ERC FUNDED PROJECTS

A central problem in developmental biology is to understand how tissues are patterned in time and space - how do identical cells differentiate to form the adult body plan? Patterns often arise from prior asymmetries in developing embryos, but there is also increasing evidence for self-organizing mechanisms that can break the symmetry of an initially homogeneous cell population. These patterning processes are mediated by a small number of signaling molecules, including the TGF-β superfamily members BMP and Nodal. While we have begun to analyze how biophysical properties such as signal diffusion and stability contribute to axis formation and tissue allocation during vertebrate embryogenesis, three key questions remain. First, how does signaling cross-talk control robust patterning in developing tissues? Opposing sources of Nodal and BMP are sufficient to produce secondary zebrafish axes, but it is unclear how the signals interact to orchestrate this mysterious process. Second, how do signaling systems self-organize to pattern tissues in the absence of prior asymmetries? Recent evidence indicates that axis formation in mammalian embryos is independent of maternal and extra-embryonic tissues, but the mechanism underlying this self-organized patterning is unknown. Third, what are the minimal requirements to engineer synthetic self-organizing systems? Our theoretical analyses suggest that self-organizing reaction-diffusion systems are more common and robust than previously thought, but this has so far not been experimentally demonstrated. We will address these questions in zebrafish embryos, mouse embryonic stem cells, and bacterial colonies using a combination of quantitative imaging, optogenetics, mathematical modeling, and synthetic biology. In addition to providing insights into signaling and development, this high-risk/high-gain approach opens exciting new strategies for tissue engineering by providing asymmetric or temporally regulated signaling in organ precursors.

1 997 750 €

Start date: 2020-07-01, End date: 2025-06-30

Cytokinesis completes cell division by partitioning the contents of the mother cell to the two daughter cells. This process is accomplished through the assembly and constriction of a contractile ring, a complex actomyosin network that remains poorly understood on the molecular level. Research in cytokinesis has overwhelmingly focused on signaling mechanisms that dictate when and where the contractile ring is assembled. By contrast, the research I propose here addresses fundamental questions about the structural and functional properties of the contractile ring itself. We will use the nematode C. elegans to exploit the power of quantitative live imaging assays in an experimentally tractable metazoan organism. The early C. elegans embryo is uniquely suited to the study of the contractile ring, as cells dividing perpendicularly to the imaging plane provide a full end-on view of the contractile ring throughout constriction. This greatly facilitates accurate measurements of constriction kinetics, ring width and thickness, and levels as well as dynamics of fluorescently-tagged contractile ring components. Combining image-based assays with powerful molecular replacement technology for structure-function studies, we will 1) determine the contribution of branched and non-branched actin filament populations to contractile ring formation; 2) explore its ultra-structural organization in collaboration with a world expert in electron microcopy; 3) investigate how the contractile ring network is dynamically remodeled during constriction with the help of a novel laser microsurgery assay that has uncovered a remarkably robust ring repair mechanism; and 4) use a targeted RNAi screen and phenotype profiling to identify new components of actomyosin contractile networks. The results from this interdisciplinary project will significantly enhance our mechanistic understanding of cytokinesis and other cellular processes that involve actomyosin-based contractility.

1 499 989 €

Start date: 2015-07-01, End date: 2021-06-30

Integrins are transmembrane cell adhesion receptors controlling cell proliferation and migration. Our objective is to gain fundamentally novel mechanistic insight into the emerging new roles of integrins in cancer and to generate a road map of integrin dependent pathways critical in mammary gland development and integrin signalling thus opening new targets for therapeutic interventions. We will combine an in vivo based translational approach with cell and molecular biological studies aiming to identify entirely novel concepts in integrin function using cutting edge techniques and synthetic-biology tools. The specific objectives are: 1) Integrin inactivation in branching morphogenesis and cancer invasion. Integrins regulate mammary gland development and cancer invasion but the role of integrin inactivating proteins in these processes is currently completely unknown. We will investigate this using genetically modified mice, ex-vivo organoid models and human tissues with the aim to identify beneficial combinational treatments against cancer invasion. 2) Endosomal adhesomes – cross-talk between integrin activity and integrin “inside-in signaling”. We hypothesize that endocytosed active integrins engage in specialized endosomal signaling that governs cell survival especially in cancer. RNAi cell arrays, super-resolution STED imaging and endosomal proteomics will be used to investigate integrin signaling in endosomes. 3) Spatio-temporal co-ordination of adhesion and endocytosis. Several cytosolic proteins compete for integrin binding to regulate activation, endocytosis and recycling. Photoactivatable protein-traps and predefined matrix micropatterns will be employed to mechanistically dissect the spatio-temporal dynamics and hierarchy of their recruitment. We will employ innovative and unconventional techniques to address three major unanswered questions in the field and significantly advance our understanding of integrin function in development and cancer.

1 887 910 €

Start date: 2014-05-01, End date: 2019-04-30

The skeleton and the sinusoidal vasculature form a functional unit with great relevance in health, regeneration, and disease. Currently, fundamental aspects of sinusoidal vessel growth, specialization, arteriovenous organization and the consequences for tissue perfusion, or the changes occurring during ageing remain unknown. Our preliminary data indicate that key principles of bone vascularization and the role of molecular regulators are highly distinct from other organs. I therefore propose to use powerful combination of mouse genetics, fate mapping, transcriptional profiling, computational biology, confocal and two-photon microscopy, micro-CT and PET imaging, biochemistry and cell biology to characterize the growth, differentiation, dynamics, and ageing of the bone vasculature. In addition to established angiogenic pathways, the role of highly promising novel candidate regulators will be investigated in endothelial cells and perivascular osteoprogenitors with sophisticated inducible and cell type-specific genetic methods in the mouse. Complementing these powerful in vivo approaches, 3D co-cultures generated by cell printing technologies will provide insight into the communication between different cell types. The dynamics of sinusoidal vessel growth and regeneration will be monitored by two-photon imaging in the skull. Finally, I will explore the architectural, cellular and molecular changes and the role of capillary endothelial subpopulations in the sinusoidal vasculature of ageing and osteoporotic mice. Technological advancements, such as new transgenic strains, mutant models or cell printing approaches, are important aspects of this proposal. AngioBone will provide a first conceptual framework for normal and deregulated function of the bone sinusoidal vasculature. It will also break new ground by analyzing the role of blood vessels in ageing and identifying novel strategies for tissue engineering and, potentially, the prevention/treatment of osteoporosis.

2 478 750 €

Start date: 2014-02-01, End date: 2019-01-31

Apoptotic cell death is essential for development, immune function or tissue homeostasis, and it is often deregulated in disease. Mitochondrial outer membrane permeabilization (MOMP) is central for apoptosis execution and plays a key role in its inflammatory outcome. Knowing the architecture of the macromolecular machineries mediating MOMP is crucial for understanding their function and for the clinical use of apoptosis. Our recent work reveals that Bax and Bak dimers form distinct line, arc and ring assemblies at specific apoptotic foci to mediate MOMP. However, the molecular structure and mechanisms controlling the spatiotemporal formation and range of action of the apoptotic foci are missing. To address this fundamental gap in our knowledge, we aim to unravel the composition, dynamics and structure of apoptotic foci and to understand how they are integrated to orchestrate function. We will reach this goal by building on our expertise in cell death and cutting-edge imaging and by developing a new analytical pipeline to: 1) Identify the composition of apoptotic foci using in situ proximity-dependent labeling and extraction of near-native Bax/Bak membrane complexes coupled to mass spectrometry. 2) Define their contribution to apoptosis and its immunogenicity and establish their assembly dynamics to correlate it with apoptosis progression by live cell imaging. 3) Determine the stoichiometry and structural organization of the apoptotic foci by combining single molecule fluorescence and advanced electron microscopies. This multidisciplinary approach offers high chances to solve the long-standing question of how Bax and Bak mediate MOMP. APOSITE will provide textbook knowledge of the mitochondrial contribution to cell death and inflammation. The implementation of this new analytical framework will open novel research avenues in membrane and organelle biology. Ultimately, understanding of Bax and Bak structure/function will help develop apoptosis modulators for medicine.

2 000 000 €

Start date: 2019-04-01, End date: 2024-03-31

Cellular organelles are continuously remodelled by numerous cytosolic proteins that associate transiently with their lipid membrane. Some distort the bilayer, others change its composition, extract lipids or bridge membranes at distance. Previous works from my laboratory have underlined the importance of membrane sensors, i.e. elements within proteins that help to organize membrane-remodelling events by sensing the physical and chemical state of the underlying membrane. A membrane sensor is not necessarily of well-folded domain that interacts with a specific lipid polar head: some intrinsically unfolded motifs harboring deceptively simple sequences can display remarkable membrane adhesive properties. Among these are some amphipathic helices: the ALPS motif with a polar face made mostly by small uncharged polar residues, the Spo20 helix with several histidines in its polar face and, like a mirror image of the ALPS motif, the alpha-synuclein helix with very small hydrophobic residues. Using biochemistry and molecular dynamics, we will compare the membrane binding properties of these sequences (effect of curvature, charge, lipid unsaturation); using bioinformatics we will look for new motifs, using cell biology we will assess the adaptation of these motifs to the physical and chemical features of organelle membranes. Concurrently, we will use reconstitution approaches on artificial membranes to dissect how membrane sensors contribute to the organization of vesicle tethering by golgins and sterol transport by ORP proteins. We surmise that the combination of a molecular ¿switch¿, a small G protein of the Arf family, and of membrane sensors permit to organize these complex reactions in time and in space.

1 997 321 €

Start date: 2011-05-01, End date: 2015-04-30

It is a basic textbook notion that the plasma membranes of virtually all organisms display an asymmetric lipid distribution between inner and outer leaflets far removed from thermodynamic equilibrium. As a fundamental biological principle, lipid asymmetry has been linked to numerous cellular processes. However, a clear mechanistic justification for the continued existence of lipid asymmetry throughout evolution has yet to be established. We propose here that lipid asymmetry serves as a store of potential energy that is used to fuel energy-intense membrane remodelling and signalling events for instance during membrane fusion and fission. This implies that rapid, local changes of trans-membrane lipid distribution rather than a continuously maintained out-of-equilibrium situation are crucial for cellular function. Consequently, new methods for quantifying the kinetics of lipid trans-bilayer movement are required, as traditional approaches are mostly suited for analysing quasi-steady-state conditions. Addressing this need, we will develop and employ novel photochemical lipid probes and lipid biosensors to quantify localized trans-bilayer lipid movement. We will use these tools for identifying yet unknown protein components of the lipid asymmetry regulating machinery and analyse their function with regard to membrane dynamics and signalling in cell motility. Focussing on cell motility enables targeted chemical and genetic perturbations while monitoring lipid dynamics on timescales and in membrane structures that are well suited for light microscopy. Ultimately, we aim to reconstitute lipid asymmetry as a driving force for membrane remodelling in vitro. We expect that our work will break new ground in explaining one of the least understood features of the plasma membrane and pave the way for a new, dynamic membrane model. Since the plasma membrane serves as the major signalling hub, this will have impact in almost every area of the life sciences.

1 500 000 €

Start date: 2018-01-01, End date: 2022-12-31

The phytohormone auxin has profound importance for plant development. The extracellular AUXIN BINDING PROTEIN1 (ABP1) and the nuclear AUXIN F-BOX PROTEINs (TIR1/AFBs) auxin receptors perceive fast, non-genomic and slow, genomic auxin responses, respectively. Despite the fact that ABP1 mainly localizes to the endoplasmic reticulum (ER), until now it has been proposed to be active only in the extracellular matrix (reviewed in Sauer and Kleine-Vehn, 2011). Just recently, ABP1 function was also linked to genomic responses, modulating TIR1/AFB-dependent processes (Tromas et al., 2013). Intriguingly, the genomic effect of ABP1 appears to be at least partially independent of the endogenous auxin indole 3-acetic acid (IAA) (Paque et al., 2014). In this proposal my main research objective is to unravel the importance of the ER for genomic auxin responses. The PIN-LIKES (PILS) putative carriers for auxinic compounds also localize to the ER and determine the cellular sensitivity to auxin. PILS5 gain-of-function reduces canonical auxin signaling (Barbez et al., 2012) and phenocopies abp1 knock down lines (Barbez et al., 2012, Paque et al., 2014). Accordingly, a PILS-dependent substrate could be a negative regulator of ABP1 function in the ER. Based on our unpublished data, an IAA metabolite could play a role in ABP1-dependent processes in the ER, possibly providing feedback on the canonical nuclear IAA-signaling. I hypothesize that the genomic auxin response may be an integration of auxin- and auxin-metabolite-dependent nuclear and ER localized signaling, respectively. This proposed project aims to characterize a novel auxin-signaling paradigm in plants. We will employ state of the art interdisciplinary (biochemical, biophysical, computational modeling, molecular, and genetic) methods to assess the projected research. The identification of the proposed auxin conjugate-dependent signal could have far reaching plant developmental and biotechnological importance.

1 441 125 €

Start date: 2015-06-01, End date: 2020-11-30

Bacteria can generate mechanical forces that are important for the colonization of surfaces, formation of biofilms, and infection of host cells. This proposal addresses the fundamental question of how bacteria can control their force generation to robustly respond to chemo-mechanical cues on complex surfaces. Currently, a knowledge gap exists between the molecular regulation pathways on the one hand and the mechanical behavior on the other hand. One major impediment for understanding of how behavior is connected to control is, to date, the impossibility of studying bacterial force directly in unconstrained situations. Based on an initial study, I propose employing new methods for the unperturbed, high-resolution measurement of bacterial traction forces on wide spatiotemporal scales. Thus, the force-generation linking behavior to control can be investigated directly. The objectives are to (A) gain access to nanoscopic mechanical phenomena through the development of cutting-edge super-resolution traction force microscopy, (B) employ the methods to characterize how Pseudomonas aeruginosa controls pilus-generated forces while responding to chemical cues, and (C) establish how surface rigidity affects force generation by P. aeruginosa during biofilm formation. In an interdisciplinary approach, I will combine traction measurements with genetic perturbations, molecule labeling, and computer simulations to produce functional models of the mechanocontrol strategies. Altogether, I will establish a novel technique, opening up the possibility of studying nanoscopic force generation in many types of cells. Through these advances, I will characterize a set of mechanoregulation strategies in P. aeruginosa that are paradigmatic for diverse Gram-negative pathogens employing the same type of pili. Broadly, I expect that the studied bacterial control strategies have a generic, minimal nature and can appear as basic motives throughout development, homeostasis, and disease.

1 498 864 €

Start date: 2020-08-01, End date: 2025-07-31

One of the ultimate goals in cell biology is to understand how cells determine their shape. In bacteria, the cell wall and the actin-like (MreB) cytoskeleton are major determinants of cell shape. As a hallmark of microbial life, the external cell wall is the most conspicuous macromolecule expanding in concert with cell growth and one of the most prominent targets for antibiotics. Despite decades of study, the mechanism of cell wall morphogenesis remains poorly understood. In rod-shaped bacteria, actin-like MreB proteins assemble into disconnected membrane-associated structures (patches) that move processively around the cell periphery and are thought to control shape by spatiotemporally organizing macromolecular machineries that effect sidewall elongation. However, the ultrastructure of MreB assemblies and the mechanistic details underlying their morphogenetic function remain to be elucidated. The aim of this project is to combine ground-breaking light microscopy and spectroscopy techniques with cutting-edge genetic, biochemical and systems biology approaches available in the model rod-shaped bacterium Bacillus subtilis to elucidate how MreB and cell wall biosynthetic enzymes collectively act to build a cell. Within this context, new features of MreB assemblies will be determined in vivo and in vitro, and a “toolbox” of approaches to determine the modes of action of antibiotics targeting cell wall processes will be developed. Parameters measured by the different approaches will be used to refine a mathematical model aiming to quantitatively describe the features of bacterial cell wall growth. The long-term goals of BActin are to understand general principles of bacterial cell morphogenesis and to provide mechanistic templates and new reporters for the screening of novel antibiotics.

1 902 195 €

Start date: 2019-02-01, End date: 2024-01-31

Most of the lymphomas diagnosed in the western world are originated from mature B cells. The hallmark of these malignancies is the presence of recurrent chromosome translocations that usually involve the immunoglobulin loci and a proto-oncogene. As a result of the translocation event the proto-oncogene becomes deregulated under the influence of immunoglobulin cis sequences thus playing an important role in the etiology of the disease. Upon antigen encounter mature B cells engage in the germinal center reaction, a complex differentiation program of critical importance to the development of the secondary immune response. The germinal center reaction entails the somatic remodelling of immunoglobulin genes by the somatic hypermutation and class switch recombination reactions, both of which are triggered by Activation Induced Deaminase (AID). We have previously shown that AID also initiates lymphoma-associated c-myc/IgH chromosome translocations. In addition, the germinal center reaction involves a fine-tuned balance between intense B cell proliferation and program cell death. This environment seems to render B cells particularly vulnerable to malignant transformation. We aim at studying the molecular events responsible for B cell susceptibility to lymphomagenesis from two perspectives. First, we will address the role of AID in the generation of lymphomagenic lesions in the context of AID specificity and transcriptional activation. Second, we will approach the regulatory function of microRNAs of AID-dependent, germinal center events. The proposal aims at the molecular understanding of a process that lies in the interface of immune regulation and oncogenic transformation and therefore the results will have profound implications both to basic and clinical understanding of lymphomagenesis.

1 596 000 €

Start date: 2008-12-01, End date: 2014-11-30

The goal of this proposal is to deliver a new theoretical framework to understand how transcription factors (TFs) sustain cell identity during developmental processes. Recognised as key drivers of cell fate acquisition, TFs are currently not considered to directly contribute to the mitotic inheritance of chromatin states. Instead, these are passively propagated through cell division by a variety of epigenetic marks. Recent discoveries, including by our lab, challenge this view: developmental TFs may impact the propagation of regulatory information from mother to daughter cells through a process known as mitotic bookmarking. This hypothesis, largely overlooked by mainstream epigenetic research during the last two decades, will be investigated in embryo-derived stem cells and during early mouse development. Indeed, these immature cell identities are largely independent from canonical epigenetic repression; hence, current models cannot account for their properties. We will comprehensively identify mitotic bookmarking factors in stem cells and early embryos, establish their function in stem cell self-renewal, cell fate acquisition and dissect how they contribute to chromatin regulation in mitosis. This will allow us to study the relationships between bookmarking factors and other mechanisms of epigenetic inheritance. To achieve this, unique techniques to modulate protein activity and histone modifications specifically in mitotic cells will be established. Thus, a mechanistic understanding of how mitosis influences gene regulation and of how mitotic bookmarking contributes to the propagation of immature cell identities will be delivered. Based on robust preliminary data, we anticipate the discovery of new functions for TFs in several genetic and epigenetic processes. This knowledge should have a wide impact on chromatin biology and cell fate studies as well as in other fields studying processes dominated by TFs and cell proliferation.

1 900 844 €

Start date: 2018-09-01, End date: 2023-08-31

My lab is interested in the development of the tissue that gives rise to vertebrae and skeletal muscles called the paraxial mesoderm. A striking feature of this tissue is its segmental organization and we have made major contributions to the understanding of the molecular control of the segmentation process. We identified a molecular oscillator associated to the rhythmic production of somites and proposed a model for vertebrate segmentation based on the integration of a rhythmic signaling pulse gated spatially by a system of traveling FGF and Wnt signaling gradients. We are also studying the differentiation of paraxial mesoderm precursors into the muscle, cartilage and dermis lineages. Our work identified the Wnt, FGF and Notch pathways as playing a prominent role in the patterning and differentiation of paraxial mesoderm. In this application, we largely focus on the molecular control of paraxial mesoderm development. Using microarray and high throughput sequencing-based approaches and bioinformatics, we will characterize the transcriptional network acting downstream of Wnt, FGF and Notch in the presomitic mesoderm (PSM). We will also use genetic and pharmacological approaches utilizing real-time imaging reporters to characterize the pacemaker of the segmentation clock in vivo, and also in vitro using differentiated embryonic stem cells. We further propose to characterize in detail a novel RA-dependent pathway that we identified and which controls the somite left-right symmetry. Our work is expected to have a strong impact in the field of congenital spine anomalies, currently an understudied biomedical problem, and will be of utility in elucidating the etiology and eventual prevention of these disorders. This work is also expected to further our understanding of the Notch, Wnt, FGF and RA signalling pathways which are involved in segmentation and in the establishment of the vertebrate body plan, and which play important roles in a wide array of human diseases.

2 500 000 €

Start date: 2010-04-01, End date: 2015-03-31

In contrast to mammals, newts possess exceptional capacities among vertebrates to rebuild complex structures, such as the brain. Our goal is to bridge the gap in the regenerative outcomes between newts and mammals. My group has made significant contributions towards this goal. We created a novel experimental system, which recapitulates central features of Parkinson’s disease in newts, and provides a unique model for understanding regeneration in the adult midbrain. We showed an unexpected but key feature of the newt brain that it is akin to the mammalian brain in terms of the extent of homeostatic cell turn over, but distinct in terms of its injury response, showing the regenerative capacity of the adult vertebrate brain by activating neurogenesis in normally quiescent regions. Further we established a critical role for the neurotransmitter dopamine in controlling quiescence in the midbrain, thereby preventing neurogenesis during homeostasis and terminating neurogenesis once the correct number of neurons has been produced during regeneration. Here we aim to identify key molecular pathways that regulate adult neurogenesis, to define lineage relationships between neuronal stem and progenitor cells, and to identify essential differences between newts and mammals. We will combine pharmacological modulation of neurotransmitter signaling with extensive cellular fate mapping approaches, and molecular manipulations. Ultimately we will test hypotheses derived from newt studies with mammalian systems including newt/mouse cross species complementation approaches. We expect that our findings will provide new regenerative strategies, and reveal fundamental aspects of cell fate determination, tissue growth, and tissue maintenance in normal and pathological conditions.

1 500 000 €

Start date: 2012-02-01, End date: 2017-01-31

My lab studies how cells regulate their growth and metabolism during normal development and in disease. Recent work in my lab, published last year in Nature, identified the metabolite stearic acid (C18:0) as a novel regulator of mitochondrial function. We showed that dietary C18:0 acts via a novel signaling route whereby it covalently modifies the cell-surface Transferrin Receptor (TfR1) to regulate mitochondrial morphology. We found that modification of TfR1 by C18:0 ('stearoylation') is analogous to protein palmitoylation by C16:0 - it is a covalent thio-ester link and requires a transferase enzyme. This work made two conceptual contributions. 1) It uncovered a novel signaling route regulating mitochondrial function. 2) Relevant to this grant application, we found by mass spectrometry multiple other proteins that are stearoylated in mammalian cells. This thereby opens a new avenue of research, suggesting that C18:0 signals via several target proteins to regulate cellular growth and metabolism. I propose here to study this C18:0 signaling. To study C18:0 signaling we will exploit tools recently developed in my lab to 1) identify as complete a set as possible of proteins that are stearoylated in human and Drosophila cells, thereby characterizing the cellular 'stearylome', 2) study how stearoylation affects the molecular function of these target proteins, and thereby cellular growth and metabolism, and 3) study how stearoylation is added, and possibly removed, from target proteins. This work will change the way we view C18:0 from simply being a metabolite to being an important dietary signaling molecule that links nutritional uptake to cellular physiology. Via unknown mechanisms, dietary C18:0 is clinically known to have special properties for cardiovascular risk. Hence this proposal, discovering how C18:0 signals to regulate cells, will have implications for both normal development and for disease.

2 000 000 €

Start date: 2017-03-01, End date: 2022-02-28

Plant and animal organs display a remarkable diversity of shapes. A major challenge in developmental and evolutionary biology is to understand how this diversity of forms is generated. Recent advances in imaging, computational modelling and genomics now make it possible to address this challenge effectively for the first time. Leaf development is a particularly tractable system because of its accessibility to imaging and preservation of connectivity during growth. Leaves also display remarkable diversity in shape and form, with perhaps the most complex form being the pitcher-shaped (epiascidiate) leaves of carnivorous plants. This form has evolved four times independently, raising the question of whether its seeming complexity may have arisen through simple modulations in underlying morphogenetic mechanisms. To test this hypothesis, I aim to develop a model system for carnivorous plants based on Utricularia gibba (humped bladderwort), which has the advantage of having one of the smallest genomes known in plants (~2/3 the size of the Arabidopsis genome) and small transparent pitcher-shaped leaves amenable to imaging. I will use this system to define the morphogenetic events underlying the formation of pitcher-shaped leaves and their molecular genetic control. I will also develop and apply computational modelling to explore hypotheses that may account for the development of U. gibba bladders and further test these hypotheses experimentally. In addition, I will investigate the relationship between U. gibba bladder development and species with simpler leaf shapes, such as Arabidopsis, or species where the epiascidiate form has evolved independently. Taken together, these studies should show how developmental rules elucidated in current model systems might be extended and built upon to account for the diversity and complexity of tissue forms, integrating evo-devo approaches with a mechanistic understanding of morphogenesis.

2 499 997 €

Start date: 2013-06-01, End date: 2018-05-31

The discovery of adult neural stem cells (NSCs) has broaden our view of the physiological plasticity of the nervous system, and has opened new perspectives on the possibility of tissue regeneration and repair in the brain. NSCs reside in specialized niches in the adult mammalian nervous system, where they are exposed to specific paracrine signals regulating their behavior. These neural progenitors are generally in a quiescent state within their niche, and they activate their proliferation depending on tissue regenerative and growth needs. Understanding the mechanisms by which NSCs enter and exit the quiescent state is crucial for the comprehension of the physiology of the adult nervous system. In this project we will study the behavior of a specific subpopulation of adult neural stem cells recently described by our group in the carotid body (CB). This small organ constitutes the most important chemosensor of the peripheral nervous system and has neuronal glomus cells responsible for the chemosensing, and glia-like sustentacular cells which were thought to have just a supportive role. We recently described that these sustentacular cells are dormant stem cells able to activate their proliferation in response to a physiological stimulus like hypoxia, and to differentiate into new glomus cells necessary for the adaptation of the organ. Due to our precise experimental control of the activation and deactivation of the CB neurogenic niche, we believe the CB is an ideal model to study fundamental questions about adult neural stem cell physiology and the interaction with the niche. We propose to study the cellular and molecular mechanisms by which these carotid body stem cells enter and exit the quiescent state, which will help us understand the physiology of adult neurogenic niches. Likewise, understanding this neurogenic process will improve the efficacy of using glomus cells for cell therapy against neurological disease, and might help us understand some neural tumors.

1 476 000 €

Start date: 2010-11-01, End date: 2015-10-31

The molecular mechanisms that mediate cell competition, cell fitness and cell selection is gaining interest. With innovative approaches, molecules and ground-breaking hypothesis, this field of research can help understand several biological processes such as development, cancer and tissue degeneration. The project has 3 clear and ambitious objectives: 1. We propose to identify all the key genes mediating cell competition and their molecular mechanisms. In order to reach this objective we will use data from two whole genome screens in Drosophila where we have identified 7 key genes. By the end of this CoG grant, we should have no big gaps in our knowledge of how slow dividing cells are recognised and eliminated in Drosophila. 2. In addition, we will explore how general the cell competition pathways are and how they can impact biomedical research, with a focus in cancer and tissue degeneration. The interest in cancer is based on experiments in Drosophila and mice where we and others have found that an active process of cell selection determines tumour growth. Preliminary results suggest that the pathways identified do not only play important roles in the elimination of slow dividing cells, but also during cancer initiation and progression. 3. We will further explore the role of cell competition in neuronal selection, specially during neurodegeneration, development of the retina and adult brain regeneration in Drosophila. This proposal is of an interdisciplinary nature because it takes a basic cellular mechanism (the genetic pathways that select cells within tissues) and crosses boundaries between different fields of research: development, cancer, regeneration and tissue degeneration. In this ERC CoG proposal, we are committed to continue our efforts from basic science to biomedical approaches. The phenomena of cell competition and its participating genes have the potential to discover novel biomarkers and therapeutic strategies against cancer and tissue degeneration.

1 968 062 €

Start date: 2014-06-01, End date: 2019-05-31

The regulation of cell migration is central in pattern formation, homeostasis and disease. The proposed research is aimed at investigating the molecular basis for cell motility and the associated polarization of the cell. In view of the dynamic nature of these processes, we have chosen to utilize the migration of Primoridal Germ Cells (PGCs) in zebrafish - a model that offers unique experimental advantages for imaging and experimental manipulations. The fact that molecules facilitating the motility of zebrafish PGCs are evolutionary conserved and the finding that the cells are directed by chemokines, molecules that control a wide range of cell trafficking events in vertebrates, make this in vivo study of particular importance. The proposed work involves both the functional analysis of previously identified candidates and the identification of molecules, which have a presently unknown effect on the migration process. For both objectives, we will employ novel experimental schemes. We will examine the role of proteins in achieving functional cell polarity compatible with efficient motility and response to directional cues, using unique techniques and analysis tools in the context of the living organism. The precise function of the identified proteins will be determined by combining mathematical tools aimed at quantitatively gauging the role of the molecules in conferring proper cell shape, biophysical methods aimed at measuring forces, rigidity and cytoplasm flow and determination of the effect on the organization of relevant structures using cryo electron tomography. Together, this approach would provide a non-conventional understanding of cell migration by correlating structural, morphological and dynamic cellular properties with the ability of cells to effectively migrate towards their target.

1 960 600 €

Start date: 2011-06-01, End date: 2017-05-31

The formation of plant organs (leaves, roots, flowers) depends on the activity of stem cells (SC), located in stem cell niches (meristems) together with adjoining organizer cells (OC) that prevent SC differentiation. Despite their importance, SC and OC have been poorly described at molecular and cellular level and mechanisms for their coordinated specification are only partially understood. We study the specification of the very first SC and OC for the root in the early Arabidopsis embryo where cell divisions are almost invariant and, in the absence of cell motility, highly predictable. Previously we have established a central role for the transcription factor MONOPTEROS (MP) in OC specification and we have recently found that MP also controls SC specification. Hence, MP offers a unique entry point into studying the genomic and cellular reprogramming that underlies coordinated SC and OC specification. Our recent identification of MP target genes has shown that its function in SC specification is cell-autonomous, while MP-dependent OC specification involves a mobile transcription factor. In recent years we have developed a set of resources to systematically study embryonic root meristem initiation, and are now in a unique position to answer the following questions in this ERC project: 1. What transcriptional reprogramming underlies the first specification of SC and OC in the plant embryo? 2. What cellular changes follow from transcriptional reprogramming and mediate elongation and asymmetric division of SC and OC? 3. What is the mechanism of directional protein transport that ensures spatiotemporal coordination between SC and OC? The project will provide genome-wide insight in the cellular reprogramming underlying the coordinated formation of a multicellular structure. Finally, this work will shed light on mechanisms of stem cell and stem cell niche formation.

1 499 070 €

Start date: 2011-10-01, End date: 2016-09-30

The difference between males and females constitutes the largest phenotypic dimorphism in most species. In humans, this variation accounts for differences seen in the risk, incidence and response to treatment for a plethora of diseases; and much of these striking differences are not explained at this time. While sex organ-derived hormones play key roles in sculpting and maintaining sex differences, my recent work highlighted the importance of cell-intrinsic mechanisms involving the sex chromosomes. In fact, using fly models I demonstrated that the sex of intestinal stem cells plays a key role in the adult gut, both for the organ size and for the sex-specific pre-disposition to tumours. While these findings establish the proof-of-principle of the influence of sex chromosomes in adult cells, essential gaps remain to be filled. Indeed, the full range of phenotypic consequences of the presence of sex chromosomes in somatic cells, the genes, the mechanisms involved and their sites of action remain entirely elusive. My research proposal aims to understand how cellular sex impacts physiology across the body using Drosophila as an in vivo model. This question has been poorly investigated in part due to the difficulties of studying sex chromosome effects. Flies will offer the remarkable possibility of generating mosaic animals in which sex chromosomes will be genetically manipulated in defined organs. Here I will combine classical fly genetics, novel genetic methods and cutting-edge genomic techniques to: 1. characterise new cellular sex pathways driving sex differences in body size and in behaviours, 2. study the role of sex determinant coding changes in sex trait evolution, 3. achieve, for the first time, organ-specific Y chromosome deletion, and use this new method to study how the Y chromosome controls sex gap in longevity. Thus, results from this research should have major impact on our understanding of the importance of cellular sex in physiology and disease.

1 498 365 €

Start date: 2020-05-01, End date: 2025-04-30

The organization and dynamics of the MT (MT) cytoskeleton underlies the morphology, polarization and division of most cells. The structural polarity of MT determines the directionality of motor proteins, which move selectively towards either the MT plus (most kinesins) or minus end (dynein) to control the transport and positioning of proteins and organelles. Understanding how different cellular MT arrays, such as the mitotic spindle or neuronal MT networks, are built and utilized to ensure proper cellular logistics is a central challenge in cell biology. Recently, our lab has introduced a new technique, motor-PAINT, to directly resolve MT polarity and the relation between MT orientations, stability and modifications. This revealed that in neurons, the mixed polarity MT network in the dendrites is much more ordered than previously anticipated. MTs with opposite orientations have different properties and are preferred by distinct kinesins, revealing an architectural principle that could explain why different plus-end directed motors move towards distinct destinations. Nevertheless, the mechanisms by which this specialized organization is established and the different ways in which it modulates intracellular transport have remained unknown. To resolve how cytoskeletal organization guides transport, I propose to explore the form, formation and functioning of the neuronal MT cytoskeleton. We will combine advanced microscopy, molecular biology, and mathematical modelling to: 1) Create a complete 3D map of the dendritic MT cytoskeleton – form. 2) Unravel the mechanisms that establish MT organization in dendrites – formation. 3) Explore how specific MT configurations modulate intracellular transport – function. This research will uncover key mechanisms of cytoskeletal organization and transport in neurons. In addition, our techniques and concepts will aid understanding intracellular transport in other cellular systems.

2 000 000 €

Start date: 2019-05-01, End date: 2024-04-30

"Centrioles are critical for the formation of cilia, flagella and centrosomes, as well as for human health. The mechanisms governing centriole formation constitute a long-standing question in cell biology. We will pursue an innovative multidisciplinary research program to gain further insight into these mechanisms, using human cells, C. elegans and Trichonympha as model systems. This program is expected to also have a major impact by contributing a novel cell free assay to the field, thus paving the way towards making synthetic centrioles. Six specific aims will be pursued: 1) Deciphering HsSAS-6/STIL distribution and dynamics. We will use super-resolution microscopy, molecular counting, photoconversion and FCS to further characterize these two key components required for centriole formation in human cells. 2) The SAS-6 ring model as a tool to redirect centriole organization. Utilizing predictions from the SAS-6 ring model, we will assay the consequences for centrioles and cilia of altering the diameter and symmetry of the structure. 3) Determining the architecture of C. elegans centrioles. We will conduct molecular counting and cryo-ET of purified C. elegans centrioles to determine if they contain a spiral or a cartwheel, as well as identify SAS-6-interacting components. 4) Comprehensive 3D map and proteomics of Trichonympha centriole. We will obtain a ~35 Å 3D map of the complete T. agilis centriole, perform proteomic analysis to identify its constituents and test their function using RNAi. 5) Regulation of cartwheel height and centriole length. We will explore whether cartwheel height is set by SAS-6 proteins and perform screens in human cells to identify novel components regulating cartwheel height and centriole length. 6) Novel cell free assay for cartwheel assembly and centriole formation. Using SAS-6 proteins on a lipid monolayer as starting point, we will develop and utilize a cell-free assay to reconstitute cartwheel assembly and centriole format"

2 499 270 €

Start date: 2014-04-01, End date: 2019-03-31

Centrioles assemble centrosomes and cilia/flagella, critical structures for cell division, polarity, motility and signalling, which are often deregulated in human disease. Centriole inheritance, in particular the preservation of their copy number and position in the cell is critical in many eukaryotes. I propose to investigate, in an integrative and quantitative way, how centrioles are formed in the right numbers at the right time and place, and how they are maintained to ensure their function and inheritance. We first ask how centrioles guide their own assembly position and centriole copy number. Our recent work highlighted several properties of the system, including positive and negative feedbacks and spatial cues. We explore critical hypotheses through a combination of biochemistry, quantitative live cell microscopy and computational modelling. We then ask how the centrosome and the cell cycle are both coordinated. We recently identified the triggering event in centriole biogenesis and how its regulation is akin to cell cycle control of DNA replication and centromere assembly. We will explore new hypotheses to understand how assembly time is coupled to the cell cycle. Lastly, we ask how centriole maintenance is regulated. By studying centriole disappearance in the female germline we uncovered that centrioles need to be actively maintained by their surrounding matrix. We propose to investigate how that matrix provides stability to the centrioles, whether this is differently regulated in different cell types and the possible consequences of its misregulation for the organism (infertility and ciliopathy-like symptoms). We will take advantage of several experimental systems (in silico, ex-vivo, flies and human cells), tailoring the assay to the question and allowing for comparisons across experimental systems to provide a deeper understanding of the process and its regulation.

2 000 000 €

Start date: 2017-01-01, End date: 2021-12-31

Centrosomes are cytoplasmic organelles found in most animal cells with important roles in polarity establishment and maintenance. Theodor Boveri s pioneering work first suggested that extra-centrosomes could contribute to genetic instability and consequently to tumourigenesis. Although many human tumours do exhibit centrosome amplification (extra centrosomes) or centrosome abnormalities, the exact contribution of centrosomes to tumour initiation in vertebrate organisms remains to be determined. I have recently showed that Drosophila flies carrying extra-centrosomes, following the over-expression of the centriole replication kinase Sak, did not exhibit chromosome segregation errors and were able to maintain a stable diploid genome over many generations. Surprisingly, however, neural stem cells fail frequently to align the mitotic spindle with their polarity axis during asymmetric division. Moreover, I have found that centrosome amplification is permissive to tumour formation in flies. So far, however, we do not know the molecular mechanisms that allow transformation when extra centrosomes are present and elucidating these mechanisms is the aim of the work presented in this proposal. Here, I describe a series of complementary approaches that will help us to decipher the link between centrosomes, stem cells and tumour biology. In addition, I wish to pursue the original observations made in Drosophila and investigate the consequences of centrosome amplification in mammals.

1 550 000 €

Start date: 2010-01-01, End date: 2015-06-30

Protein misfolding causes devastating health conditions such as neurodegeneration. Although the disease-causing protein is widely expressed, its misfolding occurs only in certain cell-types such as neurons. What governs the susceptibility of some tissues to misfolding is a fundamental question with biomedical relevance. Molecular chaperones help cellular proteins fold into their native conformation. Despite the generality of their function, chaperones are differentially expressed across various tissues. Moreover exposure to misfolding stress changes chaperone expression in a cell-type-dependent manner. Thus cell-type-specific regulation of chaperones is a major determinant of susceptibility to misfolding. The molecular mechanisms governing chaperone levels in different cell-types are not understood, forming the basis of this proposal. We will take a multidisciplinary approach to address two key questions: (1) How are chaperone levels co-ordinated with tissue-specific demands on protein folding? (2) How do different cell-types regulate chaperone genes when exposed to the same misfolding stress? Cellular chaperone levels and their response to misfolding stress are both driven by transcriptional changes and influenced by chromatin. The proposed work will bring the conceptual, technological and computational advances of chromatin/ transcription field to understand chaperone biology and misfolding diseases. Using in vivo mouse model and in vitro differentiation model, we will investigate molecular mechanisms that control chaperone levels in relevant tissues. Our work will provide insights into functional specialization of chaperones driven by tissue-specific folding demands. We will develop a novel and ambitious approach to assess protein-folding capacity in single cells moving the chaperone field beyond state-of-the-art. Thus by implementing genetic, computational and biochemical approaches, we aim to understand cell-type-specificity of chaperone regulation.

1 992 500 €

Start date: 2020-01-01, End date: 2024-12-31

One of the most dramatic transitions in biology is the oocyte-to-zygote transition. This refers to the maturation of the female germ cell or oocyte, which undergoes two rounds of meiotic chromosome segregation and, following fertilization, is converted to a mitotically dividing embryo. We aim to establish an innovative research program that addresses fundamental questions about the molecular processes controlling the mammalian oocyte-to-zygote transition to ensure faithful inheritance of genomes from one generation to the next. We are taking an interdisciplinary approach combining germ cell and chromosome biology with cell cycle and epigenetic studies to understand how maternal factors regulate chromosome segregation in oocytes and chromatin organization in the zygote. A molecular understanding of key players regulating these processes is a requisite step for investigating how their deterioration contributes to maternal age-related aneuploidy and infertility. Aneuploidy is the leading cause of mental retardation and spontaneous miscarriage. The current trend towards advanced maternal age has increased the frequency of trisomic fetuses by 71% in the past ten years. A better understanding of mammalian meiosis is therefore relevant to human reproductive health. A special feature of the female germ line is that meiotic DNA replication occurs in the embryo but oocytes remain arrested until the first meiotic division is triggered months (mouse) or decades (human) later. The longevity of oocytes poses a challenge for the cohesin complex that must hold together sister chromatids from DNA synthesis until chromosome segregation. We specifically aim to: 1) elucidate how sister chromatid cohesion is maintained in mammalian oocytes, 2) identify mechanisms regulating cohesion in young and aged oocytes, and 3) investigate how the inheritance of genetic and resetting of epigenetic information is coordinated with cell cycle progression at the oocyte-to-zygote transition.

1 499 738 €

Start date: 2014-02-01, End date: 2019-01-31

Eukaryotic cells inherit much of their genomic information in the form of chromosomes during cell division. Centimetre-long DNA molecules are packed into micrometer-sized chromosomes to enable this process. How DNA is organised within mitotic chromosomes is still largely unknown. A key structural protein component of mitotic chromosomes, implicated in their compaction, is the condensin complex. In this proposal, we aim to elucidate the molecular architecture of mitotic chromosomes, taking advantage of new genomic techniques and the relatively simple genome organisation of yeast model systems. We will place particular emphasis on elucidating the contribution of the condensin complex, and the cell cycle regulation of its activities, in promoting chromosome condensation. Our previous work has provided genome-wide maps of condensin binding to budding and fission yeast chromosomes. We will continue to decipher the molecular determinants for condensin binding. To investigate how condensin mediates DNA compaction, we propose to generate chromosome-wide DNA/DNA proximity maps. Our approach will be an extension of the chromosome conformation capture (3C) technique. High throughput sequencing of interaction points has provided a first glimpse of the interactions that govern chromosome condensation. The role that condensin plays in promoting these interactions will be investigated. The contribution of condensin s ATP-dependent activities, and cell cycle-dependent post-translational modifications, will be studied. This will be complemented by mathematical modelling of the condensation process. In addition to chromosome condensation, condensin is required for resolution of sister chromatids in anaphase. We will develop an assay to study the catenation status of sister chromatids and how condensin may contribute to their topological resolution.

2 076 126 €

Start date: 2010-04-01, End date: 2015-03-31

All animal life starts with the fertilization of an egg. A haploid egg and a haploid sperm fuse and together they form a new genetically unique embryo. But surprisingly, eggs frequently contain an incorrect number of chromosomes. Depending on the age of the woman, 10-50% of eggs are chromosomally abnormal. This high percentage of abnormal eggs results from chromosome segregation errors during oocyte maturation, the process by which a diploid oocyte matures into a haploid egg. Thus, errors during meiosis in human oocytes are the most common cause of pregnancy losses and contribute to approximately 95% of human aneuploidy such as Down’s syndrome. Surprisingly, we still know very little about how mammalian oocytes mature into eggs, and it is still unclear why chromosome segregation during meiosis is so much more error-prone than during mitosis. My proposal combines three innovative and complementary approaches towards understanding how homologous chromosomes are segregated and why oocyte maturation in mammals is so error-prone. Specifically, we will work towards the following three aims: 1. We will complete the first large scale screen for genes required for accurate progression through meiosis in mammalian oocytes and characterize the function of a few selected genes in detail. 2. We will analyse meiosis and investigate potential causes of chromosome segregation errors directly in live human oocytes. 3. We will study the function of an F-actin spindle and a chromosome-associated myosin that might be required for chromosome segregation in mammalian oocytes. Because errors during oocyte maturation lead to pregnancy loss, birth defects and infertility, this work will not only provide important insights into fundamental cellular mechanisms, but will also have important implications for human health.

1 487 611 €

Start date: 2014-02-01, End date: 2019-01-31

The mechanisms regulating neural stem cells and their progression to neurogenesis are important not only to understand brain development and evolution, but also to elicit neurogenesis after brain injury. Our recent findings imply novel chromatin-associated proteins in the regulation of neural stem cell fate and neurogenesis. Therefore this project aims to understand the molecular mechanisms of how these factors regulate neurogenesis in developing and adult mice (Aim1) and implement this knowledge for reprogramming glia into neurons after brain injury (Aim2). This will be pursued in mouse models in vivo (2.1) and with human glial cells derived from patient brain resections in vitro (2.2). It is well known that transcription factors need to alter the chromatin structure to achieve transcriptional regulation, but the factors involved in this regulation in neural stem and progenitor cells are still ill understood. Therefore the molecular function of the novel chromatin interacting protein Trnp1 with essential roles in neural stem cell (NSC) fate and the chromatin conformation mediated at neurogenic target genes by Pax6/Brg1-containing BAF complexes will be addressed in Aim1. Combined with genome-wide approaches to determine changes in chromatin conformation at neurogenic target gene sites this will greatly further our understanding of key roles of chromatin conformation in neural stem cells and neurogenesis. In Aim2 Trnp1 promoting neural stem cells fate and later acting neurogenic transcription factors will be used to improve neuronal reprogramming after stab wound injury in the murine brain in vivo and in patient-derived glial cells in vitro. Together with novel strategies to induce chromatin looping in a sequence-specific manner this project will not only advance our knowledge at the frontier of transcriptional regulation in neurogenesis, but also implement highly innovative approaches to utilize this knowledge for neuronal repair by direct reprogramming.

2 376 560 €

Start date: 2014-02-01, End date: 2019-01-31

Motile cilia are tiny microtubule-based projections which create fluid flow and are essential to human health. Cilia movement is powered by coordinated action of complex macromolecular motors, the axonemal dyneins. During differentiation, as cells produce hundreds of motile cilia, millions of dynein subunits must be pre-assembled in the cytoplasm into very large complexes in the correct stoichiometry which are then trafficked into growing cilia. This poses a sizeable challenge for the cell in terms of allocation of a significant fraction of the global translational machinery for streamlined assembly of dyneins within a crowded cellular space. The key question remains: How does the cell know how much is enough? This is an extreme example of a common problem in cell biology. Responsive and adaptive mechanisms must exist to prevent futile expenditure of cellular resources in making a surplus of large molecules like dyneins that may also pose a risk of toxic aggregation. While a well-defined transcriptional code for induction of cilia motility genes exists, the translational dynamics and subsequent feedback circuitry coordinating dynein pre-assembly with ciliogenesis remain unexplored. The molecular logic underlying the construction of motile cilia assembly are still not fully understood. The ambitious nature of CiliaCircuits proposes to use super-resolution and systems approaches to elucidate key mechanisms regulating this process in health and disease. Human genetics tells us that making cilia motile is a complex process. To date, almost 40 genes have been implicated in primary ciliary dyskinesia (PCD), the disease of motile cilia, for which there is no cure. The long-term vision is to understand this dynamic control operating over a specialized proteome in time and space in order to develop effective PCD therapeutics and identify additional candidate genes involved in this translation regulation.

1 965 460 €

Start date: 2020-08-01, End date: 2025-07-31

Specific recruitment of different proteins to distinct intracellular membranes is fundamental in the biology of eukaryotic cells, but the molecular basis for specificity is incompletely understood. This proposal investigates the hypothesis that coincidence detection of proteins and lipids constitutes a major mechanism for specific recruitment of proteins to intracellular membranes in order to control cellular membrane dynamics. CODE will establish and validate mathematical models for coincidence detection, identify and functionally characterise novel coincidence detectors, and engineer artificial coincidence detectors as novel tools in cell biology and biotechnology.

2 500 000 €

Start date: 2019-01-01, End date: 2023-12-31

How cells retain, lose, and regain developmental plasticity is poorly understood due to ignorance of the molecular mechanisms regulating gene expression. Each gene is regulated by a unique set of factors and as a consequence the trans-acting factors and cis-acting chromatin modification states regulating a given gene are extremely rare. Transcription is affected by events taking place many thousands of base pairs away from the start, a property enabling developmental and evolutionary plasticity, presumably made possible by DNA looping or translocation of factors along chromatin. Most factors regulating a given gene function at many other genes, complicating interpretation of the consequences of altering the activity of such factors. It is difficult to exclude the possibility that phenotypes are knock-on effects. This could be surmounted if it were possible to observe individual genes in real time in three-dimensional space and to analyse the immediate consequences of altering the activity of regulatory factors. Of these, those capable of inter-connecting DNAs or of translocating large distances along chromatin are of interest. Cohesin is such a factor, composed of three core subunits, a pair of Smc proteins and a kleisin subunit, that interact with each other to form a huge tripartite ring, within which it is thought chromatin fibres are entrapped. In proliferating cells, cohesin’s primary function is to connect sister chromatids during DNA replication until the onset of anaphase, possibly by virtue of co-entrapment within a single ring. However, cohesin is present in most quiescent cells and it is becoming clear that it also regulates gene expression and recombination. This proposal has two goals: To image gene expression on polytene chromosomes and to investigate cohesin’s role during ecdysone-induced transcription. The advantage of this system is that we can use micro-injection of TEV protease to inactivate cohesin. A second goal is to develop the TEV system to

2 421 212 €

Start date: 2012-05-01, End date: 2018-04-30

Colorectal cancer (CRC) is one of the most common cancers of the western world. The underlying initiating mutation for the majority of CRC is within the Adenomatous Polyposis Coli (Apc) gene. The APC protein performs an important role in controlling the levels of Wnt signalling by targeting beta-catenin for degradation. Loss of the APC protein leads to the activation of Wnt signaling target genes such as c-Myc which is required for phenotypes causes by Apc loss. However, despite the clear importance of APC loss and deregulated Wnt signalling, additional events are required for the development of CRC such as KRAS and P53 mutations.The impact of these changes on the development of CRC and response to therapy is not well understood. Furthermore, identification and testing of potential novel targets and therapies is hampered by lack of a preclinical model that faithfully recapitulates the course of the human disease. This proposal has two aims: 1. Assess the impact of cooperating mutations with Apc and assess how they alter sensitivities of Apc deficient cells. 2. Develop mouse models of invasive and metastatic colorectal cancer that recapitulate the human disease. We will use ‘state of the art’ methodologies to identify the changes in signaling output conferred by these cooperating mutations. Genetic mouse models of invasive and metastatic colorectal cancers will be generated through the acquisition of additional mutations and genomic instability. These studies will produce predictions on therapeutic combinations that will be tested in mouse models in vitro and in vivo that may identify new treatment regimens for patients with late stage CRC.

1 499 045 €

Start date: 2012-11-01, End date: 2017-10-31

Cell division is fundamental for development. In the early mammalian embryo it drives the rapid proliferation of totipotent cells, the basis for forming the fetus. Given its crucial importance, it is surprising that cell division is particularly error-prone at the beginning of mammalian life, resulting in spontaneous abortion or severe developmental retardation, the incidence of which is increasing with age of the mother. Why aneuploidy is so prevalent and how early embryonic development nevertheless achieves robustness is largely unknown. The goal of this project is a comprehensive analysis of cell divisions in the mouse preimplantation embryo to determine the molecular mechanisms underlying aneuploidy and its effects on normal development. Recent technological breakthroughs, including light sheet microscopy and rapid loss-of-function approaches in the mouse embryo will allow us for the first time to tackle the molecular mechanisms of aneuploidy generation and establish the preimplantation mouse embryo as a standard cell biological model system. For that purpose we will develop next generation light sheet microscopy to enable automated chromosome tracking in the whole embryo. Mapping of cell division errors will reveal when, where, and how aneuploidy occurs, what the fate of aneuploid cells is in the embryo, and how this changes with maternal age. We will then perform high resolution functional imaging assays to identify the mitotic pathways responsible for aneuploidy and understand why they do not fully function in early development. Key proteins will be functionally characterised in detail integrating light sheet imaging with single molecule biophysics in embryos from young and aged females to achieve a mechanistic understanding of the unique aspects of cell division underlying embryonic aneuploidy. The achieved knowledge gain will have an important impact for our understanding of mammalian, including human infertility.

2 497 156 €

Start date: 2017-01-01, End date: 2021-12-31

Developing tissues have a remarkable plasticity illustrated by their capacity to regenerate and form normal organs despite strong perturbations. This requires the adjustment of single cell behaviour to their neighbours and to tissue scale parameters. The modulation of cell growth and proliferation was suggested to be driven by mechanical inputs, however the mechanisms adjusting cell death are not well known. Recently it was shown that epithelial cells could be eliminated by spontaneous live-cell delamination following an increase of cell density. Studying cell delamination in the midline region of the Drosophila pupal notum, we confirmed that local tissue crowding is necessary and sufficient to drive cell elimination and found that Caspase 3 activation precedes and is required for cell delamination. This suggested that a yet unknown pathway is responsible for crowding sensing and activation of caspase, which does not involve already known mechanical sensing pathways. Moreover, we showed that fast growing clones in the notum could induce neighbouring cell elimination through crowding-induced death. This suggested that crowding-induced death could promote tissue invasion by pretumoural cells. Here we will combine genetics, quantitative live imaging, statistics, laser perturbations and modelling to study crowding-induced death in Drosophila in order to: 1) find single cell deformations responsible for caspase activation; 2) find new pathways responsible for density sensing and apoptosis induction; 3) test their contribution to adult tissue homeostasis, morphogenesis and cell elimination coordination; 4) study the role of crowding induced death during competition between different cell types and tissue invasion 5) Explore theoretically the conditions required for efficient space competition between two cell populations. This project will provide essential information for the understanding of epithelial homeostasis, mechanotransduction and tissue invasion by tumoural cells

1 489 147 €

Start date: 2018-01-01, End date: 2022-12-31

Colour patterns are prominent features of many animals and have important functions in communication such as camouflage, kin recognition and mate selection. Colour patterns are highly variable and evolve rapidly leading to large diversities even within a single genus. As targets for natural as well as sexual selection, they are of high evolutionary significance. The zebrafish (Danio rerio), a vertebrate model organism for the study of development and disease, displays a conspicuous pattern of alternating blue and golden stripes on the body and on the anal- and tailfins. Mutants with spectacularly altered patterns have been analysed, and novel approaches in lineage tracing have provided first insights into the cellular and molecular basis of colour patterning. These studies revealed that the mechanisms at play are novel and of fundamental interest to the biology of pattern formation. Closely related Danio species have very divergent colour patterns in body and fins offering the unique opportunity to study development and evolution of colour patterns in vertebrates building on the thorough analysis of one model species. Our research in zebrafish will explore the basis of direct interactions between chromatophores mediated by channels and junctions. We will investigate the divergent mode of stripe formation in the fins and the molecular influence of the cellular environment on chromatophore interactions. In closely related Danio species, we will investigate the cellular interactions during pattern formation. We will analyse transcriptomes and genome sequences to identify candidate genes providing the molecular basis for pigment pattern diversity. These candidate genes will be tested by creating mutants and exchanging allelic variants using the CRISPR/Cas9 system. The work will lay the foundation to understand not only the genetic basis of variation in colour pattern formation between Danio species, but also the evolution of biodiversity in other vertebrates.

2 250 000 €

Start date: 2016-11-01, End date: 2021-04-30

Dendritic cells (DCs) play a crucial role as gatekeepers of the immune system, coordinating the balance between protective immunity and tolerance to self antigens. What determines the switch between immunogenic versus tolerogenic antigen presentation remains one of the most puzzling questions in immunology. My team recently discovered an unanticipated link between a conserved stress response in the endoplasmic reticulum (ER) and tolerogenic DC maturation, thereby setting the stage for new insights in this fundamental branch in immunology. Specifically, we found that one of the branches of the unfolded protein response (UPR), the IRE1/XBP1 signaling axis, is constitutively active in murine dendritic cells (cDC1s), without any signs of an overt UPR gene signature. Based on preliminary data we hypothesize that IRE1 is activated by apoptotic cell uptake, orchestrating a metabolic response from the ER to ensure tolerogenic antigen presentation. This entirely novel physiological function for IRE1 entails a paradigm shift in the UPR field, as it reveals that IRE1’s functions might stretch far from its well-established function induced by chronic ER stress. The aim of my research program is to establish whether IRE1 in DCs is the hitherto illusive switch between tolerogenic and immunogenic maturation. To this end, we will dissect its function in vivo both in steady-state conditions and in conditions of danger (viral infection models). In line with our data, IRE1 has recently been identified as a candidate gene for autoimmune disease based on Genome Wide Association Studies (GWAS). Therefore, I envisage that my research program will not only have a large impact on the field of DC biology and apoptotic cell clearance, but will also yield new insights in diseases like autoimmunity, graft versus host disease or tumor immunology, all associated with disturbed balances between tolerogenic and immunogenic responses.

1 999 196 €

Start date: 2019-02-01, End date: 2024-01-31

The bacterial microflora has always been regarded as beneficial for the host but recent studies have shown that this symbiosis has risks as well as benefits. Although active mechanisms allow tolerating the commensal flora, the physiological stress that is associated with the symbionts’ metabolism can exhaust the intestinal barrier resulting in serious effects on the health of the host. Protracted immune deregulations can lead to severe disorders including diabetes, cancer and inflammatory bowel disease (IBD). Several mechanisms and players are involved in the maintenance of intestinal immune homeostasis, including T regulatory cells and Immunoglobulin (Ig)-A. In this proposal we focus our attention on dendritic cells (DCs) for their ability to induce both tolerance and immunity by regulating B and T cell responses. We have recently shown that DC function is controlled by intestinal epithelial cell (EC) derived factors and in particular by Thymic stromal lymphopoietin (TSLP). EC-conditioned DCs acquire a ‘mucosal’ phenotype as they are prone to activate T regulatory cells and IgA responses. Three major issues related to the maintenance and disruption of intestinal immune homeostasis will be explored in this project: 1) What are the mediators and mechanisms that regulate the interaction between intestinal epithelial cells and dendritic cells? What is the function of TSLP? 2) Which are the sites and players for the activation of an IgA response to pathogenic and commensal bacteria? Can we visualize them in vivo? 3) Can prolonged infections or bacterial products promote intestinal tumour development? Are there different bacterial constituents acting as inducers or protectors of carcinogenesis? What is the role of Toll-like receptors?

1 195 680 €

Start date: 2008-07-01, End date: 2013-06-30

The global incidence of kidney disease is on the rise, but little progress has been made to develop novel therapies or preventative measures. New methods to generated renal tissue in vitro hold great promise for regenerative medicine and the prospect of organ replacement. Most of the strategies employed differentiate induced pluripotent stem cells (iPSCs) into kidney organoids, which can be derived from patient tissue. Direct reprogramming is an alternative approach to convert one cell type into another using cell fate specifying transcription factors. We were the first to develop a method to directly reprogram mouse and human fibroblasts to kidney cells (induced renal tubular epithelial cells - iRECs) without the need for pluripotent cells. Morphological, transcriptomic and functional analyses found that directly reprogrammed iRECs are remarkably similar to native renal tubular cells. Direct reprogramming is fast, technically simple and scalable. This proposal aims to establish direct reprogramming in nephrology and develop novel in vitro models for kidney diseases that primarily affect the renal tubules. We will unravel the mechanics of how only four transcription factors can change the morphology and function of fibroblasts towards a renal tubule cell identity. These insights will be used to identify alternative routes to directly reprogram tubule cells with increased efficiency and accuracy. We will identify cell type specifying factors for reprogramming of tubular segment specific cell types. Finally, we will use of reprogrammed kidney cells to establish new in vitro models for autosomal dominant polycystic kidney disease and nephronophthisis. Direct reprogramming holds enormous potential to deliver patient specific disease models for diagnostic and therapeutic applications in the age of personalized and targeted medicine.

1 499 917 €

Start date: 2019-03-01, End date: 2024-02-29

The activities of the Polycomb group (PcG) of repressive chromatin modifiers are required to maintain correct transcriptional identity during development and differentiation. These activities are altered in a variety of tumours by gain- or loss-of-function mutations, whose mechanistic aspects still remain unclear. PcGs can be classified in two major repressive complexes (PRC1 and PRC2) with common pathways but distinct biochemical activities. PRC1 catalyses histone H2A ubiquitination of lysine 119, and PRC2 tri-methylation of histone H3 lysine 27. However, PRC1 has a more heterogeneous composition than PRC2, with six mutually exclusive PCGF subunits (PCGF1–6) essential for assembling distinct PRC1 complexes that differ in subunit composition but share the same catalytic core. While up to six different PRC1 forms can co-exist in a given cell, the molecular mechanisms regulating their activities and their relative contributions to general PRC1 function in any tissue/cell type remain largely unknown. In line with this biochemical heterogeneity, PRC1 retains broader biological functions than PRC2. Critically, however, no molecular analysis has yet been published that dissects the contribution of each PRC1 complex in regulating transcriptional identity. We will take advantage of newly developed reagents and unpublished genetic models to target each of the six Pcgf genes in either embryonic stem cells or mouse adult tissues. This will systematically dissect the contributions of the different PRC1 complexes to chromatin profiles, gene expression programs, and cellular phenotypes during stem cell self-renewal, differentiation and adult tissue homeostasis. Overall, this will elucidate some of the fundamental mechanisms underlying the establishment and maintenance of cellular identity and will allow us to further determine the molecular links between PcG deregulation and cancer development in a tissue- and/or cell type–specific manner.

2 000 000 €

Start date: 2017-11-01, End date: 2022-10-31

Oriented cell divisions dictate morphogenesis by shaping tissues and organs of multicellular organisms. Oriented cell divisions have profound influence in plants because their cell positions are locked by shared cell walls. A relay of cell divisions involving precise division plane switches determines embryonic body plan, organ layout and organ architecture in plants. Cell division planes in plants are specified by reorganization of premitotic cortical microtubule array and how this occurs is a long-standing key question. My recent results establish, for the first time in plants, an in vivo inducible and traceable, precise 90º cell division plane switch system. With this system I identified a pathway that proceeds from transcriptional activation through a signaling module all the way to the activation of microtubule regulators that orchestrate switches in premitotic microtubule organization and cell division planes. My findings provide a first paradigm in plants of how genetic circuitry patterns cell division planes via feeding onto cellular machinery and pave the way for unraveling mechanistic control of cell division plane switch. By establishing a precise cell division plane switch system I am in a unique position to answer: 1. What transcriptional program and molecular players control premitotic microtubule reorganization? 2. Which mechanisms switch premitotic microtubule array? 3. What influence do identified players and mechanisms have on different types of oriented cell divisions in plants? For this I propose a systematic research plan combining (i) forward genetics and expression profile screens for identifying a suite of microtubule regulators, (ii) state-of-the-art microscopy and modeling approaches for uncovering mechanisms of their actions and (iii) their tissue-specific manipulations to modify plant form. By unraveling players and mechanisms this proposal shall resolve regulation of oriented cell divisions and expand plant engineering toolbox.

1 500 000 €

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

Cell motility is essential for many biological processes, including development and pathogenesis. Thus, the molecular mechanisms underlying this process have been intensively studied in many cell systems, for example, leukocytes, amoeba and even bacteria. Intriguingly, bacteria are also able to move across solid surfaces (gliding motility) like eukaryotic cells by a process that has remained largely mysterious. The emergence of bacterial cell biology: the discovery that the bacterial cell also has a dynamic cytoskeleton and specialized subcellular regions now provides new research angles to study the motility mechanism. Using cell biology approaches, we previously suggested that the mechanism may be akin to acto-myosin-based motility in eukaryotic cells and proposed that bacterial focal adhesion complexes also power locomotion. In this project, we propose two complementary research axes to define both the mechanism and its spatial regulation in the cell at molecular resolution. Using the model motility bacterium Myxococcus xanthus, we first propose to develop a “toolbox” of biophysical and cell biology assays to analyze the motility process. Specifically, we will construct a Traction Force Microscopy assay designed to image the motility forces directly by live moving cells and use microfluidics to quantitate the secretion of a mucus that may participate directly in the motility process. These assays, combined with a newly developed laser trap system to visualize dynamic focal adhesions in the cell envelope, will be instrumental not only to define new features of the motility process, but also to study the function of novel motility genes which may encode the components of the elusive motility engine. This way, we hope to establish the mechanism and structure function relationships within an entirely novel motility machinery. In a second part, we propose to investigate the mechanism that controls a polarity switch, allowing M. xanthus cells to change their direction of movement. We have previously shown that dynamic motility protein pole-to-pole oscillations convert the initial leading cell pole into the lagging pole. Here, we propose that like in a eukaryotic cells, a bacterial counterpart of small GTPases of the Ras superfamily, MglA controls the polarity cycle. To test this hypothesis, we will study both the MglA upstream regulation and the MglA downstream effectors. We thus hope to establish a model of dynamic polarity control in a bacterial

1 437 693 €

Start date: 2011-01-01, End date: 2015-12-31

Organisms and cells face a myriad of environmental changes with periods of nutrient surplus and shortage. It is therefore not surprising that in all kingdoms of life, cells have evolved the means to store energy and thereby minimize the effects of environmental fluctuations. While the capability for energy storage has obvious advantages, deregulated energy accumulation can also be detrimental and is the hallmark of many diseases such as obesity. In most cells energy is stored as neutral lipids in a dedicated cellular compartment, the lipid droplets (LDs). LDs are found in virtually every eukaryotic cell and play a central role in cellular lipid and energy metabolism. Despite their ubiquitous presence and importance, the physiology of LDs is poorly understood. LDs are composed of a single lipid layer and therefore distinct from all other cellular compartments. How do LDs originate at the endoplasmic reticulum (ER) and what is the machinery involved? How is the size, number and the storage capacity of the LDs regulated? How are specific proteins and lipids targeted to LDs? Addressing these questions is fundamental for understanding the “life cycle” of LDs and for a global picture of the cellular energy homeostasis. The main goal of this proposal is to reveal the molecular mechanisms controlling neutral lipid dynamics and their storage in LDs. We will focus specifically on the role of the endoplasmic reticulum in the biogenesis of LDs. First, we will identify the ER protein complexes required for LD formation and regulation. Second, we will develop an assay to dissect the targeting of proteins to LDs. Finally, we will develop a cell-free system that recapitulates the biogenesis of LDs in vitro. Altogether, our strategy constitutes a systematic, in-depth analysis of LD dynamics and will lead to significant insight on the mechanisms of cellular energy storage. Our findings will likely offer a better understanding of human pathologies such as obesity and lipodistrophies

1 475 282 €

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

The discovery of RNA interference (RNAi) has revolutionized biology. As a technology it opened up new experimental and therapeutic avenues. As a biological phenomenon it changed our view on a diverse array of cellular processes. Among those are the control of gene expression, the suppression of viral replication, the formation of heterochromatin and the protection of the genome against selfish genetic elements such as transposons. I propose to study the molecular mechanism and the biological impact of a recently discovered RNAi pathway, the Piwi interacting RNA pathway (piRNA pathway). The piRNA pathway is an evolutionarily conserved small RNA pathway acting in the animal germline. It is the key genome surveillance system that suppresses the activity of transposons. Recent work has provided a conceptual framework for this pathway: According to this, the genome stores transposon sequences in heterochromatic loci called piRNA clusters. These provide the RNA substrates for the biogenesis of 23-29 nt long piRNAs. An amplification cycle steers piRNA production predominantly to those cluster regions that are complementary to transposons being active at a given time. Finally, piRNAs guide a protein complex centered on Piwi-proteins to complementary transposon RNAs in the cell, leading to their silencing. In contrast to other RNAi pathways, the mechanistic framework of the piRNA pathway is largely unknown. Moreover, the spectrum of biological processes impacted by it is only poorly understood. piRNAs are for example not only derived from transposon sequences but also from various other genomic repeats that are enriched at telomeres and in heterochromatin. We will systematically dissect the piRNA pathway regarding its molecular architecture as well as its biological functions in Drosophila. Our studies will be a combination of fly genetics, proteomics and genomics approaches. Throughout we aim at linking our results back to the underlying biology of germline development.

1 500 000 €

Start date: 2010-09-01, End date: 2015-08-31

Cells in intestinal epithelia turn over rapidly due to aging, damage, and toxins produced by the enteric microbiota. Gut homeostasis is maintained by intestinal stem cells (ISCs) that divide to renew the intestinal epithelium, but little is known about how ISC division and differentiation are coordinated with the loss of spent gut epithelial cells. This proposal addresses the mechanisms of dynamic self-renewal in the intestine of Drosophila. Our recent work has outlined a paradigm explaining intestinal homeostasis in Drosophila that could apply also to humans. A new lab is being established in Heidelberg where we wish to extend these studies. Our objectives are to understand: 1) How intestinal stem cell pool sizes are regulated; 2) How the cytokines and growth factors that mediate gut homeostasis are controlled; and 3) How these signals regulate the ISC cell cycle. Established genetic and cell biological methods will be applied, supported by molecular assays (microarrays, qPCR, ChIP/Seq) of gene control. New pathways of ISC control will be discovered via comprehensive genetic screens using transgenic RNAi and gene over-expression. In vitro culture of ISCs will be developed and used for live imaging and molecular analysis of the mechanisms controlling ISC proliferation and differentiation. These studies should elaborate a paradigm explaining intestinal homeostasis in flies that can guide studies in mammals, eventually contributing to the diagnosis and treatment for diseases in which gut homeostasis is disrupted, such as colorectal cancer and inflammatory bowel disease. Because stem cell biology is so highly relevant to wound healing, regeneration, cancer, aging and degenerative disease, this research could impact human health at many levels.

2 682 080 €

Start date: 2011-05-01, End date: 2016-04-30

Different morphologies evolve in different organisms in response to changing environments. As land plants evolved, developmental mechanisms were either generated de novo, or were recruited from existing toolkits and adapted to facilitate changes in form. Some of these changes occurred once, others on multiple occasions, and others were gained and then subsequently lost in a subset of lineages. Why have certain forms survived and others not? Why does a fern look different from a flowering plant, and why should developmental biologists care? By determining how many different ways there are to generate a particular morphology, we gain an understanding of whether a particular transition is constrained. This basic information allows an assessment of the extent to which genetic variation can modify developmental mechanisms and an indication of the degree of developmental plasticity that is possible and/or tolerated both within and between species. This proposal aims to characterize the developmental mechanisms that underpin the diverse shoot forms seen in extant plant species. The main goal is to compare developmental mechanisms that operate in vegetative shoots of bryophytes, lycophytes, ferns and angiosperms, with a view to understanding the constraints that limit morphological variation. Specifically, we will investigate the developmental basis of three major innovations that altered the morphology of vegetative shoots during land plant evolution: 1) formation of a multi-cellular embryo; 2) organization of apical growth centres and 3) patterning of leaves in distinct spatial arrangements along the shoot. To facilitate progress we also aim to develop transgenic methods, create mutant populations and generate digital transcriptomes for model species at key phylogenetic nodes. The proposed work will generate scenarios to explain how land plant form evolved and perhaps more importantly, how it could change in the future.

2 230 732 €

Start date: 2009-07-01, End date: 2015-06-30

The majority of animals show an external bilateral symmetry, precluding the observation of multiple internal left-right (L/R) asymmetries which are fundamental for organ packaging and function. A prominent molecular pathway converging on and downstream of the Pitx2 transcription factor confers left-handed information in the left side of the embryo, with players expressed on the right ensuring that the left determinants are excluded. Therefore, conferring or excluding left identity in left and right hand sides, respectively, drives L/R asymmetry. Some indications suggest that a program actively specifying right–handed information could exist on the right. Our recent findings support this view. In a screening for novel regulators of the epithelial to mesenchymal transition (EMT), we have identified a transcription factor, EMT2, which similarly to well known factor Snail, it is an EMT inducer. The EMT is crucial for the development of tissues during embryonic development and for the progression of carcinomas to the invasive state. Strikingly, again as Snail, in addition to promote EMT, the EMT2 factor is predominantly expressed on the right side and may operate instructing L/R identity on the right-hand side of the embryo. With this background, our knowledge of the EMT and a series of genome-wide high-throughput approaches and a comprehensive functional analysis using the chick, the fish and the mouse as model systems we propose to reveal the putative molecular pathways conveying right-handed information and to reveal commonalities between L/R pathways and the EMT. In the long run, we aim at better understanding human pathologies that involve these morphogenetic and cellular processes, including pathological situs conditions (i.e. altered organ positioning) and cancer progression.

2 460 000 €

Start date: 2013-05-01, End date: 2018-12-31

In addition to maintaining homeostasis within their cells, multicellular organisms also need to control their inner, extracellular spaces between cells. In order to do so, epithelia have developed, bearing ring-like paracellular barriers, with specialised membrane surfaces facing either the environment or the inner space of the organism. In animals, such polarised epithelia use specialised protein assemblies, called tight junctions, to seal the extracellular space, which have been a topic of active research for decades. Plant roots need to extract inorganic elements from the soil. A plethora of transporters are expressed in plant roots, yet, as in animals, transporter action is contingent upon the presence of efficient paracellular (apoplastic) barriers. Therefore, an understanding of the development, structure and function of the root apoplastic barrier is crucial for mechanistic models of root nutrient uptake. The endodermis is the main apoplastic barrier in roots, but, in contrast to animals, molecular data about endodermal differentiation and function has been virtually absent. We recently gained insights into the factors that drive endodermal differentiation, largely due to efforts from my research team. Our work has led a foundation of mutants, markers and protocols that provide an unprecented opportunity to test the many supposed roles of the root endodermis. Our preliminary insights indicate that generally accepted views of endodermal function have been overly simplistic. The topic of this proposal is to develop better tools and much more precise molecular analysis of nutrient uptake, centered around the endodermis. I propose to investigate our specific barrier mutants with new tools that allow visualisation of changes in nutrient transport at cellular resolution. The results from this project will provide a new foundation for models of plant nutrition and help us to understand how plants manage, and sometimes fail, to extract what they need from the soil.

1 985 443 €

Start date: 2014-06-01, End date: 2019-05-31