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