ERC FUNDED PROJECTS

For many years, the ubiquitin-26S proteasome degradation pathway was considered the primary route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by a ubiquitin-independent mechanism mediated by the core 20S proteasome itself. Although initially believed to be limited to rare exceptions, degradation by the 20S proteasome is now understood to have a wide range of substrates, many of which are key regulatory proteins. Despite its importance, little is known about the mechanisms that control 20S proteasomal degradation, unlike the extensive knowledge acquired over the years concerning degradation by the 26S proteasome. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route. To achieve this goal we will carry out a comprehensive research program characterizing three distinct levels of 20S proteasome regulation: Intra-molecular regulation- Revealing the intrinsic molecular switch that activates the latent 20S proteasome. Inter-molecular regulation- Identifying novel proteins that bind the 20S proteasome to regulate its activity and characterizing their mechanism of function. Cellular regulatory networks- Unraveling the cellular cues and multiple pathways that influence 20S proteasome activity using a novel systematic and unbiased screening approach. Our experimental strategy involves the combination of biochemical approaches with native mass spectrometry, cross-linking and fluorescence measurements, complemented by cell biology analyses and high-throughput screening. Such a multidisciplinary approach, integrating in vitro and in vivo findings, will likely provide the much needed knowledge on the 20S proteasome degradation route. When completed, we anticipate that this work will be part of a new paradigm – no longer perceiving the 20S proteasome mediated degradation as a simple and passive event but rather a tightly regulated and coordinated process.

1 500 000 €

Start date: 2015-04-01, End date: 2020-03-31

Homologous recombination (HR) is a conserved DNA double-strand breaks (DSB) repair pathway that uniquely uses an intact DNA molecule as a template. Genome-wide homology search is carried out by a nucleoprotein filament (NPF) assembled on the ssDNA flanking the DSB, and whose product is a “D-loop” joint molecule. Beyond accurate DSB repair, this capacity of HR to spatially associates homologous molecules is also harnessed for homolog pairing in meiosis. The goal of “3D-loop” is to tackle two long lasting conundrums: the fundamental homology search mechanism that achieves accurate and efficient identification of a single homologous donor in the vastness of the genome and nucleus, and how this mechanism is adapted for the purpose of homologs attachment in meiosis. I overcame the main hurdle to study these core steps of HR by developing a suite of proximity ligation-based methodologies and experimental systems to physically detect joint molecules in yeast cells. It revealed elaborate regulation controlling D-loop dynamics and a novel class of joint molecules. This proposal builds upon these methodologies and findings to first address basic properties of the homology sampling process by the NPF and the role of D-loop dynamics, with the long-term goal to establish a quantitative framework of homology search in mitotic cells (WP1). Second, the meiosis-specific regulation of homology search leading to homolog pairing likely integrates chromosomal-scale information. Genome re-synthesis and engineering approaches will be deployed to (i) achieve a quantitative and dynamic cartography of the cytological and molecular events of meiosis over a large chromosomal region, (ii) probe cis-acting regulations at the chromosomal scale, and (iii) revisit the molecular paradigm for crossover formation (WP2). We expect this project to shed light on the fundamental process of homology search and its involvement in the chromosome pairing phenomenon lying at the basis of sexual reproduction.

1 499 779 €

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

Antigenic variation is a widely employed strategy to evade the host immune response. It has similar functional requirements even in evolutionarily divergent pathogens. These include the mutually exclusive expression of antigens and the periodic, nonrandom switching in the expression of different antigens during the course of an infection. Despite decades of research the mechanisms of antigenic variation are not fully understood in any organism. The recent development of high-throughput sequencing-based assays to probe the 3D genome architecture (Hi-C) has revealed the importance of the spatial organization of DNA inside the nucleus. 3D genome architecture plays a critical role in the regulation of mutually exclusive gene expression and the frequency of translocation between different genomic loci in many eukaryotes. Thus, genome architecture may also be a key regulator of antigenic variation, yet the causal links between genome architecture and the expression of antigens have not been studied systematically. In addition, the development of CRISPR-Cas9-based approaches to perform nucleotide-specific genome editing has opened unprecedented opportunities to study the influence of DNA sequence elements on the spatial organization of DNA and how this impacts antigen expression. I have adapted both Hi-C and CRISPR-Cas9 technology to the protozoan parasite Trypanosoma brucei, one of the most important model organisms to study antigenic variation. These techniques will enable me to bridge the field of antigenic variation research with that of genome architecture. I will perform the first systematic analysis of the role of genome architecture in the mutually exclusive and hierarchical expression of antigens in any pathogen. The experiments outlined in this proposal will provide new insight, facilitating a new view of antigenic variation and may eventually help medical intervention in T. brucei and in other pathogens relying on antigenic variation for their survival.

1 498 175 €

Start date: 2017-04-01, End date: 2022-03-31

Epigenetic inheritance entails transmission of phenotypic traits not encoded in the DNA sequence and, in the most extreme case, Transgenerational Epigenetic Inheritance (TEI) involves transmission of memory through multiple generations. Very little is known on the mechanisms governing TEI and this is the subject of the present proposal. By transiently enhancing long-range chromatin interactions, we recently established isogenic Drosophila epilines that carry stable alternative epialleles, defined by differential levels of the Polycomb-dependent H3K27me3 mark. Furthermore, we extended our paradigm to natural phenotypes. These are ideal systems to study the role of Polycomb group (PcG) proteins and other components in regulating nuclear organization and epigenetic inheritance of chromatin states. The present project conjugates genetics, epigenomics, imaging and molecular biology to reach three critical aims. Aim 1: Analysis of the molecular mechanisms regulating Polycomb-mediated TEI. We will identify the DNA, protein and RNA components that trigger and maintain transgenerational chromatin inheritance as well as their mechanisms of action. Aim 2: Role of 3D genome organization in the regulation of TEI. We will analyze the developmental dynamics of TEI-inducing long-range chromatin interactions, identify chromatin components mediating 3D chromatin contacts and characterize their function in the TEI process. Aim 3: Identification of a broader role of TEI during development. TEI might reflect a normal role of PcG components in the transmission of parental chromatin onto the next embryonic generation. We will explore this possibility by establishing other TEI paradigms and by relating TEI to the normal PcG function in these systems and in normal development. This research program will unravel the biological significance and the molecular underpinnings of TEI and lead the way towards establishing this area of research into a consolidated scientific discipline.

2 500 000 €

Start date: 2018-11-01, End date: 2023-10-31

This project investigates the two-way relationship between spatio-temporal genome organization and coordinated gene regulation, through an approach at the interface between physics, computer science and biology. In the nucleus, preferred positions are observed from chromosomes to single genes, in relation to normal and pathological cellular states. Evidence indicates a complex spatio-temporal coupling between co-regulated genes: e.g. certain genes cluster spatially when responding to similar factors and transcriptional noise patterns suggest domain-wide mechanisms. Yet, no individual experiment allows probing transcriptional coordination in 4 dimensions (FISH, live locus tracking, Hi-C...). Interpreting such data also critically requires theory (stochastic processes, statistical physics…). A lack of appropriate experimental/analytical approaches is impairing our understanding of the 4D genome. Our proposal combines cutting-edge single-molecule imaging, signal-theory data analysis and physical modeling to study how genes coordinate in space and time in a single nucleus. Our objectives are to understand (a) competition/recycling of shared resources between genes within subnuclear compartments, (b) how enhancers communicate with genes domain-wide, and (c) the role of local conformational dynamics and supercoiling in gene co-regulation. Our organizing hypothesis is that, by acting on their microenvironment, genes shape their co-expression with other genes. Building upon my expertise, we will use dual-color MS2/PP7 RNA labeling to visualize for the first time transcription and motion of pairs of hormone-responsive genes in real time. With our innovative signal analysis tools, we will extract spatio-temporal signatures of underlying processes, which we will investigate with stochastic modeling and validate through experimental perturbations. We expect to uncover how the functional organization of the linear genome relates to its physical properties and dynamics in 4D.

1 499 750 €

Start date: 2018-04-01, End date: 2023-03-31

Chromosomal DNA is continuously subjected to exogenous and endogenous damaging insults. In the presence of DNA damage cells activate a multi-faceted checkpoint response that delays cell cycle progression and promotes DNA repair. Failures in this response lead to genomic instability, the main feature of cancer cells. Several cancer-prone human syndromes including the Ataxia teleangiectasia (A-T), the A-T Like Disorder (ATLD) and the Seckel Syndrome reflect defects in the specific genes of the DNA damage response such as ATM, MRE11 and ATR. DNA damage response pathways are poorly understood at biochemical level in vertebrate organisms. We have established a cell-free system based on Xenopus laevis egg extract to study molecular events underlying DNA damage response. This is the first in vitro system that recapitulates different aspects of the DNA damage response in vertebrates. Using this system we propose to study the biochemistry of the ATM, ATR and the Mre11 complex dependent DNA damage response. In particular we will: 1) Dissect the signal transduction pathway that senses DNA damage and promotes cell cycle arrest and DNA damage repair; 2) Analyze at molecular level the role of ATM, ATR, Mre11 in chromosomal DNA replication and mitosis during normal and stressful conditions; 3) Identify substrates of the ATM and ATR dependent DNA damage response using an innovative screening procedure.

1 000 000 €

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

Cell volume regulation is crucial for any living cell because changes in volume determine the metabolic activity through e.g. changes in ionic strength, pH, macromolecular crowding and membrane tension. These physical chemical parameters influence interaction rates and affinities of biomolecules, folding rates, and fold stabilities in vivo. Understanding of the underlying volume regulatory mechanisms has immediate application in biotechnology and health, yet these factors are generally ignored in systems analyses of cellular functions. My team has uncovered a number of mechanisms and insights of cell volume regulation. The next step forward is to elucidate how the components of a cell volume regulatory circuit work together and control the physicochemical conditions of the cell. I propose construction of a synthetic cell in which an osmoregulatory transporter and mechanosensitive channel form a minimal volume regulatory network. My group has developed the technology to reconstitute membrane proteins into lipid vesicles (synthetic cells). One of the challenges is to incorporate into the vesicles an efficient pathway for ATP production and maintain energy homeostasis while the load on the system varies. We aim to control the transmembrane flux of osmolytes, which requires elucidation of the molecular mechanism of gating of the osmoregulatory transporter. We will focus on the glycine betaine ABC importer, which is one of the most complex transporters known to date with ten distinct protein domains, transiently interacting with each other. The proposed synthetic metabolic circuit constitutes a fascinating out-of-equilibrium system, allowing us to understand cell volume regulatory mechanisms in a context and at a level of complexity minimally needed for life. Analysis of this circuit will address many outstanding questions and eventually allow us to design more sophisticated vesicular systems with applications, for example as compartmentalized reaction networks.

2 247 231 €

Start date: 2015-07-01, End date: 2020-06-30

Abiogenesis, the transition from non-living to living matter, is at the core of the origin of life question. However, the dynamical processes underlying abiogenesis remain unknown. The AbioEvo project aims to test the hypothesis that RNA-catalysed RNA recombination, if coupled with template-based mechanisms, provides a gradual route for the emergence of evolution by natural selection, starting from collective autocatalysis, toward template-based replication. Indeed, recombination allows both self-reproduction and shuffling of other sequences, thus, once combined with templating, provides the basic ingredients of reproduction, heredity and variation required for Darwinian evolution. The project decomposes the problem into five steps: (WP1) the study of molecular-level mechanisms to generate and stabilize novel sequences by recombination and templating; (WP2) collective dynamics integrating these mechanisms into the properties of reproduction with heredity, variation, and selection, in order to establish proof-of-concepts of evolutionary modes; (WP3) viability thresholds of recombination-based replicators from increasingly random substrates; (WP4) conditions for open-ended evolution toward template-based replication; (WP5) experimentally informed theoretical estimates of the probability of the proposed evolutionary transitions. The project would provide first demonstrations of evolution by natural selection in a purely chemical system, gradual and experimentally accessible paths from oligomers to template-based replication, and a method to evaluate prebiotic plausibility from sequence-to-function relationships, kinetics and evolutionary dynamics.

2 000 000 €

Start date: 2021-06-01, End date: 2026-05-31

The centriole is the largest evolutionary conserved macromolecular structure responsible for building centrosomes and cilia or flagella in many eukaryotes. Centrioles are critical for the proper execution of important biological processes ranging from cell division to cell signaling. Moreover, centriolar defects have been associated to several human pathologies including ciliopathies and cancer. This state of facts emphasizes the importance of understanding centriole biogenesis. The study of centriole formation is a deep-rooted question, however our current knowledge on its molecular organization at high resolution remains fragmented and limited. In particular, exquisite details of the overall molecular architecture of the human centriole and in particular of its central core region are lacking to understand the basis of centriole organization and function. Resolving this important question represents a challenge that needs to be undertaken and will undoubtedly lead to groundbreaking advances. Another important question to tackle next is to develop innovative methods to enable the nanometric molecular mapping of centriolar proteins within distinct architectural elements of the centriole. This missing information will be key to unravel the molecular mechanisms behind centriolar organization. This research proposal aims at building a cartography of the human centriole by elucidating its molecular composition and architecture. To this end, we will combine the use of innovative and multidisciplinary techniques encompassing spatial proteomics, cryo-electron tomography, state-of-the-art microscopy and in vitro assays and to achieve a comprehensive molecular and structural view of the human centriole. All together, we expect that these advances will help understand basic principles underlying centriole and cilia formation as well as might have further relevance for human health.

1 498 965 €

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

"Eukaryotic nuclei are organised into functional compartments, – local microenvironments that are enriched in certain molecules or biochemical activities and therefore specify localised functional outputs. Our study seeks to unveil fundamental principles of co-regulation of genes in a chromo¬somal compartment and the preconditions for homeostasis of such a compartment in the dynamic nuclear environment. The dosage-compensated X chromosome of male Drosophila flies satisfies the criteria for a functional com¬partment. It is rendered structurally distinct from all other chromosomes by association of a regulatory ribonucleoprotein ‘Dosage Compensation Complex’ (DCC), enrichment of histone modifications and global decondensation. As a result, most genes on the X chromosome are co-ordinately activated. Autosomal genes inserted into the X acquire X-chromosomal features and are subject to the X-specific regulation. We seek to uncover the molecular principles that initiate, establish and maintain the dosage-compensated chromosome. We will follow the kinetics of DCC assembly and the timing of association with different types of chromosomal targets in nuclei with high spatial resolution afforded by sub-wavelength microscopy and deep sequencing of DNA binding sites. We will characterise DCC sub-complexes with respect to their roles as kinetic assembly intermediates or as representations of local, functional heterogeneity. We will evaluate the roles of a DCC- novel ubiquitin ligase activity for homeostasis. Crucial to the recruitment of the DCC and its distribution to target genes are non-coding roX RNAs that are transcribed from the X. We will determine the secondary structure ‘signatures’ of roX RNAs in vitro and determine the binding sites of the protein subunits in vivo. By biochemical and cellular reconstitution will test the hypothesis that roX-encoded RNA aptamers orchestrate the assembly of the DCC and contribute to the exquisite targeting of the complex."

2 482 770 €

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

MAL: an actin-regulated SRF transcriptional coactivator Recent years have seen a revitalised interest in the role of actin in nuclear processes, but the molecular mechanisms involved remain largely unexplored. We will elucidate the molecular basis for the actin-based control of the SRF transcriptional coactivator, MAL. SRF controls transcription through two families of coactivators, the actin-binding MRTFs (MAL, Mkl2), which couple its activity to cytoskeletal dynamics, and the ERK-regulated TCFs (Elk-1, SAP-1, Net). MAL subcellular localisation and transcriptional activity responds to signal-induced changes in G-actin concentration, which are sensed by its actin-binding N-terminal RPEL domain. Members of a second family of RPEL proteins, the Phactrs, also exhibit actin-regulated nucleocytoplasmic shuttling. The proposal addresses the following novel features of actin biology: ¿ Actin as a transcriptional regulator ¿ Actin as a signalling molecule ¿ Actin-binding proteins as targets for regulation by actin, rather than regulators of actin function We will analyse the sequences and proteins involved in actin-regulated nucleocytoplasmic shuttling, using structural biology and biochemistry to analyse its control by changes in actin-RPEL domain interactions. We will characterise the dynamics of shuttling, and develop reporters for changes in actin-MAL interaction for analysis of pathway activation in vivo. We will identify genes controlling MAL itself, and the balance between the nuclear and cytoplasmic actin pools. The mechanism by which actin represses transcriptional activation by MAL in the nucleus, and its relation to MAL phosphorylation, will be elucidated. Finally, we will map MRTF and TCF cofactor recruitment to SRF targets on a genome-wide scale, and identify the steps in transcription controlled by actin-MAL interaction.

1 889 995 €

Start date: 2011-10-01, End date: 2017-09-30

The cell cortex is a highly dynamic layer of crosslinked actin filaments and myosin molecular motors beneath the cell membrane. It plays a central role in large scale rearrangements that occur inside cells. Many molecular mechanisms contribute to cortex structure and dynamics. However, cell scale physical properties of the cortex are difficult to grasp. This is problematic because for large scale rearrangements inside a cell, such as coherent flow of the cell cortex, it is the cell scale emergent properties that are important for the realization of such events. I will investigate how the actomyosin cytoskeleton behaves at a coarse grained and cellular scale, and will study how this emergent active behaviour is influenced by molecular mechanisms. We will study the cell cortex in the one cell stage C. elegans embryo, which undergoes large scale cortical flow during polarization and cytokinesis. We will combine theory and experiment. We will characterize cortex structure and dynamics with biophysical techniques such as cortical laser ablation and quantitative photobleaching experiments. We will develop and employ novel theoretical approaches to describe the cell scale mechanical behaviour in terms of an active complex fluid. We will utilize genetic approaches to understand how these emergent mechanical properties are influenced by molecular activities. A central goal is to arrive at a coarse grained description of the cortex that can predict future dynamic behaviour from the past structure, which is conceptually similar to how weather forecasting is accomplished. To date, systematic approaches to link molecular scale physical mechanisms to those on cellular scales are missing. This work will open new opportunities for cell biological and cell biophysical research, by providing a methodological approach for bridging scales, for studying emergent and large-scale active mechanical behaviours and linking them to molecular mechanisms.

1 500 000 €

Start date: 2011-12-01, End date: 2017-08-31

The mechanism of cell division is conserved in many eukaryotes, from yeast to man. A contractile ring of filamentous actin and myosin II motors generates the force to bisect a mother cell into two daughters. The actomyosin ring is among the most complex cellular machines, comprising over 150 proteins. Understanding how these proteins organize themselves into a functional ring with appropriate contractile properties remains one of the great challenges in cell biology. Efforts to generate a comprehensive understanding of the mechanism of actomyosin ring assembly have been hampered by the lack of structural information on the arrangement of actin, myosin II, and actin modulators in the ring in its native state. Fundamental questions such as how actin filaments are assembled and organized into a ring remain actively debated. This project will investigate key issues pertaining to cytokinesis in the fission yeast Schizosaccharomyces pombe, which divides employing an actomyosin based contractile ring, using the methods of genetics, biochemistry, cellular imaging, DNA origami, genetic code expansion, and click chemistry. Specifically, we will (1) attempt to visualize actin filament assembly in live cells expressing fluorescent actin generated through synthetic biological approaches, including genetic code expansion and click chemistry (2) decipher actin filament polarity in the actomyosin ring using total internal reflection fluorescence microscopy of labelled dimeric and multimeric myosins V and VI generated through DNA origami approaches (3) address when, where, and how actin filaments for cytokinesis are assembled and organized into a ring and (4) reconstitute actin filament and functional actomyosin ring assembly in permeabilized spheroplasts and in supported bilayers. Success in the project will provide major insight into the mechanism of actomyosin ring assembly and illuminate principles behind cytoskeletal self-organization.

2 863 705 €

Start date: 2015-11-01, End date: 2021-04-30

What processes shape vertebrate diversity through deep time? Approaches to this question can focus on many different factors, from life history and ecology to large-scale environmental change and extinction. To date, the majority of studies on the evolution of vertebrate diversity have focused on relatively simple metrics, specifically taxon counts or univariate measures, such as body size. However, multivariate morphological data provides a more complete picture of evolutionary and palaeoecological change. Morphological data can also bridge deep-time palaeobiological analyses with studies of the genetic and developmental factors that shape variation and must also influence large-scale patterns of evolutionary change. Thus, accurately reconstructing the patterns and processes underlying evolution requires an approach that can fully represent an organism’s phenome, the sum total of their observable traits. Recent advances in imaging and data analysis allow large-scale study of phenomic evolution. In this project, I propose to quantitatively analyse the deep-time evolutionary diversity of tetrapods (amphibians, reptiles, birds, and mammals). Specifically, I will apply and extend new imaging, morphometric, and analytical tools to construct a multivariate phenomic dataset for living and extinct tetrapods from 3-D scans. I will use these data to rigorously compare extinction selectivity, timing, pace, and shape of adaptive radiations, and ecomorphological response to large-scale climatic shifts across all tetrapod clades. To do so, I will quantify morphological diversity (disparity) and rates of evolution spanning over 300 million years of tetrapod history. I will further analyse the evolution of phenotypic integration by quantifying not just the traits themselves, but changes in the relationships among traits, which reflect the genetic, developmental, and functional interactions that shape variation, the raw material for natural selection.

1 482 818 €

Start date: 2015-06-01, End date: 2021-05-31

The goal of this project is to demonstrate that novel aspects of the molecular basis of Darwinian adaptation can be discovered if the polygenic basis of adaptation is taken into account. This project will use the genome-wide distribution of cis-regulatory variants to discover the molecular pathways that are optimized during adaptation via accumulation of small effect mutations. Current approaches include scans for outlier genes with strong population genetics signatures of selection, or large effect QTL associating with fitness. They can only reveal a small subset of the molecular changes recruited along adaptive paths. Here, instead, the distribution of small effect mutations will be used to make inferences on the targets of polygenic adaptation across divergent populations in each of the two closely related species, A. thaliana and A. lyrata. These species are both found at diverse latitudes and show sign of local adaptation to climatic differences. Mutations affecting cis-regulation will be identified in leaves of plants exposed to various temperature regimes triggering phenotypic responses of adaptive relevance. Their distribution in clusters of functionally connected genes will be quantified. The phylogeographic differences in the distribution of the mutations will be used to disentangle neutral from adaptive clusters of functionally connected genes in each of the two species. This project will identify the molecular pathways subjected collectively to natural selection and provide a completely novel view on adaptive landscapes. It will further examine whether local adaptation occurs by convergent evolution of molecular systems in plants. This approach has the potential to find broad applications in ecology and agriculture.

1 683 120 €

Start date: 2015-09-01, End date: 2021-02-28

Obesity and its metabolic co-morbidities have given rise to a rapidly expanding ‘metabolic syndrome’ pandemic affecting hundreds of millions of individuals worldwide. The integrative genetic and environmental causes of the obesity pandemic remain elusive. White adipose tissue (WAT)-resident immune cells have recently been highlighted as important factors contributing to metabolic complications. However, a comprehensive understanding of the regulatory circuits governing their function and the cell type-specific mechanisms by which they contribute to the development of metabolic syndrome is lacking. Likewise, the gut microbiome has been suggested as a critical regulator of obesity, but the bacterial species and metabolites that influence WAT inflammation are entirely unknown. We propose to use our recently developed high-throughput genomic and gnotobiotic tools, integrated with CRISPR-mediated interrogation of gene function, microbial culturomics, and in-vivo metabolic analysis in newly generated mouse models, in order to achieve a new level of molecular understanding of how WAT immune cells integrate environmental cues into their crosstalk with organismal metabolism, and to explore the microbial contributions to the molecular etiology of WAT inflammation in the pathogenesis of diet-induced obesity. Specifically, we aim to (a) decipher the global regulatory landscape and interaction networks of WAT hematopoietic cells at the single-cell level, (b) identify new mediators of WAT immune cell contributions to metabolic homeostasis, and (c) decode how host-microbiome communication shapes the development of WAT inflammation and obesity. Unraveling the principles of WAT immune cell regulation and their amenability to change by host-microbiota interactions may lead to a conceptual leap forward in our understanding of metabolic physiology and disease. Concomitantly, it may generate a platform for microbiome-based personalized therapy against obesity and its complications.

2 000 000 €

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

Our tissues are constantly renewed by stem cells. Over time, stem cells accumulate cellular damage that will compromise renewal and results in aging. As stem cells can divide asymmetrically, segregation of harmful factors to the differentiating daughter cell could be one possible mechanism for slowing damage accumulation in the stem cell. However, current evidence for such mechanisms comes mainly from analogous findings in yeast, and studies have concentrated only on few types of cellular damage. I hypothesize that the chronological age of a subcellular component is a proxy for all the damage it has sustained. In order to secure regeneration, mammalian stem cells may therefore specifically sort old cellular material asymmetrically. To study this, I have developed a novel strategy and tools to address the age-selective segregation of any protein in stem cell division. Using this approach, I have already discovered that stem-like cells of the human mammary epithelium indeed apportion chronologically old mitochondria asymmetrically in cell division, and enrich old mitochondria to the differentiating daughter cell. We will investigate the mechanisms underlying this novel phenomenon, and its relevance for mammalian aging. We will first identify how old and young mitochondria differ, and how stem cells recognize them to facilitate the asymmetric segregation. Next, we will analyze the extent of asymmetric age-selective segregation by targeting several other subcellular compartments in a stem cell division. Finally, we will determine whether the discovered age-selective segregation is a general property of stem cell in vivo, and it's functional relevance for maintenance of stem cells and tissue regeneration. Our discoveries may open new possibilities to target aging associated functional decline by induction of asymmetric age-selective organelle segregation.

1 500 000 €

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

Understanding how proteins respond to mutations is of paramount importance to biology and disease. While protein stability and misfolding have been instrumental in rationalizing the impact of mutations, we recently discovered that an alternative route is also frequent, where mutations at the surface of symmetric proteins trigger novel self-interactions that lead to infinite self-assembly. This mechanism can be involved in disease, as in sickle-cell anemia, but may also serve in adaptation. Importantly, it differs fundamentally from aggregation, because misfolding does not drive it. Thus, we term it “agglomeration”. The ease with which agglomeration can occur, even by single point mutations, shifts the paradigm of how quickly new protein assemblies can emerge, both in health and disease. This prompts us to determine the basic principles of protein agglomeration and explore its implications in cell physiology and human disease. We propose an interdisciplinary research program bridging atomic and cellular scales to explore agglomeration in three aims: (i) Map the landscape of protein agglomeration in response to mutation in endogenous yeast proteins; (ii) Characterize how yeast physiology impacts agglomeration by changes in gene expression or cell state, and, conversely, how protein agglomerates impact yeast fitness. (iii) Analyze agglomeration in relation to human disease via two approaches. First, by predicting single nucleotide polymorphisms that trigger agglomeration, prioritizing them using knowledge from Aims 1 & 2, and characterizing them experimentally. Second, by providing a proof-of-concept that agglomeration can be exploited in drug design, whereby drugs induce its formation, like mutations can do. Overall, through this research, we aim to establish agglomeration as a paradigm for protein assembly, with implications for our understanding of evolution, physiology, and disease.

2 574 819 €

Start date: 2019-04-01, End date: 2024-09-30

As global climates warmed ca. 10,000 years ago came a remarkable convergent transformation of human lifestyles that occurred independently in multiple continents and human populations. This transition from hunter-gatherer subsistence to food-production catalysed large-scale population growth, offering the opportunity for increased rates of adaptation, but also rapidly presented a large number of independent human populations with a new evolutionary challenge. This project will use ancient population genomics—the only way to directly reconstruct human genetic evolution—to study whether evolutionary processes during the agricultural transition differed in differed regions. Which genomic adaptations were associated with the agricultural transition? Did adaptation to hunter-gatherer and agricultural lifestyles act on similar genetic architecture in different instances? To which extent did adaptation in domestic dogs—the only species domesticated prior to the agricultural transition—occur in convergence with humans? To answer these questions, the project will generate ancient genomic data from pre-agricultural and early agricultural populations from multiple human- and domestic dog populations from Africa, Central America, and Southeast Asia. This will be achieved with direct sequencing as well as a new human ~850,000 SNP capture panel designed to avoid bias towards Eurasian ancestry. We will also develop new computational methods robust to the challenges posed by ancient genomes to identify adaptive admixture, analyse copy number variation, test continuous population models, and statistically assess convergence in the genomic architecture of adaptation. Leveraging cutting-edge ancient genomics and two model organisms for the genomic basis of phenotypic variation, this project aims to reconstruct the universal evolutionary phenomena underpinning a watershed evolutionary episode that shapes global biodiversity and the human condition to this day.

1 500 000 €

Start date: 2019-11-01, End date: 2024-10-31

Host cytokines, chemokines and type I IFNs are critical effectors of the innate immune response to viral and bacterial pathogens. Several classes of germ-line encoded pattern recognition receptors have been identified, which sense non-self nucleic acids and trigger these responses. Recently NLRP-3, a member of the NOD-like receptor (NLR) family, has been shown to sense endogenous danger signals, environmental insults and the DNA viruses adenovirus and HSV. Activation of NLRP-3 induces the formation of a large multiprotein complex in cells termed inflammasome , which controls the activity of pro-caspase-1 and the maturation of pro-IL-1² and pro-IL18 into their active forms. NLRP-3, however, does not regulate these responses to double stranded cytosolic DNA. We identified the cytosolic protein AIM2 as the missing receptor for cytosolic DNA. AIM2 contains a HIN200 domain, which binds to DNA and a pyrin domain, which associates with the adapter molecule ASC to activate both NF-ºB and caspase-1. Knock down of AIM2 down-regulates caspase-1-mediated IL-1² responses following DNA stimulation or vaccinia virus infection. Collectively, these observations demonstrate that AIM2 forms an inflammasome with the DNA ligand and ASC to activate caspase-1. Our underlying hypothesis for this proposal is that AIM2 plays a central role in host-defence to cytosolic microbial pathogens and also in DNA-triggered autoimmunity. The goals of this research proposal are to further characterize the DNA ligand for AIM2, to explore the molecular mechanisms of AIM2 activation, to define the contribution of AIM2 to host-defence against viral and bacterial pathogens and to assess its function in nucleic acid triggered autoimmune disease. The characterization of AIM2 and its role in innate immunity could open new avenues in the advancement of immunotherapy and treatment of autoimmune disease.

1 727 920 €

Start date: 2009-12-01, End date: 2014-11-30

This project focuses on two questions about host/parasite interactions: how do biotrophic plant pathogens suppress host defence? and, what is the basis for pathogen specialization on specific host species? A broadly accepted model explains resistance and susceptibility to plant pathogens. First, pathogens make conserved molecules ( PAMPS ) such as flagellin, that plants detect via cell surface receptors, leading to PAMP-Triggered Immunity (PTI). Second, pathogens make effectors that suppress PTI. Third, plants carry 100s of Resistance (R) genes that detect an effector, and activate Effector-Triggered Immunity (ETI). One effector is sufficient to trigger resistance. Albugo candida (Ac) (white rust) strongly suppresses host defence; Ac-infected Arabidopsis are susceptible to pathogen races to which they are otherwise resistant. Ac is an oomycete, not a fungus. Arabidopsis is resistant to races of Ac that infect brassicas. The proposed project involves three programs. First ( genomics, transcriptomics and bioinformatics ), we will use next-generation sequencing (NGS) methods (Solexa and GS-Flex), and novel transcriptomics methods to define the genome sequence and effector set of three Ac strains, as well as carrying out >40- deep resequencing of 7 additional Ac strains. Second, ( effectoromics ), we will carry out functional assays using Effector Detector Vectors (Sohn Plant Cell 19:4077 [2007]), with the set of Ac effectors, screening for enhanced virulence, for suppression of defence, for effectors that are recognized by R genes in disease resistant Arabidopsis and for host effector targets. Third, ( resistance diversity ), we will characterize Arabidopsis germplasm for R genes to Ac, both for recognition of Arabidopsis strains of Ac, and for recognition in Arabidopsis of effectors from Ac strains that infect brassica. This proposal focuses on Ac, but will establish methods that could discover new R genes in non-hosts against many plant diseases.

2 498 923 €

Start date: 2009-01-01, End date: 2014-06-30

The Anthropocene, the current geological epoch, is characterised by human-induced ecological changes, which have prompted a global biodiversity crisis. Human-introduced alien plants could help to offset native species loss, augmenting diversity and maintaining the services and capital that humans derive from nature. However, alien species that become invasive are themselves a key threat to biodiversity. Alien species thus presents a huge challenge for biodiversity conservation in the Anthropocene: should their arrival and establishment be inhibited or disregarded as they can potentially both exacerbate and ameliorate biodiversity loss? Coupling empirical and theoretical approaches, ALIENIMPACTS will directly address this challenge by developing an approach for accurately predicting impacts of alien plant invasions on plant community diversity and identifying the circumstances under which negative impacts will occur. Using temperate grasslands as a model system, ALIENIMPACTS will use innovative field experiments and global observations to systematically quantify – for the first time – how often, for how long, to what extent, under what conditions and in what ways alien plants can impact plant community diversity. ALIENIMPACTS will develop mechanistic niche models, validated with empirical data from grasslands in North America, Europe and Australia, that will enable realistic scenarios of invasion biodiversity impacts to be forecast, now and in the future. Developing empirically accurate mechanistic models that predict invasions and their biodiversity impact is a highly ambitious goal. Its achievement will mark a step-change in ecological theory and understanding, will inform environmental policy and management, and address a critical research challenge of the Anthropocene: how to conserve the biodiversity of plants – the dominant life form on earth – under global environmental change.

1 999 997 €

Start date: 2021-04-01, End date: 2026-03-31

Currently, more than 30% of all Europeans suffer from one or more allergic disorder but treatment is still mostly symptomatic due to a lack of understanding the underlying causality. Allergies are caused by type 2 immune responses triggered by recognition of harmless antigens. Both genetic and environmental factors have been proposed to favour allergic predisposition and both factors have a huge impact on the symbiotic microbiota and the intestinal immune system. Recently we and others showed that the transcription factor ROR(γt) seems to play a key role in mucosal tolerance in the gut and also regulates intestinal type 2 immune responses. Based on these results I postulate two major events in the gut for the development of an allergy in the lifetime of an individual: First, a failure to establish mucosal tolerance or anergy constitutes a necessity for the outbreak of allergic symptoms and allergic disease. Second, a certain ‘core’ microbiome or pathway of the intestinal microbiota predispose certain individuals for the later development of allergic disorders. Therefore, I will address the following aims: 1) Influence of ROR(γt) on mucosal tolerance induction and allergic disorders 2) Elucidate the T cell receptor repertoire of intestinal Th2 and ROR(γt)+ Tregs and assess the role of alternative NFκB pathway for induction of mucosal tolerance 3) Identification of ‘core’ microbiome signatures or metabolic pathways that favour allergic predisposition ALLERGUT will provide ground-breaking knowledge on molecular mechanisms of the failure of mucosal tolerance in the gut and will prove if the resident ROR(γt)+ T(reg) cells can function as a mechanistic starting point for molecular intervention strategies on the background of the hygiene hypothesis. The vision of ALLERGUT is to diagnose mucosal disbalance, prevent and treat allergic disorders even before outbreak and thereby promote Public Health initiative for better living.

1 498 175 €

Start date: 2017-07-01, End date: 2022-06-30

DNA repair pathways evolved as an intricate network that senses DNA damage and resolves it in order to minimise genetic lesions and thus preventing tumour formation. Gaining in recognition the last few years, the alternative end-joining (alt-EJ) DNA repair pathway was recently shown to be up-regulated and required for cancer cell viability in the absence of homologous recombination-mediated repair (HR). Despite this integral role, the alt-EJ repair pathway remains poorly characterised in humans. As such, its molecular composition, regulation and crosstalk with HR and other repair pathways remain elusive. Additionally, the contribution of the alt-EJ pathway to tumour progression as well as the identification of a mutational signature associated with the use of alt-EJ has not yet been investigated. Moreover, the clinical relevance of developing small-molecule inhibitors targeting players in the alt-EJ pathway, such as the polymerase Pol Theta (Polθ), is of importance as current anticancer drug treatments have shown limited effectiveness in achieving cancer remission in patients with HR-deficient (HRD) tumours. Here, we propose a novel, multidisciplinary approach that aims to characterise the players and mechanisms of action involved in the utilisation of alt-EJ in cancer. This understanding will better elucidate the changing interplay between different DNA repair pathways, thus shedding light on whether and how the use of alt-EJ contributes to the pathogenic history and survival of HRD tumours, eventually paving the way for the development of novel anticancer therapeutics. For all the abovementioned reasons, we are convinced this project will have important implications in: 1) elucidating critical interconnections between DNA repair pathways, 2) improving the basic understanding of the composition, regulation and function of the alt-EJ pathway, and 3) facilitating the development of new synthetic lethality-based chemotherapeutics for the treatment of HRD tumours.

1 498 750 €

Start date: 2017-07-01, End date: 2022-06-30

Despite the discovery of amyloidosis more than a century ago, the molecular and cellular mechanisms of these devastating human disorders remain obscure. In addition to their involvement in disease, amyloid fibrils perform physiological functions, whilst others have potentials as biomaterials. To realise their use in nanotechnology and to enable the development of amyloid therapies, there is an urgent need to understand the molecular pathways of amyloid assembly and to determine how amyloid fibrils interact with cells and cellular components. The challenges lie in the transient nature and low population of aggregating species and the panoply of amyloid fibril structures. This molecular complexity renders identification of the culprits of amyloid disease impossible to achieve using traditional methods. Here I propose a series of exciting experiments that aim to cast new light on the molecular and cellular mechanisms of amyloidosis by exploiting approaches capable of imaging individual protein molecules or single protein fibrils in vitro and in living cells. The proposal builds on new data from our laboratory that have shown that amyloid fibrils (disease-associated, functional and created from de novo designed sequences) kill cells by a mechanism that depends on fibril length and on cellular uptake. Specifically, I will (i) use single molecule fluorescence and non-covalent mass spectrometry and to determine why short fibril samples disrupt biological membranes more than their longer counterparts and electron tomography to determine, for the first time, the structural properties of cytotoxic fibril ends; (ii) develop single molecule force spectroscopy to probe the interactions between amyloid precursors, fibrils and cellular membranes; and (iii) develop cell biological assays to discover the biological mechanism(s) of amyloid-induced cell death and high resolution imaging and electron tomography to visualise amyloid fibrils in the act of killing living cells.

2 498 465 €

Start date: 2013-05-01, End date: 2019-04-30

Our ability to predict adaptation and the response of populations to selection is limited. Solving this issue is a fundamental challenge of evolutionary ecology with implications for applied sciences such as conservation, and agronomy. Non genetic inheritance (NGI; e.g., ecological niche transmission) is suspected to play a foremost role in adaptive evolution but such hypothesis remains untested. Using quantitative genetics in wild plant populations, experimental evolution, and epigenetics, we will assess the role of NGI in the adaptive response to selection of plant populations. The ANGI project will follow the subsequent research program: (1) Using long-term survey data, we will measure natural selection in wild populations of Antirrhinum majus within its heterogeneous array of micro-habitats. We will calculate the fitness gain provided by multiple traits and stem elongation to plants growing in bushes where they compete for light. Stem elongation is known to depend on epigenetic variation. (2) Using a statistical approach that we developed, we will estimate the quantitative genetic and non genetic heritability of traits. (3) We will identify phenotypic changes caused by fitness that are based on genetic variation and NGI and assess their respective roles in adaptive evolution. (4) In controlled conditions, we will artificially select for increased stem elongation in clonal lineages, thereby excluding DNA variation. We will quantify the non genetic response to selection and test for a quantitative epigenetic signature of selection. (5) We will build on our results to generate an inclusive theory of genetic and non genetic natural selection. ANGI builds on a confirmed expertise in selection experiments, quantitative genetics and NGI. In addition, the availability of survey data provides a solid foundation for the achievement of this project. Our ambition is to shed light on original mechanisms underlying adaptation that are an alternative to genetic selection.

1 999 970 €

Start date: 2016-03-01, End date: 2022-02-28

What does the fossil record tell us about the evolution of colour in animals through deep time? Evidence of colour in fossils can inform on the visual signalling strategies used by ancient animals. Research to date often has a narrow focus, lacks a broad phylogenetic and temporal context, and rarely incorporates information on taphonomy. This proposal represents a bold new holistic approach to the study of fossil colour: it will couple powerful imaging- and chemical analytical techniques with a rigorous programme of fossilisation experiments simulating decay, burial, and transport, and analysis of fossils and their sedimentary context, to construct the first robust models for the evolution of colour in animals through deep time. The research will resolve the original integumentary colours of fossil higher vertebrates, and the original colours of fossil hair; the fossil record of non-melanin pigments in feathers and insects; the biological significance of monotonal patterning in fossil insects; and the evolutionary history of scales and 3D photonic crystals in insects. Critically, the research will test, for the first time, whether evidence of fossil colour can solve broader evolutionary questions, e.g. the true affinities of enigmatic Cambrian chordate-like metazoans, and feather-like integumentary filaments in dinosaurs. The proposal entails construction of a dedicated experimental maturation laboratory for simulating the impact of burial on tissues. This laboratory will form the core of the world’s first integrated ‘experimental fossilisation facility’, consolidating the PI’s team as the global hub for fossil colour research. The research team comprises the PI, three postdoctoral researchers, and three PhD students, and will form an extensive research network via collaborations with 13 researchers from Europe and beyond. The project will reach out to diverse scientists and will inspire a positive attitude to science among the general public and policymakers alike.

1 562 000 €

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

Malaria parasite infection in humans has been called “the strongest known force for evolutionary selection in the recent history of the human genome”, and I hypothesize that a similar statement may apply to the mosquito vector, which is the definitive host of the malaria parasite. We previously discovered efficient malaria-resistance mechanisms in natural populations of the African malaria vector, Anopheles gambiae. Aim 1 of the proposed project will implement a novel genetic mapping design to systematically survey the mosquito population for common and rare genetic variants of strong effect against the human malaria parasite, Plasmodium falciparum. A product of the mapping design will be living mosquito families carrying the resistance loci. Aim 2 will use the segregating families to functionally dissect the underlying molecular mechanisms controlled by the loci, including determination of the pathogen specificity spectra of the host-defense traits. Aim 3 targets arbovirus transmission, where Anopheles mosquitoes transmit human malaria but not arboviruses such as Dengue and Chikungunya, even though the two mosquitoes bite the same people and are exposed to the same pathogens, often in malaria-arbovirus co-infections. We will use deep-sequencing to detect processing of the arbovirus dsRNA intermediates of replication produced by the RNAi pathway of the mosquitoes. The results will reveal important new information about differences in the efficiency and quality of the RNAi response between mosquitoes, which is likely to underlie at least part of the host specificity of arbovirus transmission. The 3 Aims will make significant contributions to understanding malaria and arbovirus transmission, major global public health problems, will aid the development of a next generation of vector surveillance and control tools, and will produce a definitive description of the major genetic factors influencing host-pathogen interactions in mosquito immunity.

2 307 800 €

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

Humans diverged from our closest living relatives, chimpanzees and other great apes, 6-10 million years ago. Since this divergence, our ancestors acquired genetic changes that enhanced cognition, altered metabolism, and endowed our species with an adaptive capacity to colonize the entire planet and reshape the biosphere. Through genome comparisons between modern humans, Neandertals, chimpanzees and other apes we have identified genetic changes that likely contribute to innovations in human metabolic and cognitive physiology. However, it has been difficult to assess the functional effects of these genetic changes due to the lack of cell culture systems that recapitulate great ape organ complexity. Human and chimpanzee pluripotent stem cells (PSCs) can self-organize into three-dimensional (3D) tissues that recapitulate the morphology, function, and genetic programs controlling organ development. Our vision is to use organoids to study the changes that set modern humans apart from our closest evolutionary relatives as well as all other organisms on the planet. In ANTHROPOID we will generate a great ape developmental cell atlas using cortex, liver, and small intestine organoids. We will use single-cell transcriptomics and chromatin accessibility to identify cell type-specific features of transcriptome divergence at cellular resolution. We will dissect enhancer evolution using single-cell genomic screens and ancestralize human cells to resurrect pre-human cellular phenotypes. ANTHROPOID utilizes quantitative and state-of-the-art methods to explore exciting high-risk questions at multiple branches of the modern human lineage. This project is a ground breaking starting point to replay evolution and tackle the ancient question of what makes us uniquely human?

1 500 000 €

Start date: 2019-06-01, End date: 2024-05-31

Interferons (IFNs), which are signalling proteins produced by infected cells, are the first line of defence against viral infections. IFNs induce, in infected and neighbouring cells, the expression of hundreds of IFN-stimulated genes (ISGs). The ISGs in turn induce in cells a potent antiviral state, capable of preventing replication of most viruses, including Human Immunodeficiency Virus type 1 (HIV-1) and influenza A virus (FLUAV). Identifying the antiviral ISGs and understanding their mechanisms of action is therefore crucial to progress in the fight against viruses. ISGs playing a role in the antiviral state have been identified, such as human MX1, a well-known antiviral factor able to restrict numerous viruses including FLUAV, and MX2, an HIV-1 inhibitor. Both proteins bind to viral components but their detailed mechanisms of action, as well as the consequences of restriction on the activation of the innate immune system, remain unclear. Moreover, our preliminary work shows that additional anti-HIV-1 and anti-FLUAV ISGs remain to identify. In this context, this proposal seeks an ERC StG funding to explore 3 major aims: 1) unravelling the mechanisms of antiviral action of MX proteins, by taking advantage of their similar structure and engineered chimeric proteins, and by using functional genetic screens to identify their cofactors; 2) investigating the consequences of incoming virus recognition by MX proteins on innate immune signalling, by altering their expression in target cells and measuring the cell response in terms of gene induction and cytokine production; 3) identifying and characterizing new ISGs able to inhibit viral replication with a combination of powerful approaches, including a whole-genome CRISPR/Cas9 knock-out screen. Overall, this proposal will provide a better understanding of the molecular mechanisms involved in the antiviral effect of IFN, and may guide future efforts to identify novel therapeutic targets against major pathogenic viruses.

1 499 794 €

Start date: 2017-12-01, End date: 2023-05-31

RNA interference (RNAi) is a conserved sequence-specific, gene-silencing mechanism that is induced by double-stranded RNA (dsRNA). One of the functions of this pathway is the defense against parasitic nucleic acids: transposons and viruses. Previous results demonstrated that viral infections in Drosophila melanogaster are fought by an antiviral RNAi response and that components of the endocytic pathway are required for dsRNA entry to initiate the RNAi response. Recently we have shown that infected insect cells spread a systemic silencing signal that elicits a protective RNAi-dependent immunity throughout the organism. This suggests that the cell-autonomous RNAi response is insufficient to control a viral infection and that flies also rely on systemic immune response to fight against such infections. As a junior group leader, I will study the mechanisms that mediate the RNAi-based antiviral response in insects. By combining biochemical, cellular, molecular and genomic approaches, both in vivo and in cell culture, I will analyze the mechanisms underlying viral tropism, systemic propagation of the antiviral signal and the basis of the persistence of the antiviral state. Furthermore, I will examine whether the dsRNA-uptake pathway is conserved in mosquitoes and its relationship with viral immunity in that host. This comprehensive approach will tackle how this nucleic acid-based immunity works in insects to generate an anti-viral stage. A better understanding of the role of RNA silencing in insects during virus infection will allow the exploitation of this pathway for improvement of public health related problems such as arbovirus infection and disease.

1 900 000 €

Start date: 2009-10-01, End date: 2014-12-31

"The attine fungus-growing ants are prime models for understanding phenotypic adaptations in social evolution and symbiosis. The mutualism has many hallmarks of advanced cooperation in its mating system commitments and functional complementarity between multiple symbiont partners, but potential conflicts between sexes and castes over reproductive priorities, and between hosts and symbionts over symbiont mixing have also been documented. With collaborators at BGI-Shenzhen and the Smithsonian Institution my group has obtained six reference genomes representing all genus-level branches of the higher attine ants and a lower attine outgroup. With collaborators in Denmark and Australia we have pioneered proteomic approaches to understand the preservation of sperm viability in spite of sperm competition and the enzymatic decomposition of plant substrates that the ants use to make their fungus gardens grow. Here, I propose an integrated study focusing on four major areas of attine ant biology that are particularly inviting for in depth molecular approaches: 1. The protein-level networks that secure life-time (up to 20 years) sperm storage in specialized ant-queen organs and the genetic mechanisms that shape and adjust these “sexual symbiome” networks. 2. The ant-fungal symbiome, i.e. the dynamics of fungal enzyme production for plant substrate degradation and the redistribution of these enzymes in fungus gardens through fecal deposition after they are ingested but not digested by the ants. 3. The microbial symbiome of ant guts and other tissues with obligate bacterial mutualists, of which we have identified some and will characterize a wider collection across the different branches of the attine ant phylogeny. 4. The genome-wide frequency of genomic imprinting and the significance of these imprints for the expression of caste phenotypes and the regulation of potential reproductive conflicts."

2 290 102 €

Start date: 2013-05-01, End date: 2018-04-30

In our quest to fully understand the processes that shape the genomic variation of species, describing variation of the past is a fundamental objective. However, the origins and the extent of great ape variation, the genomic description of extinct primate species and the genomic footprints of introgression events all remain unknown. Even today, and in contraposition to human evolutionary biology, the almost null presence of ancient great ape samples has precluded a comprehensive exploration of such diversity. Here, I present two approaches that will expose great ape diversity throughout time and will allow me to compare the genomic impact of introgression events across lineages. First, I would like to take advantage of ancient ape samples that will provide us with a direct view of the genomes of extinct populations. Second, I would like to exploit current and recent diversity to indirectly access the parts of extinct ape genomes that became hybridized with current species in the past. For the latter, we will analyse hundreds of non-invasive samples taken from present-day great apes as well as historical specimens. Altogether, this information will enable me to decipher novel genomes that until now have been lost in time. In this way, I will be able to properly understand the origins and dynamics of genomic variants and to study how admixture has contributed to today´s adaptive landscape. By completing this proposal and performing analogies to the human lineage, fundamental insights will be revealed about (i) the spatial-temporal history of our closest species and (ii) the functional consequences of introgressed events. On top of that, these results will help to annotate functional consequences of novel mutations in the human genome. In so doing, a fundamental insight will be provided into the evolutionary history of these regions and into human mutations with multiple repercussions in the understanding of evolution and human biology.

1 896 875 €

Start date: 2020-06-01, End date: 2025-05-31

Work conducted in my laboratory on the trypanosome killing factor of human serum led to the identification of the primate-specific Apolipoprotein L1 (APOL1) as a novel pore-forming protein with striking similarities with proteins of the apoptotic BCL2 family. APOL1 belongs to a family of proteins induced under inflammatory conditions in myeloid and endothelial cells. APOL1 is efficiently neutralized by the SRA protein of Trypanosoma rhodesiense, accounting for the ability of this trypanosome subspecies to infect humans and cause sleeping sickness. We found that natural APOL1 variants escaping SRA neutralization and therefore conferring human resistance to T. rhodesiense are associated with chronic kidney disease. Moreover, transgenic mice expressing these APOL1 variants exhibit an obese phenotype. Our unpublished results also indicate that APOLs control the lifespan of dendritic cells and podocytes activated by viral stimuli. Therefore, we propose that the pathology of APOL variants is due to their deregulated activity on the control of the cellular lifespan in myeloid/endothelial cells activated by pathogen detection. This project aims at characterizing (i) the molecular mechanism by which APOLs control the lifespan of activated dendritic cells and podocytes, which has direct impact on innate immunity and inflammation, and (ii) the mechanism by which APOL1 variants cause pathology. In addition, we plan to detail the physiological function of APOLs by studying the phenotype of transgenic mice either expressing human APOL1 (wild-type and variants) or devoid of APOL genes, which we have recently generated. Finally, we propose to exploit the extraordinary potential of trypanosomes for antigenic variation in order to produce SRA variants able to neutralize the pathogenic APOL1 variants. Preliminary experiments suggest that in podocytes SRA antagonizes APOL1 induction by viral stimulus and subsequent cell death, opening new perspectives to treat kidney disease.

2 250 000 €

Start date: 2015-09-01, End date: 2021-06-30

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

Arboviruses infect millions of people each year, however, mechanisms that drive viral emergence and maintenance remain largely unknown. A combination of host factors (e.g., human mobility), mosquito factors (e.g., abundance) and viral factors (e.g., transmissibility) interconnect to drive spread. Further, for endemic arboviruses, complex patterns of population immunity, built up over many years, appear key to the emergence of particular lineages. To disentangle the contribution of these different drivers, we need detailed data from the same pathogen system over a long time period from the same location. In addition, we need new methods, which can integrate these different data sources and allow appropriate mechanistic inferences. In this project, I will use the most globally prevalent arbovirus, dengue virus, as a case study. I will focus on Thailand where all four dengue serotypes have circulated endemically for decades and excellent long-term data and isolates exist, to address two fundamental questions: i) How do population-level patterns of immunity evolve over time and what is their impact on strain dynamics? I will use mechanistic models applied to historic serotype-specific case data to reconstruct the evolving immune profile of the population and explore the impact of immunity on viral diversity using sequences from archived isolates from each year over a 50-year period. ii) How do human behaviors, vector densities interact with immunity to dictate spread? I will work with geolocated full genome sequences from across Thailand and use detailed data on how people move, their contact patterns, their immunity profiles and mosquito distributions to study competing hypotheses of how arboviruses spread. I will compare the key drivers of dengue spread with that found for outbreaks of Zika and chikungunya. This proposal addresses fundamental questions about the mechanisms that drive arboviral emergence and spread that will be relevant across disease systems.

1 499 896 €

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

"Archaea constitute the third domain of life and are believed to be close to the origin of life. They comprise a diverse group of micro-organisms that combine bacterial and eukaryotic features, but also employ many novel mechanisms. They possess a unique cell envelope with a cytoplasmic membrane of ether lipids surrounded by a proteinaceous S-layer and various cell appendages such as flagella, pili and more unusual structures. Studies have shown that the archaeal flagellum is an unique structure as it functionally resembles the bacterial flagellum, but structurally it is a simple type IV pilus. Moreover, we have shown that this type IV pilus can rotate. Therefore I propose to name the archaeal flagellum, the archaellum, as it is fundamentally different from the bacterial flagellum. In this proposal I aim to understand the assembly and mechanism of rotation of the archaellum of the thermocacidophilic crenarchaen Sulfolobus acidocaldarius by using biochemical, genetic and biophysical methods. The main milestons are: - Biochemical and structural characterization of all archaellum subunits - To understand the assembly pathway of the archaellum and the interactions of its different subunits - To understand how rotation of the filament is achieved and which subunits are important for this movement This work will identify a new, relatively simple motor complex that has evolved from primordial type IV pili assembly machineries and therefore uncover general principles of macromolecular assemblies at cellular surfaces and a novel mechanism to generate mechanical force that can be translated into movement."

1 464 317 €

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

Arl (Arf-like) proteins, GTP-binding proteins of the Ras superfamily, are molecular switches that cycle between a GDP-bound inactive and GTP-bound active state. There are 16 members of the Arl subfamily in the human genome whose basic mechanistic function is unknown. The interactome of Arl2/3 includes proteins involved in retinopathies and other ciliary diseases such as Leber¿s Congenital Amaurosis (LCA) and kidney diseases such as nephronophthisis. Arl6 has been found mutated in Bardet Biedl Syndrome, another pleiotropic ciliary disease. In the proposed interdisciplinary project I want to explore the function of the protein network of Arl2/3 and Arl6 by a combination of biochemical, biophysical and structural methods and use the knowledge obtained to probe their function in live cells. As with other subfamily proteins of the Ras superfamily which have been found to mediate similar biological functions I want to derive a basic understanding of the function of Arl proteins and how it relates to the development and function of the ciliary organelle and how they contribute to ciliary diseases. The molecules in the focus of the project are: the GTP-binding proteins Arl2, 3, 6; RP2, an Arl3GAP mutated in Retinitis pigmentosa; Regulators of Arl2 and 3; PDE¿ and HRG4, effectors of Arl2/3, which bind lipidated proteins; RPGR, mutated in Retinitis pigmentosa, an interactor of PDE¿; RPGRIP and RPGRIPL, interactors of RPGR mutated in LCA and other ciliopathies; Nephrocystin, mutated in nephronophthisis, an interactor of RPGRIP and Arl6, mutated in Bardet Biedl Syndrome, and the BBS complex. The working hypothesis is that Arl protein network(s) mediate ciliary transport processes and that the GTP switch cycle of Arl proteins is an important element of regulation of these processes.

2 434 400 €

Start date: 2011-04-01, End date: 2016-03-31

Argonaute nucleases are key players of the eukaryotic RNA interference (RNAi) system. Using small RNA guides, these Argonaute (Ago) proteins specifically target complementary RNA molecules, resulting in regulation of a wide range of crucial processes, including chromosome organization, gene expression and anti-virus defence. Since 2010, my research team has studied closely-related prokaryotic Argonaute (pAgo) variants. This has revealed spectacular mechanistic variations: several thermophilic pAgos catalyse DNA-guided cleavage of double stranded DNA, but only at elevated temperatures. Interestingly, a recently discovered mesophilic Argonaute (CbAgo) can generate double strand DNA breaks at moderate temperatures, providing an excellent basis for this ARGO project. In addition, genome analysis has revealed many distantly-related Argonaute variants, often with unique domain architectures. Hence, the currently known Argonaute homologs are just the tip of the iceberg, and the stage is set for making a big leap in the exploration of the Argonaute family. Initially we will dissect the molecular basis of functional and mechanistic features of uncharacterized natural Argonaute variants, both in eukaryotes (the presence of an Ago-like subunit in the Mediator complex, strongly suggests a regulatory role of an elusive non-coding RNA ligand) and in prokaryotes (selected Ago variants possess distinct domains indicating novel functionalities). After their thorough biochemical characterization, I aim at engineering the functionality of the aforementioned CbAgo through an integrated rational & random approach, i.e. by tinkering of domains, and by an unprecedented in vitro laboratory evolution approach. Eventually, natural & synthetic Argonautes will be selected for their exploitation, and used for developing original genome editing applications (from silencing to base editing). Embarking on this ambitious ARGO expedition will lead us to many exciting discoveries.

2 177 158 €

Start date: 2019-07-01, End date: 2024-06-30

Main goal. We aim to understand the puzzling coexistence of antibiotic-resistant and antibiotic-sensitive species in natural soil environments, using novel quantitative experimental techniques and mathematical analysis. The ecological insights gained will be translated into novel treatment strategies for combating antibiotic resistance. Background. Microbial soil ecosystems comprise communities of species interacting through copious secretion of antibiotics and other chemicals. Defence mechanisms, i.e. resistance to antibiotics, are ubiquitous in these wild communities. However, in sharp contrast to clinical settings, resistance does not take over the population. Our hypothesis is that the ecological setting provides natural mechanisms that keep antibiotic resistance in check. We are motivated by our recent finding that specific antibiotic combinations can generate selection against resistance and that soil microbial strains produce compounds that directly target antibiotic resistant mechanisms. Approaches. We will: (1) Isolate natural bacterial species from individual grains of soil, characterize their ability to produce and resist antibiotics and identify the spatial scale for correlations between resistance and production. (2) Systematically measure interactions between species and identify interaction patterns enriched in co-existing communities derived from the same grain of soil. (3) Introducing fluorescently-labelled resistant and sensitive strains into natural soil, we will measure the fitness cost and benefit of antibiotic resistance in situ and identify natural compounds that select against resistance. (4) Test whether such “selection-inverting” compounds can slow evolution of resistance to antibiotics in continuous culture experiments. Conclusions. These findings will provide insights into the ecological processes that keep antibiotic resistance in check, and will suggest novel antimicrobial treatment strategies.

1 900 000 €

Start date: 2012-09-01, End date: 2018-08-31

Life on earth is sustained by the process that converts sunlight energy into chemical energy: photosynthesis. This process is operating near the boundary between life and death: if the absorbed energy exceeds the capacity of the metabolic reactions, it can result in photo-oxidation events that can cause the death of the organism. Over-excitation is happening quite often: oxygenic organisms are exposed to (drastic) changes in environmental conditions (light intensity, light quality and temperature), which influence the physical (light-harvesting) and chemical (enzymatic reactions) parts of the photosynthetic process to a different extent, leading to severe imbalances. However, daily experience tells us that plants are able to deal with most of these situations, surviving and happily growing. How do they manage? The photosynthetic membrane is highly flexible and it is able to change its supramolecular organization and composition and even the function of some of its components on a time scale as fast as a few seconds, thereby regulating the light-harvesting capacity. However, the structural/functional changes in the membrane are far from being fully characterized and the molecular mechanisms of their regulation are far from being understood. This is due to the fact that all these mechanisms require the simultaneous presence of various factors and thus the system should be analyzed at a high level of complexity; however, to obtain molecular details of a very complex system as the thylakoid membrane in action has not been possible so far. Over the last years we have developed and optimized a range of methods that now allow us to take up this challenge. This involves a high level of integration of biological and physical approaches, ranging from plant transformation and in vivo knock out of individual pigments to ultrafast-spectroscopy in a mix that is rather unique for my laboratory and will allow us to unravel the photoprotective mechanisms in algae and plants.

1 696 961 €

Start date: 2011-12-01, End date: 2017-11-30

Spontaneous protein crystallization is a rare event in biology. Eosinophilic inflammation such as seen in the airways in asthma, chronic rhinosinusitis and helminth infection is however accompanied by accumulation of large amounts of extracellular Charcot-Leyden crystals. These are made of Galectin-10, a protein of unknown function produced by eosinophils, hallmark cells of type 2 immunity. In mice, eosinophilic inflammation is also accompanied by protein crystal build up, composed of the chitinase-like proteins Ym1 and Ym2, produced by alternatively activated macrophages. Here we challenge the current view that these crystals are just markers of eosinophil demise or macrophages activation. We hypothesize that protein crystallization serves an active role in immunoregulation of type 2 immunity. On the one hand, crystallization might turn a harmless protein into a danger signal. On the other hand, crystallization might sequester and eliminate the physiological function of soluble Galectin-10 and Ym1, or prolong it via slow release elution. For full understanding, we therefore need to understand the function of the proteins in a soluble and crystalline state. Our program at the frontline of immunology, molecular structural biology and clinical science combines innovative tool creation and integrative research to investigate the structure, function, and physiology of galectin-10 and related protein crystals. We chose to study asthma as the crystallizing proteins are abundantly present in human and murine disease. There is still a large medical need for novel therapies that could benefit patients with chronic steroid-resistant disease, and are alternatives to eosinophil-depleting antibodies whose long term effects are unknown.

2 499 846 €

Start date: 2018-08-01, End date: 2023-07-31

Microbial symbioses play an essential role in most aspects of life’s evolution and enabled major evolutionary events such as the origin of eukaryotes. But in spite of the importance of symbioses, our knowledge is based on a limited number of microbial host-symbiont systems, few of which include Archaea. Notably, recent cultivation-independent approaches have revealed two hugely diverse microbial groups of putative deep-branching archaeal and bacterial symbionts referred to as DPANN and CPR, respectively. The major aims of ASymbEL are to test the hypotheses that (a) DPANN, together with CPR, have key positions in the tree of life, requiring to revise our view on the early evolution of cells and (b) that the diverse DPANN substantially shape the evolution of life through symbiont-host interactions. This will be achieved using a unique three-pronged strategy that will integrate knowledge from both micro- and macroevolutionary levels: I will place the diverse DPANN in a rooted tree of life including CPR, reconcile the history of their genome content evolution and identify novel host-symbiont systems using sophisticated phylogenomic approaches combined with microscopy. Further, I will elucidate fundamental principles of their genome evolution and interactions, for the first time using experimental evolution approaches to study two cultivated DPANN symbionts in co-culture with their hosts. Finally, I will determine the dynamics of natural populations of known and newly identified archaeal host-symbiont systems and the influence of symbionts on host population structure and genome evolution using a unique approach combining single-cell, metagenomics and population genomics. Altogether, this will allow to provide key insights into the global impact of these symbionts in the deep origins and diversification of cellular life on Earth. Thereby, ASymbEL paves the way for a deeper understanding of symbiotic evolutionary theory and the fundamental laws of cellular evolution.

1 869 946 €

Start date: 2021-02-01, End date: 2026-01-31

Autophagy is a conserved, lysosomal-mediated pathway required for cell homeostasis and survival. It is controlled by the master regulators of energy (AMPK) and growth (TORC1) and mediated by the ATG (autophagy) proteins. Deregulation of autophagy is implicated in cancer, immunity, infection, aging and neurodegeneration. Autophagosomes form and expand using membranes from the secretory and endocytic pathways but how this occurs is not understood. ATG9, the only transmembrane ATG protein traffics through the cell in vesicles, and is essential for rapid initiation and expansion of the membranes which form the autophagosome. Crucially, how ATG9 functions is unknown. I will determine how ATG9 initiates the formation and expansion of the autophagosome by amino acid starvation through a molecular dissection of proteins resident in ATG9 vesicles which modulate the composition and property of the initiating membrane. I will employ high resolution light and electron microscopy to characterize the nucleation of the autophagosome, proximity-specific biotinylation and quantitative Mass Spectrometry to uncover the proteome required for the function of the ATG9, and optogenetic tools to acutely regulate signaling lipids. Lastly, with our tools and knowledge I will develop an in vitro reconstitution system to define at a molecular level how ATG9 vesicle proteins, membranes that interact with ATG9 vesicles, and other accessory ATG components nucleate and form an autophagosome. In vitro reconstitution of autophagosomes will be assayed biochemically, and by correlative light and cryo-EM and cryo-EM tomography, while functional reconstitution of autophagy will be tested by selective cargo recruitment. The development of a reconstituted system and identification proteins and lipids which are key components for autophagosome formation will provide a means to identify a new generation of targets for translational work leading to manipulation of autophagy for disease related therapies.

2 121 055 €

Start date: 2018-07-01, End date: 2023-06-30

DNA double-strand breaks are perhaps the most harmful DNA damages and result in carcinogenic chromosome aberrations. Cells protect their genome by activating a complex signaling and repair network, collectively denoted DNA damage response (DDR). A key initial step of the DDR is the activation of the 360 kDa checkpoint kinase ATM (ataxia telangiectasia mutated) by the multifunctional DSB repair factor Mre11-Rad50-Nbs1 (MRN). MRN senses and tethers DSBs, processes DSBs for further resection, and recruits and activates ATM to trigger the DDR. A mechanistic basis for the activities of the core DDR sensor MRN has not been established, despite intense research over the past decade. Our recent breakthroughs on structures of core Mre11-Rad50 and Mre11-Nbs1 complexes enable us now address three central questions to finally clarify the mechanism of MRN in the DDR: - How does MRN interact with DNA or DNA ends in an ATP dependent manner? - How do MRN and associated factors such as CtIP process blocked DNA ends? - How do MRN and DNA activate ATM? We will employ an innovative structural biology hybrid methods approach by combining X-ray crystallography, electron microscopy and small angle scattering with crosslink mass spectrometry and combine the structure-oriented techniques with validating in vitro and in vivo functional studies. The anticipated outcome will clarify the structural mechanism of one of the most important but enigmatic molecular machineries in maintaining genome stability and also help understand the molecular defects associated with several prominent cancer predisposition and neurodegenerative disorders.

2 498 019 €

Start date: 2013-05-01, End date: 2018-04-30

Autophagy, a lysosomal degradation pathway in which the cell digests its own components, is an essential biological pathway that promotes organismal health and longevity and helps combat cancer and neurodegenerative diseases. Accordingly, the 2016 Nobel Prize in Physiology or Medicine was awarded for research in autophagy. Although autophagy has been extensively studied from yeast to mammals, the molecular events that underlie its induction and progression remain elusive. A highly conserved protein kinase, Atg1, plays a unique and essential role in initiating autophagy, yet despite this pivotal importance it has taken over twenty years for its first downstream target to be discovered. However, whilst our identification of the autophagy related membrane protein Atg9 as the first Atg1 substrate is an important advance, the molecular mechanisms that enable the extensive remodelling of cellular membranes that occurs during autophagy is still completely undefined. A detailed knowledge of the inputs and outputs of the Atg1 kinase will enable us to provide a definitive mechanistic understanding of autophagy. We have devised a novel permeabilized cell assay that reconstitutes the pathway in vitro, allowing us to recapitulate key steps in the autophagic process and thereby determine how the individual steps that lead up to autophagy are controlled. We will use this system to dissect the functional role of Atg1 kinase in autophagosome-vacuole fusion (Objective 1), and to determine the origin of the autophagic membrane and the role of Atg1 in expanding these (Objective 2). To reveal how Atg1/ULK1 kinase is activated in mammalian cells, we will apply the unique and carefully tailored synthetic in vivo approaches that we have recently developed (Objective 3). By focusing on the activation of the Atg1 kinase and the molecular events that it executes, we will be able to explain its central role in regulating the autophagic process and define the mechanistic steps in the pathway.

1 955 666 €

Start date: 2018-06-01, End date: 2023-05-31

The proposed project aims at investigating the molecular mechanisms that activate B cell antigen receptor (BCR) signalling in chronic lymphocytic leukaemia (CLL). While it is widely accepted that the unbroken BCR expression in CLL cells is indicative for a key role in disease development, the mechanisms that induce BCR activation and survival of malignant cells are still elusive. Using a unique reconstitution system, we have recently shown that CLL-derived BCRs possess the exceptional capacity for cell-autonomous signalling independent of external antigen. Crystallographic analyses confirmed our model that CLL-BCRs bind to intrinsic motifs in nearby BCRs on the very same cell. In addition to the BCR, several pathogenic factors influence the biological behaviour of CLL cells, but the functional hierarchy and the effect on BCR signalling are insufficiently understood. Here, we aim at investigating the structural cause of autonomous signalling as well as the characterization of important signalling pathways and their mechanistic action in CLL pathogenesis. By combining crystallography with the measurement of autonomous signalling of wild type and mutated receptors in our unique reconstitution system, we will generate a structure-function relationship for CLL-BCRs. By generating new animal models and by employing classical as well as cutting-edge approaches of biochemistry and molecular/cellular immunology, we will comprehensively characterize the signalling pathways that are activated by autonomous signalling and might be important for CLL pathogenesis. These systematic efforts are necessary to understand how various biological mechanisms operate and ultimately activate downstream pathways that result in a lymphoproliferative disease. In addition, a cohesive model of CLL pathogenesis, which elucidates the hierarchical order of pathogenic factors and their interaction with BCR signalling, may well lead to novel disease-specific preventive or therapeutic intervention.

2 256 250 €

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

I propose to study how eukaryotic cells generate autophagosomes, organelles bounded by a double membrane. These are formed during autophagy and mediate the degradation of cytoplasmic substances within the lysosomal compartment. Autophagy thereby protects the organism from pathological conditions such as neurodegeneration, cancer and infections. Many core factors required for autophagosome formation have been identified but the order in which they act and their mode of action is still unclear. We will use a combination of biochemical and cell biological approaches to elucidate the choreography and mechanism of these core factors. In particular, we will focus on selective autophagy and determine how the autophagic machinery generates an autophagosome that selectively contains the cargo. To this end we will focus on the cytoplasm-to-vacuole-targeting pathway in S. cerevisiae that mediates the constitutive delivery of the prApe1 enzyme into the vacuole. We will use cargo mimetics or prApe1 complexes in combination with purified autophagy proteins and vesicles to reconstitute the process and so determine which factors are both necessary and sufficient for autophagosome formation, as well as elucidating their mechanism of action. In parallel we will study selective autophagosome formation in human cells. This will reveal common principles and special adaptations. In particular, we will use cell lysates from genome-edited cells in combination with purified autophagy proteins to reconstitute selective autophagosome formation around ubiquitin-positive cargo material. The insights and hypotheses obtained from these reconstituted systems will be validated using cell biological approaches. Taken together, our experiments will allow us to delineate the major steps of autophagosome formation during selective autophagy. Our results will yield detailed insights into how cells form and shape organelles in a de novo manner, which is major question in cell- and developmental biology.

1 999 640 €

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

It has long been understood that genes contribute to phenotypes that are then the basis of selection. However, the nature and process of this relationship remains largely theoretical, and the relative contribution of change in gene expression and coding sequence to phenotypic diversification is unclear. The aim of this proposal is to fuse information about sexually dimorphic phenotypes, the mating systems and sexually antagonistic selective agents that shape sexual dimorphism, and the sex-biased gene expression patterns that encode sexual dimorphisms, in order to create a cohesive integrated understanding of the relationship between evolution, the genome, and the animal form. The primary approach of this project is to harnesses emergent DNA sequencing technologies in order to measure evolutionary change in gene expression and coding sequence in response to different sex-specific selection regimes in a clade of birds with divergent mating systems. Sex-specific selection pressures arise in large part as a consequence of mating system, however males and females share nearly identical genomes, especially in the vertebrates where the sex chromosomes house very small proportions of the overall transcriptome. This single shared genome creates sex-specific phenotypes via different gene expression levels in females and males, and these sex-biased genes connect sexual dimorphisms, and the sexually antagonistic selection pressures that shape them, with the regions of the genome that encode them. The Galloanserae (fowl and waterfowl) will be used to in the proposed project, as this clade combines the necessary requirements of both variation in mating systems and a well-conserved reference genome (chicken). The study species selected from within the Galloanserae for the proposal exhibit a range of sexual dimorphism and sperm competition, and this will be exploited with next generation (454 and Illumina) genomic and transcriptomic data to study the gene expression patterns that underlie sexual dimorphisms, and the evolutionary pressures acting on them. This work will be complemented by the development of mathematical models of sex-specific evolution that will be tested against the gene expression and gene sequence data in order to understand the mechanisms by which sex-specific selection regimes, arising largely from mating systems, shape the phenotype via the genome.

1 350 804 €

Start date: 2011-01-01, End date: 2016-07-31