. Over the past 5 years, we established state-of-the-art models of human hepatocytes; oxygen nanosensors; and cutting-edge liver-on-chip devices, making us uniquely suited to address this challenge. We aim to develop a platform capable of tracking the metabolism of tissue engineered livers in real time, enabling an accurate assessment of chronic liver toxicity (e.g. repeated dose response) and the deconstruction of complex metabolic regulation during nutritional events. Our approach is to integrate liver-on-chip devices, with real time measurements of oxygen uptake, infrared microspectroscopy, and continuous MS/MS analysis. This innovative endeavour capitalizes on advances in nanotechnology and chemical characterization offering the ability to non-invasively monitor the metabolic state of the cells (e.g. steatosis) while tracking minute changes in metabolic pathways. This project has the short-term potential to replace animal models in toxicity studies and long-term potential to elucidate critical aspects in metabolic homeostasis.

2 118 175 €

Start date: 2016-10-01, End date: 2021-09-30

As any other obligate parasite, Plasmodium depends on its hosts and on the nutrients they provide to survive and complete its life cycle. Surprisingly, nothing is know about Plasmodium’s capacity to sense nutrients or its host’s nutritional status and thereby reprogram its metabolism. Our preliminary data provides unequivocal evidence that Plasmodium has the ability to sense the host low-nutrient status and adapt to it by decreasing its multiplication rate. Thus, the overall goal of the present proposal is to unveil the molecular mechanisms by which parasites are capable to sense and adapt to environmental signals originated from nutrients and to determine its impact on the course and virulence of infection. To that end we propose to: (i) Identify Plasmodium pathway(s) that sense (host) nutritional changes; (ii) Uncover which molecules are sensed by Plasmodium during its intracellular development; (iii) Study the impact of the parasite’s nutrient sensing pathways activity on the course of infection; and (iv) Evaluate host nutritional status sensing as a common feature in parasites. The present proposal moves towards a change of paradigm on how host-parasite interactions are viewed. By definition, since a parasite requires a host in order to survive, a decrease in the availability of an essential molecule obtained from the host will weaken the parasite and render it incapable of succeeding in its life cycle. The rationale behind this proposal is that parasites monitor host nutritional environment and, prior to any nutrient(s) becoming limiting, are able to respond and adapt to the sensed alteration(s). Multidisciplinary approaches that combine genetic, genomic, cell biological and physiological methodologies will be used. Results arising from the present proposal will provide novel insights into the cell biology of these parasites and will increase our understanding of the interactions that these parasites maintain with their hosts.

1 500 000 €

Start date: 2012-12-01, End date: 2017-11-30

Cyanobacteria play a key role in marine photosynthesis, contributing almost 50% of primary production in oligotrophic regions of the ocean. Marine cyanophages were recently discovered to carry photosystem II (PSII) genes, and it was suggested that these genes increase phage fitness by helping the phages to maintain photosynthesis in the infected bacterial cells. We recently showed evidence for the presence of photosystem I (PSI) genes in genomes of marine cyanophages [Sharon et al. 2009 Nature 461, 258-262]. Cyanobacterial core PSI gene cassettes, containing psaJFABCDEK, or psaDCAB gene cassettes forms unique clusters in cyanophage genomes, suggestive of selection for a distinct function in virus reproduction. Potentially, the proteins encoded by the viral genes are sufficient for forming intact monomeric PSI complexes. Projection of viral predicted peptides on the cyanobacterial PSI crystal structure suggests that the viral PSI components provide a unique way for funneling reducing power from respiratory and other electron transfer chains to PSI, therefore bypassing the need to rely solely on reducing power from the photosystem electron transfer chain. The main goals of this proposal are: (1) To determine how much of oceanic photosynthesis is actually performed with viral proteins. (2) To establish a model system to understand the role of modified photosynthetic viral proteins in photosynthesis We hypothesize that viral photosynthetic peptides are integrated into the bacterial photosynthetic membranes in order to maintain photosynthesis in infected cells, that otherwise stop to photosynthesize, and that changes are introduced to the system as a whole. The proposed research will integrate concepts and techniques from metagenomics, metaproteomics and bioinformatics techniques to explore the interaction of viral PSII and PSI proteins with their host reaction center complexes, and to examine their influence on global marine photosynthesis production

1 933 800 €

Start date: 2013-02-01, End date: 2018-01-31

During infection, the host immune system interacts with the bacterial cell surface, a complex structure made of peptidoglycan, wall teichoic acids, lipoteichoic acids, capsule polysaccharide and peptidoglycan-attached proteins. A lot is known about the metabolic pathways for the synthesis of each individual cell surface component. Almost nothing is known about the coordination between the synthesis of the peptidoglycan, the major structural component of the cell surface and the main inflammatory component of gram-positive bacteria, and the synthesis of the other molecules present at the surface. However, this coordination is essential for the construction of a surface capable not only of performing its biological functions in cell protection and morphology, but also of masking its inflammatory components for evasion from host recognition. Using the clinical pathogen Staphylococcus aureus as a model organism, we propose to investigate the temporal and spatial regulation of the enzymes responsible for the synthesis of the cell surface components, as well as their dependence on the underlying divisome. We will (i) use state-of –the art fluorescence microscopy to localize fluorescent derivatives of enzymes required for cell surface synthesis; (ii) use libraries of antibiotics, of antisense RNA expression plasmids, and of transposon mutants to identify the order of assembly and requirements for the localization of cell surface synthesis enzymes; (iii) identify the exact metabolic compound/protein/geometric cue responsible for the localization of key enzymes; (iv) determine if cells with impaired surface synthesis due to protein delocalization are more susceptible to host recognition and therefore less capable of causing infections. This project will result in the identification of new mechanisms of protein localization, a fundamental question in cell biology, and in a better understanding of the assembly of the bacterial cell surface of successful bacterial pathogens

1 656 960 €

Start date: 2013-03-01, End date: 2018-02-28

The direct conversion approach and the generation of induced pluripotent stem cells (iPSCs) provide an invaluable resource of cells for disease modelling, drug screening, and patient-specific cell-based therapy. However, the directly converted cells are not stable, and the vast majority of iPSCs exhibit poor developmental potential as measured by stringent pluripotency tests. This suggests that the prevailing method of reprogramming is not ideal and leads to aberrant/incomplete conversion. To improve the quality of the converted cells, efforts should be focused on uncovering the molecular mechanisms that characterize the nuclear reprogramming process. There are two critical hurdles that hinder the progress of deciphering the elements that dictate successful reprogramming: (1) The ability to detect and capture solely the rare cells that eventually will be converted and (2) to monitor the transcriptional profile of cells at the single-cell level. Single-cell technology is in its infancy and many of the methods used today are characterized by high noise to signal ratio. In this grant proposal we intend to overcome these limitations by (1) establishing a complex fluorescent knock-in reporter system using the CRISPR/Cas9 method to capture the early rare reprogrammable cells and by (2) employing several cutting-edge single-cell technologies, RNA-Seq, Fluidigm BioMark and single-molecule mRNA-FISH, to segregate the real signal from the noise. To identify common and more global elements that facilitate nuclear reprogramming at large, we will trace in parallel, reprogrammable cells from two different somatic cell conversion models that reach high degree of nuclear reprogramming, and analyse their transcriptome using sophisticated bioinformatic tools. This study will provide a general overview of the changes that occur during the conversion of various cell types and will uncover the basic features that are essential to reach safe and complete conversion.

1 500 000 €

Start date: 2016-03-01, End date: 2021-02-28

Mitochondria at the synapse have a pivotal role in neurotransmitter release, but almost nothing is known about synaptic mitochondria composition or specific functions. Synaptic mitochondria compared to mitochondria in other cells, need to cope with increased calcium load, more oxidative stress, and high demands of energy generation during synaptic activity. My hypothesis is that synaptic mitochondria have acquired specific mechanisms to manage local stress and that disruption of these mechanisms contributes to neurodegeneration. How mitochondria sense their dysfunction is unclear. Even more intriguing is the question how they decide whether their failure should lead to removal of the organelle or dismissal of the complete neuron via cell death. We anticipate that these decisions are not only operational during disease, but might constitute a fundamental mechanism relevant for maintenance of synaptic activity and establishment of new synapses. Recent studies have revealed several genes implicated in neurodegenerative disorders involved in mitochondrial maintenance. However the function of these genes at the synapse, where the initial damage occurs, remains to be clarified. These genes provide excellent starting points to decipher the molecular mechanisms discussed above. Furthermore I propose to use proteomic approaches to identify the protein fingerprint of synaptic mitochondria and to compare them to mitochondria from other tissues. I plan to identify key players of the proposed regulatory pathways involved in intrinsic mitochondria quality control. In a complimentary approach, I will exploit our findings and use in vitro and in vivo experimental approaches to measure mitochondrial function of synaptic versus non-synaptic mitochondria and the relevance of those changes for synaptic function. Our work will unravel the specific properties of synaptic mitochondria and provide much needed insight in their hypothesized predominant role in neurodegenerative disorders.

1 300 000 €

Start date: 2016-09-01, End date: 2021-08-31

We walk, run and jump using the complex and ingenious musculoskeletal system. It is therefore puzzling that although each of its components has been extensively studied, research of the musculoskeleton as an integrated system and, in particular, of its assembly has been scarce. In recent years, studies conducted in my lab have demonstrated the centrality of cross regulation between musculoskeletal tissues in skeletogenesis. These works have provided me with the inspiration for a revolutionary hypothesis on the way tendons connect to bones, along with sufficient preliminary data on which to base it. The critical component in the assembly of the musculoskeleton is the formation of an attachment unit, where a tendon is inserted into a bone. Instead of two tissues that attach to each other, my novel hypothesis suggests that the entire attachment unit originates from a single pool of progenitor cells, which following differentiation diverges to form a tendon attached to cartilage. With the support of the ERC scheme, I will uncover the previously uncharacterized cellular origin of the attachment unit and the genetic program underlying its development. The attachment unit is a compound tissue, as it is composed of chondrocytes at one end and of tenocytes at the other end. We will investigate the mechanisms that facilitate in situ differentiation of mesenchymal progenitor cells into two distinct cell fates, under one defined niche. In addition, I will identify the contribution of both mechanical stimuli and molecular signals to the development of the attachment unit. The ultimate goal of this program is to provide a complete picture of attachment unit development, in order to promote understanding of musculoskeletal assembly. The acquired knowledge may provide the basis for new therapies for enthesopathies, through tissue engineering or repair.

1 499 999 €

Start date: 2013-01-01, End date: 2017-12-31

Organisms across all kingdoms share several systems that are essential to life, one of the most central being protein synthesis. Living in a continuously changing environment, cells need to constantly respond to various environmental cues and change their protein landscape. In extreme cases, cells globally shut down protein synthesis and upregulate stress-protective proteins. Mechanisms of translational repression or selective enhancement of stress-induced proteins have been characterized, but their effects were demonstrated on an individual mRNA basis. Which target mRNAs are translationally regulated in response to different environmental cues, and what are the cis-regulatory elements involved, largely remain as open questions. Using ribosome footprint profiling, I recently discovered a novel mode of translational control in stress, underscoring the potential of new technologies to uncover novel regulatory mechanisms. But while transcription cis-regulatory elements have been thoroughly mapped in the past decade, and splicing regulatory elements are accumulating, the identification of translation cis-regulatory elements is lagging behind. Here I propose to crack the mammalian translation regulatory code, and close this long-standing gap. I present a novel interdisciplinary framework to comprehensively identify translation cis-regulatory elements, and map their mRNAs targets in a variety of cellular perturbations. Importantly, we plan to explore mechanisms underlying novel cis-regulatory elements, and create the first genome-wide functionally annotated translation regulatory code. The translation regulatory code will map targets of existing mechanisms and shed light on newly identified pathways that play a role in stress-induced translational control. The proposed project is an imperative stepping stone to understanding translational regulation by cis-regulatory elements, opening new avenues in the functional genomics research of translational control.

1 587 500 €

Start date: 2016-03-01, End date: 2021-02-28

Post-translational modifications (PTMs) of proteins are a major tool that the cell uses to monitor events and initiate appropriate responses. While a protein is defined by its backbone of amino acid sequence, its function is often determined by PTMs, which specify stability, activity, or cellular localization. Among PTMs, ubiquitin and ubiquitin-like (Ubl) modifications were shown to regulate a variety of fundamental cellular processes such as cell division and differentiation. Aberrations in these pathways have been implicated in the pathogenesis of cancer. Over the past decade high-throughput genomic and transcriptional analyses have profoundly broadened our understanding of the processes underlying cancer development and progression. Yet, proteomic analyses and the PTM landscape in cancer, remained relatively unexplored. Our goal is to decipher molecular mechanisms of Ubl regulation in cancer. We will utilize the PTM profiling technology that I developed and further develop it to allow for subsequent MS analysis. Together with cutting-edge genomic, imaging and proteomic technologies, we will analyze novel aspects of PTM regulation at the level of the enzymatic machinery, the substrates and the downstream cellular network. We will rely on ample in-vitro and in-vivo characterization of Ubl conjugation to:a. Elucidate the regulatory principles of substrate specificity and recognition. b. Understand signalling dynamics in the ubiquitin system. c. Reveal how aberrations in these pathways may lead to diseases such as cancer. Identifying both the Ubl modifying enzymes and the modified substrates will form the basis for deciphering the molecular pathways in which they operate in the cell and the principles of their dynamic regulation. Revealing the PTM regulatory code presents a unique opportunity for the development of novel therapeutics. More broadly, our approaches may provide a new paradigm for addressing other complex biological questions involving PTM regulation.

1 500 000 €

Start date: 2016-05-01, End date: 2021-04-30

Phytoplankton blooms are ephemeral events of exceptionally high primary productivity that regulate the flux of carbon across marine food webs. The cosmopolitan coccolithophore Emiliania huxleyi (Haptophyta) is a unicellular eukaryotic alga responsible for the largest oceanic algal blooms covering thousands of square kilometers. These annual blooms are frequently terminated by a specific large dsDNA E. huxleyi virus (EhV). Despite the huge ecological importance of host-virus interactions, the ability to assess their ecological impact is limited to current approaches, which focus mainly on quantification of viral abundance and diversity. On the molecular basis, a major challenge in the current understanding of host-virus interactions in the marine environment is the ability to decode the wealth of “omics” data and translate it into cellular mechanisms that mediate host susceptibility and resistance to viral infection. In the current proposal we intend to provide novel functional insights into molecular mechanisms that regulate host-virus interactions at the single-cell level by unravelling phenotypic heterogeneity within infected populations. By using physiological markers and single-cell transcriptomics, we propose to discern between host subpopulations and define their different “metabolic states”, in order to map them into different modes of susceptibility and resistance. By using advanced metabolomic approaches, we also aim to define the infochemical microenvironment generated during viral infection and examine how it can shape host phenotypic plasticity. Mapping the transcriptomic and metabolic footprints of viral infection will provide a meaningful tool to assess the dynamics of active viral infection during natural E. huxleyi blooms. Our novel approaches will pave the way for unprecedented quantification of the “viral shunt” that drives nutrient fluxes in marine food webs, from a single-cell level to a population and eventually ecosystem levels.

2 749 901 €

Start date: 2016-11-01, End date: 2021-10-31