ERC FUNDED PROJECTS

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

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

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

Calcium-activated chloride channels (CaCCs) play key roles in a range of physiological processes such as the control of membrane excitability, photoreception and epithelial secretion. Although the importance of these channels has been recognized for more than 30 years their molecular identity remained obscure. The recent discovery of two protein families encoding for CaCCs, Anoctamins and Bestrophins, was a scientific breakthrough that has provided first insight into two novel ion channel architectures. Within this proposal we aim to determine the first high resolution structures of members of both families and study their functional behavior by an interdisciplinary approach combining biochemistry, X-ray crystallography and electrophysiology. The structural investigation of eukaryotic membrane proteins is extremely challenging and will require us to investigate large numbers of candidates to single out family members with superior biochemical properties. During the last year we have made large progress in this direction. By screening numerous eukaryotic Anoctamins and prokaryotic Bestrophins we have identified well-behaved proteins for both families, which were successfully scaled-up and purified. Additional family members will be identified within the course of the project. For these stable proteins we plan to grow crystals diffracting to high resolution and to proceed with structure determination. With first structural information in hand we will perform detailed functional studies using electrophysiology and complementary biophysical techniques to gain mechanistic insight into ion permeation and gating. As the pharmacology of both families is still in its infancy we will in later stages also engage in the identification and characterization of inhibitors and activators of Anoctamins and Bestrophins to open up a field that may ultimately lead to the discovery of novel therapeutic strategies targeting calcium-activated chloride channels.

2 176 000 €

Start date: 2014-02-01, End date: 2020-01-31

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

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

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 is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Cytotoxic stresses and starvation, however, induces the formation of larger autophagosomes that capture cargo unselectively. Autophagosomes are being generated from a cup-shaped precursor membrane, the isolation membrane, which expands to engulf cytoplasmic components. Sealing of this structure gives rise to the double-membrane surrounded autophagosomes. Two interconnected ubiquitin (Ub)-like conjugation systems coordinate the expansion of autophagosomes by conjugating the autophagy related (Atg)-protein Atg8 to the isolation membrane. In an effort to unravel the function of Atg8, we reconstituted the system on model membranes in vitro and found that Atg8 forms together with the Atg12–Atg5-Atg16 complex a membrane scaffold which is required for productive autophagy in yeast. Humans possess seven Atg8-homologs and two mutually exclusive Atg16-variants. Here, we propose to investigate the function of the human Ub-like conjugation system using a fully reconstituted in vitro system. The spatiotemporal organization of recombinant fluorescent-labeled proteins with synthetic model membranes will be investigated using confocal and TIRF-microscopy. Structural information will be obtained by atomic force and electron microscopy. Mechanistic insights, obtained from the in vitro work, will be tested in vivo in cultured human cells. We belief that revealing 1) the function of the human Ub-like conjugation system in autophagy, 2) the functional differences of Atg8-homologs and the two Atg16-variants Atg16L1 and TECPR1 and 3) how Atg16L1 coordinates non-canonical autophagy will provide essential insights into the pathophysiology of cancer, neurodegenerative, and autoimmune diseases.

1 499 726 €

Start date: 2015-04-01, End date: 2020-09-30

"The main objective of this application is to study the molecular basis of cellular infection by bacterial ABC-type toxins (Tc). Tc complexes are important virulence factors of a range of bacteria, including Photorhabdus luminescens and Yersinia pseudotuberculosis that infect insects and humans. Belonging to the class of pore-forming toxins, tripartite Tc complexes perforate the host membrane by forming channels that translocate toxic enzymes into the host. In our previous cryo-EM work on the P. luminescens Tc complex we discovered that Tcs use a special syringe-like device for cell entry. Building on these results, we now intend to unravel the molecular mechanism through which this unusual and complicated injection system allows membrane permeation and protein translocation. We will use a hybrid approach, including biochemical reconstitution, structural analysis by cryo-EM and X-ray crystallography, fluorescence-based assays and site-directed mutagenesis to provide a comprehensive description of the molecular mechanism of infection at an unprecedented level of molecular detail. Our results will be paradigmatic for understanding the mechanism of action of ABC-type toxins and will shed new light on the interactions of bacterial pathogens with their hosts."

1 999 992 €

Start date: 2014-07-01, End date: 2019-06-30

The functioning of a single cell or organism is governed by the laws of chemistry and physics. The bridge from biology to chemistry and physics is provided by structural biology: to understand the functioning of a cell, it is necessary to know the atomic structure of macromolecular assemblies, which may contain hundreds of components. To characterise the structures of the increasingly large and often flexible complexes, high resolution structure determination (as was possible for example for the ribosome) will likely stay the exception, and multiple sources of structural data at multiple resolutions are employed. Integrating these data into one consistent picture poses particular difficulties, since data are much more sparse than in high resolution methods, and the data sets from heterogeneous sources are of highly different and unknown quality and may be mutually inconsistent, and that data are in general averaged over large ensembles and long times. Molecular modelling, a crucial element of any structure determination, plays an even more important role in these multi-scale and multi-technique approaches, not only to obtain structures from the data, but also to evaluate their reliability. This proposal is to develop a consistent framework for this highly complex data integration problem, principally based on Bayesian probability theory. Appropriate models for the major types data types used in hybrid approaches will be developed, as well as representations to include structural knowledge for the components of the complexes, at multiple scales. The new methods will be applied to a series of problems with increasing complexity, going from the determination of protein complexes with high resolution information, over low resolution structures based on protein-protein interaction data such as the nuclear pore, to the genome organisation in the nucleus.

2 130 212 €

Start date: 2012-05-01, End date: 2017-04-30

Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.

1 602 600 €

Start date: 2009-10-01, End date: 2014-09-30

The biomembrane is a prerequisite of life. It enables the cell to maintain a controlled environment and to establish electrochemical gradients as rapidly accessible energy stores. Biomembranes also provide scaffold for organisation and spatial definition of signal transmission in the cell. Crystal structures of membrane proteins are determined with an increasing pace. Along with functional studies integral studies of individual membrane proteins are now widely implemented. The BIOMEMOS proposal goes a step further and approaches the function of the biomembrane at the higher level of membrane protein complexes. Through a combination of X-ray crystallography, electrophysiology, general biochemistry, biophysics and bioinformatics and including also the application of single-particle cryo-EM and small-angle X-ray scattering, the structure and function of membrane protein complexes of key importance in life will be investigated. The specific targets for investigation in this proposal include: 1) higher-order complexes of P-type ATPase pumps such as signalling complexes of Na+,K+-ATPase, and 2) development of methods for structural studies of membrane protein complexes Based on my unique track record in structural studies of large, difficult structures (ribosomes and membrane proteins) in the setting of a thriving research community in structural biology and biomembrane research in Aarhus provides a critical momentum for a long-term activity. The activity will take advantage of the new possibilities offered by synchrotron sources in Europe. Furthermore, a single-particle cryo-EM research group formed on my initiative in Aarhus, and a well-established small-angle X-ray scattering community provides for an optimal setting through multiple cues in structural biology and functional studies

2 444 180 €

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

We aim to understand host cell entry of enveloped viruses at molecular level. A crucial step in this process is when the viral membrane fuses with the cell membrane. Similarly to cell–cell fusion, this step is mediated by fusion proteins (classes I–III). Several medically important viruses, notably dengue and many bunyaviruses, harbour a class II fusion protein. Class II fusion protein structures have been solved in pre- and post-fusion conformation and in some cases different factors promoting fusion have been determined. However, questions about the most important steps of this key process remain unanswered. I will focus on the entry mechanism of bunyaviruses by using cutting-edge, high spatial and temporal resolution bio-imaging techniques. These viruses have been chosen as a model system to maximise the significance of the project: they form an emerging viral threat to humans and animals, no approved vaccines or antivirals exist for human use and they are less studied than other class II fusion protein systems. Cryo-electron microscopy and tomography will be used to solve high-resolution structures (up to ~3 Å) of viruses, in addition to virus–receptor and virus–membrane complexes. Advanced fluorescence microscopy techniques will be used to probe the dynamics of virus entry and fusion in vivo and in vitro. Deciphering key steps in virus entry is expected to contribute to rational vaccine and drug design. During this project I aim to establish a world-class laboratory in structural and cellular biology of emerging viruses. The project greatly benefits from our unique biosafety level 3 laboratory offering advanced bio-imaging techniques. Furthermore it will also pave way for similar projects on other infectious viruses. Finally the novel computational image processing methods developed in this project will be broadly applicable for the analysis of flexible biological structures, which often pose the most challenging yet interesting questions in structural biology.

1 998 375 €

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

Eukaryotic cells constantly convert signals between biochemical energy and mechanical work to timely accomplish many key functions such as migration, division or development. Filopodia are essential finger-like structures that emerge at the cell front to orient the cell in response to its chemical and mechanical environment. Yet, the molecular interactions that make the filopodia mechanosensitive are not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity. Here we will focus on how the underlying actin filament bundle regulates filopodium growth and retraction cycles at the micrometer and seconds scales. These parallel actin filaments are mainly elongated at their barbed-end by formins and cross-linked by bundling proteins such as fascins. We aim to: 1) Elucidate how formin and fascin functions are regulated by mechanics at the single filament level. We will investigate how formin partners and competitors present in filopodia affect formin processivity; how fascin affinity for the side of filaments is modified by filament tension and formin presence at the barbed-end. 2) Reconstitute filopodium-like actin bundles in vitro to understand how actin bundle size and fate are regulated down to the molecular scale. Using a unique experimental setup that combines microfluidics and optical tweezers, we will uncover for the first time actin bundles mechanosensitive capabilities, both in tension and compression. 3) Decipher in vivo the mechanics of actin bundles in filopodia, using fascins and formins with integrated fluorescent tension sensors. This framework spanning from in vitro single filament to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.

1 499 190 €

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

Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear. My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry. Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes. Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.

2 332 500 €

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

Cancer progression, characterized by uncontrolled proliferation and motility of cells, is a complex and deadly process. Integrins, a major cell surface adhesion receptor family, are transmembrane proteins known to regulate cell behaviour by transducing extracellular signals to cytoplasmic protein complexes. We and others have shown that recruitment of specific protein complexes by the cytoplasmic domains of integrins is important in tumorigenesis. Here our aim is to study three interrelated processes in cancer progression which involve integrin signalling, but which have not been elucidated earlier at all. 1) Integrins in cell division (cytokinesis). Since coordinated action of the cytoskeleton and membranes is needed both for cell division and motility, shared integrin functions can regulate both events. 2) Dynamic integrin signalosomes at the leading edge of invading cells. Spatially and temporally regulated, integrin-protein complexes at the front of infiltrating cells are likely to dictate the movement of cancer cells in tissues. 3) Transmembrane segments of integrins as scaffolds for integrin signalling. In addition to cytosolic proteins, integrins most likely interact with proteins within the membrane resulting into new signalling modalities. In this proposal we will use innovative, modern and even unconventional techniques (such as RNAi and live-cell arrays detecting integrin traffic, cell motility and multiplication, laser-microdissection, proteomics and bacterial-two-hybrid screens) to unravel these new integrin functions, for which we have preliminary evidence. Each project will give fundamentally novel mechanistic insight into the role of integrins in cancer. Moreover, these interdisciplinary new openings will increase our understanding in cancer progression in general and will open new possibilities for therapeutic intervention targeting both cancer proliferation and dissemination in the body.

1 529 369 €

Start date: 2008-08-01, End date: 2013-07-31

Mammalian cells express thousands of RNA molecules structurally similar to protein coding genes –they are large, spliced, poly-adenylated, transcribed by RNA Pol II, with conserved promoters and exonic structures –however lack coding capacity. Although thousands exist, only few of these large intergenic non-coding RNAs (lincRNAs) have been characterized. The few that have, show powerful biological roles as regulators of gene expression by diverse epigenetic and non-epigenetic mechanisms. Significantly, their expression patterns suggest that some lincRNAs are involved in cellular pathways critical in cancer, like the p53 pathway. I explored this association demonstrating that p53 induces the expression of many lincRNAs. One them, named lincRNA-p21, is directly induced by p53 to play a critical role in the p53 response, being required for the global repression of genes that interfere with p53 induction of apoptosis. My results, together with the emerging evidence in the field, suggest that lincRNAs may play key roles in numerous tumor-suppressor and oncogenic pathways, representing an unknown paradigm in cellular transformation. However, their mechanisms of function and biological roles remain largely unexplored. The goal of this project is to decipher the functional and biological roles of lincRNAs in the context of oncogenic pathways to better understand the cellular mechanisms of gene regulation at the epigenetic and non-epigenetic levels, and be able to implement lincRNA use for diagnostics and therapies. In order to accomplish these goals we will integrate molecular and cell biology techniques with functional genomics approaches and in vivo studies. Importantly, the profiling of patient samples will reveal the relevance of our findings in human disease. Together, the functional study of lincRNAs will not only be crucial for developing improved diagnostics and therapies, but also will help a better understanding of the mechanisms that govern cellular network.

1 500 000 €

Start date: 2012-01-01, End date: 2017-12-31

Biofuel from wood and waste will be a substantial share of our future energy mix. The conversion of lignocellulose to fermentable polysaccharides is the current bottleneck. We propose to use single molecule cut and paste technology to assemble designer cellulosoms and combine enzymes from different species with nanocatalysts.

2 351 450 €

Start date: 2012-03-01, End date: 2018-02-28

Cell polarity is fundamental to many aspects of cell and developmental biology and it is implicated in differentiation, proliferation and morphogenesis in both unicellular and multi-cellular organisms. We study the mechanisms that regulate cell polarity during both asymmetric cell division and epithelial cell polarization in Drosophila. To understand these fundamental processes, we are currently using two complementary approaches. Firstly, we are coupling genetic tools to state of the art time-lapse microscopy to genetically dissect the mechanisms of cortical cell polarization and mitotic spindle orientation. Secondly, we are introducing two innovative inter-disciplinary methodologies into the fields of cell and developmental biology: 1) single molecule imaging during asymmetric cell division, to unravel the mechanism of polarized protein distribution within the cell; 2) multi-scale tensor analysis of epithelial tissues to describe and understand how epithelial tissues grow, acquire and maintain their shape and organization during development. Using both conventional and innovative methodologies, our goals over the next four years are to better understand how molecules and protein complexes move and are activated at different locations within the cell and how cell polarization impacts on cell identities and on epithelial tissue growth and morphogenesis. Since the mechanisms underlying cell polarization are conserved throughout evolution, the proposed experiments will improve our understanding of these processes not only in Drosophila, but in all animals.

1 159 000 €

Start date: 2008-09-01, End date: 2013-08-31

Cellular respiration provides energy to power essential processes of life. Respiratory complexes are macromolecular batteries coupling electron flow through a wire of metal clusters and cofactors with proton transfer across the inner membrane of mitochondria and bacteria. Waste products of these cellular factories are reactive oxygen species causing ageing and diseases. Assembly and maturation mechanisms of respiratory complexes remain enigmatic because of their membrane location, multisubunit composition and cofactor insertion. E. coli Complex I, one of the largest membrane proteins, composed of 14 conserved subunits with 9 Fe/S clusters and a flavin, is a minimal model for its 45-subunit human homologue. When proton pumping by respiratory complexes is affected, bacteria become resistant to antibiotics requiring proton gradient for uptake. Based on the latest genetic data, we realize that the huge E. coli macromolecular cage, the structure of which we recently solved by cryo-electron microscopy (cryoEM), in conjunction with a novel protein cofactor, is a specific chaperone for Fe/S cluster biogenesis and assembly of respiratory complexes. This integrated multidisciplinary project combines cryoEM and other structural, biophysical and spectroscopic techniques, to uncover the functional mechanism of this emerging chaperone. The structural plasticity of the chaperone fuelled by ATP hydrolysis, and its interaction with Fe/S cluster biogenesis systems and the main respiratory complexes as a function of stresses, will be scrutinized to gain quasiatomic insights into the way the chaperone operates on its substrates. A novel technology for synergetic in situ investigation of protein complexes in the bacterial cytoplasm by optical imaging, state-of-the-art cryogenic correlative light and electron microscopy, and subtomogram analysis, will be developed and used to obtain snapshots of the chaperone-substrate interactions in the cellular context.

1 999 956 €

Start date: 2015-10-01, End date: 2021-09-30

Striking morphological transformations are a hallmark of any cell division cycle. During nuclear division chromatin is compacted into distinctive rod-shaped chromatids in preparation of chromosome segregation by the spindle apparatus. Multi-subunit SMC protein complexes and a large number of regulatory factors are at the heart of this elementary process. SMC complexes also play key roles during other aspects of genome function such as the control of gene expression and the repair of damaged DNA. They are thought to act as chromatin linkers with exquisite specificity for certain pairs of DNA fibres. However, the underlying molecular mechanisms are not understood. Active extrusion of DNA loops by the SMC complex has been proposed to be the mechanistic basis for the establishment of long-range, intra-chromatid DNA bridges. Here, I put forward a multi-pronged research programme that aims to elucidate fundamentally conserved features of SMC protein function and action using the prokaryotic SMC condensin complex in Bacillus subtilis as a tractable model system. We will conduct a combined structural, biochemical and cell biology approach (including crystallography, electron paramagnetic resonance, ChIP-Seq and ‘native’ HiC) to uncover how the SMC complex acts at the higher levels of organization of the bacterial chromosome to promote the efficient individualization of sister DNA molecules. We will reveal the molecular and structural bases for the association between the SMC complex and the bacterial chromosome at different stages of the loading reaction – each representing a crucial intermediate in a sophisticated chromosome organization process. For the first time, we will be able to map the paths of chromosomal DNA through an SMC complex. Our in-depth mechanistic insights will likely have implications for the understanding of various pathological conditions and have the potential to contribute to the development of novel antibacterial compounds.

1 999 599 €

Start date: 2017-06-01, End date: 2022-05-31

Chromatin packaging into the nucleus of eukaryotic cells is highly sophisticated. It not only serves to condense the genomic content into restricted space, but mainly to encode epigenetic traits ensuring temporally controlled and balanced transcription of genes and coordinated DNA replication and repair. The non-random three-dimensional chromatin architecture including looped structures between genomic control elements relies on the action of architectural proteins. However, despite increasing interest in spatio-temporal chromatin organization, mechanistic details of their contributions are not well understood. With this proposal I aim at unveiling molecular mechanisms of protein–mediated chromatin organization by in vivo single molecule tracking and quantitative super-resolution imaging of architectural proteins using reflected light sheet microscopy (RLSM). I will measure the interaction dynamics, the spatial distribution and the stoichiometry of architectural proteins throughout the nucleus and at specific chromatin loci within single cells. In complement single molecule force spectroscopy experiments using magnetic tweezers (MT), I will study mechanisms of DNA loop formation in vitro by structure-mediating proteins. Integrating these spatio-temporal and mechanical single molecule information, I will in the third sup-project measure the dynamics of relative end-to-end movements and the forces acting within a looped chromatin structure in living cells. Taken together, my experiments will greatly enhance our mechanistic understanding of three-dimensional chromatin architecture and inspire future experiments on its regulatory effects on nuclear functions and potential therapeutic utility upon controlled modification.

1 486 578 €

Start date: 2015-05-01, End date: 2021-04-30

The packaging of genetic information into chromatin regulates a wide range of vital processes that depend on direct access to the DNA template. Many chromatin-interacting complexes impact chromatin structure and their aberrant regulation or dysfunction has been implicated in various cancers and severe developmental disorders. A better understanding of the roles of chromatin-interacting complexes in such disease states requires a detailed mechanistic study. Many chromatin-interacting complexes modify chromatin structure, yet understanding the underlying mechanisms remains a major challenge in the field. Furthermore, how chromatin-interacting complexes are regulated to enable their various functions is incompletely understood. We will address these longstanding questions in two specific aims. Aim I: Building on our expertise in single-molecule biology, we will develop powerful single-molecule imaging approaches to monitor the action of chromatin-interacting complexes in real time. We will further probe how the diverse activities of the chromatin-associated complexes are coordinated and coupled to conformational transitions. Aim II: Drawing on our expertise in structural biology, we will use a range of structural techniques in combination with biochemical approaches to study the vital regulation of chromatin-interacting complexes by their regulatory subunits as well as by chromatin features. We expect to obtain ground-breaking insights into the mechanisms and regulation of disease-related chromatin-associated complexes, which may open up new horizons for developing therapeutic intervention strategies. Furthermore, the approaches developed here will enable the investigation of a large number of chromatin-related processes.

1 498 954 €

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

Chromatin undergoes fascinating structural and functional changes during the metazoan cell cycle. It massively condenses at the beginning of mitosis with a degree of compaction up to fiftyfold higher than in interphase. At the end of mitosis, mitotic chromosomes decondense to re-establish their interphase chromatin structure. This process is indispensable for reinitiating transcription and treplication, and is thus of central importance in the cellular life cycle. Despite its significance to basic research as well as its potential medical implications, postmitotic chromatin decondensation is only poorly understood. It has been well described cytologically, but we lack an understanding of the underlying molecular events. We are ignorant about the proteins that mediate chromatin decondensation, the distinct steps in this multi-step procedure and their regulation. Using a novel in vitro assay, which recapitulates the process in the simplicity of a cell free reaction, we will identify the molecular machinery mediating postmitotic chromatin decondensation and define the different steps of the process. The cell free assay offers the unique possibility to isolate and purify activities responsible for individual steps in chromatin decondensation, to identify their molecular composition and to analyse the molecular changes they induce on chromatin. Accompanied by live cell imaging in mammalian tissue culture cells, the proposed approach will not only facilitate the elucidation of the factors involved in chromatin decondensation, but will also provide insight into how this process is integrated into mitotic exit and nuclear reformation and linked to other concomitant processes such as nuclear envelope assembly or nuclear body formation. Thus, using an unprecedented approach to study the ill-defined but important cell biological process of postmitotic chromatin decondensation, we aim to expand the frontiers in our knowledge on this topic.

1 499 880 €

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

A PubMed search for ‘epigenetic’ identifies nearly 35,000 entries, yet the molecular mechanisms by which chromatin modification and gene expression patterns are actually inherited during chromosome replication — mechanisms which lie at the heart of epigenetic inheritance of gene expression — are still largely uncharacterised. Understanding these mechanisms would be greatly aided if we could reconstitute the replication of chromosomes with purified proteins. The past few years have seen great progress in understanding eukaryotic DNA replication through the use of cell-free replication systems and reconstitution of individual steps in replication with purified proteins and naked DNA. We will use these in vitro replication systems together with both established and novel chromatin assembly systems to understand: a) how chromatin influences replication origin choice and timing, b) how nucleosomes on parental chromosomes are disrupted during replication and are distributed to daughter chromatids, and c) how chromatin states and gene expression patterns are re-established after passage of the replication fork. We will begin with simple, defined templates to learn basic principles, and we will use this knowledge to reconstitute genome-wide replication patterns. The experimental plan will exploit our well-characterised yeast systems, and where feasible explore these questions with human proteins. Our work will help explain how epigenetic inheritance works at a molecular level, and will complement work in vivo by many others. It will also underpin our long-term research goals aimed at making functional chromosomes from purified, defined components to understand how DNA replication interacts with gene expression, DNA repair and chromosome segregation.

1 983 019 €

Start date: 2015-11-01, End date: 2021-10-31

DNA replication represents a dangerous moment in the life of the cell as endogenous and exogenous events challenge genome integrity by interfering with the progression, stability and restart of the replication fork. Failure to protect stalled forks or to process the replication fork appropriately contribute to the pathological mechanisms giving rise to cancer, therefore an understanding of the intricate mechanisms that ensure fork integrity can provide targets for new chemotherapeutic assays. Smc5-Smc6 is a multi-subunit complex with a poorly understood function in DNA replication and repair. One of its subunits, Nse2, is able to promote the addition of a small ubiquitin-like protein modifier (SUMO) to specific target proteins. Recent work has revealed that the Smc5-Smc6 complex is required for the progression of replication forks through damaged DNA and is recruited de novo to forks that undergo collapse. In addition, Smc5-Smc6 mediate repair of DNA breaks by homologous recombination between sister-chromatids. Thus, Smc5-Smc6 is anticipated to promote recombinational repair at stalled/collapsed replication forks. My laboratory proposes to develop molecular techniques to study replication at the level of single replication forks. We will employ these assays to identify and dissect the function of factors involved in replication fork stability and repair. We will place an emphasis on the study of the Smc5-Smc6 complex in these processes because of its potential roles in recombination-dependent fork repair and restart. We also propose to identify novel Nse2 substrates involved in DNA repair using yeast model systems. Specifically, we will address the following points: (1) Development of assays for analysis of factors involved in stabilisation, collapse and re-start of single-forks, (2) Analysis of the roles of Smc5-Smc6 in fork biology using developed techniques, (3) Isolation and functional analysis of novel Nse2 substrates.

893 396 €

Start date: 2008-09-01, End date: 2013-08-31

This project aims at understanding of the role of the tubulin code for self-organization of complex microtubule based structures. Cilia turn out to be the ideal structures for the proposed research. A cilium is a sophisticated cellular machine that self-organizes from many protein complexes. It plays motility, sensory, and signaling roles in most eukaryotic cells, and its malfunction causes pathologies. The assembly of the cilium requires intraflagellar transport (IFT), a specialized bidirectional motility process that is mediated by adaptor proteins and direction specific molecular motors. Work from my lab shows that anterograde and retrograde IFT make exclusive use of the B-tubules and A-tubules, respectively. This insight answered a long standing question and shows that functional differentiation of tubules exists and is important for IFT. Tubulin post-translational modifications (PTMs) contribute to a tubulin code, making microtubules suitable for specific functions. Mutation of tubulin-PTM enzymes can have dramatic effects on cilia function and assembly. However, we do not understand of the role of tubulin-PTMs in cilia. Therefore, I propose to address the hypotheses that the tubulin code contributes to regulating bidirectional IFT motility, and more generally, that the tubulin code is a key player in structuring complex cellular assembly processes in space and time. This proposal aims at (i) understanding if tubulin-PTMs are necessary and/or sufficient to regulate the bidirectionality of IFT (ii) examining how the tubulin code regulates the assembly of cilia and (iii) generating a high-resolution atlas of tubulin-PTMs and their respective enzymes. We will combine advanced techniques encompassing state-of-the-art cryo-electron tomography, biochemical imaging, fluorescent microscopy, and in vitro assays to achieve molecular and structural understanding of the role of the tubulin code in the self-organization of cilia and of microtubule based cellular structures.

1 986 406 €

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

The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose and xenobiotic metabolism. Impairment of this rhythm has been show to lead to diverse pathologies including metabolic syndrome. At present, it is supposed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level. We have now collected evidence that post-transcriptional regulations play also an important role in this regulation. Particularly, recent results from our laboratory show that the circadian clock can synchronize mRNA translation in mouse liver through rhythmic activation of the Target Of Rapamycin Complex 1 (TORC1) with a 12-hours period. Based on this unexpected observation, we plan to identify the genes rhythmically translated in the mouse liver as well as the mechanisms involved in this translation. Indeed, our initial observations suggest a cap-independent translation during the day and a cap-dependent translation during the night. Identification of the different complexes involved in translation at this two different times and their correlation with the sequence, structure, and/or function of the translated genes will provide new insight into the action of the circadian clock on animal metabolism. In parallel, we will identify the signalling pathways involved in the rhythmic activation of TORC1 in mouse liver. Finally, we will study the consequences of a deregulated rhythmic translation in circadian clock-deficient mice on the metabolism and the longevity of these animals. Perturbations of the circadian clock have been linked to numerous pathologies, including obesity, type 2 diabetes and cancer. Our project on the importance of circadian clock-coordinated translation will likely reveal new findings in the field of regulation of animal metabolism by the circadian clock.

1 475 831 €

Start date: 2011-03-01, End date: 2016-02-29

An important question of modern neurobiology is how neurons regulate synaptic function in response to excitation. In particular, the roles of alternative pre-mRNA splicing and mRNA translation regulation in this response are poorly understood. We will study the RNA-binding proteins (RBPs) that control these post-transcriptional changes using a UV crosslinking-based purification method (CLIP) and ultra-high throughput sequencing. Computational analysis of the resulting data will define the sequence and structural features of RNA motifs recognized by each RBP. Splicing microarrays and translation reporter assays will then allow us to examine the regulatory functions of RBPs and RNA motifs. By integrating the biochemical and functional datasets, we will relate the position of RNA motifs to the activity of bound RBPs, and predict the interactions that act as central nodes in the regulatory network. The physiological role of these core RBP-RNA interactions will then be tested using antisense RNAs. Together, these projects will provide insights to the regulatory mechanisms underlying neuronal activity-dependent changes, and provide new opportunities for future treatments of neurodegenerative disorders.

900 000 €

Start date: 2008-09-01, End date: 2013-08-31

During S-phase newly synthesized “sister” DNA molecules become physically connected. This sister chromatid cohesion resists the pulling forces of the mitotic spindle and thereby enables the bi-orientation and subsequent symmetrical segregation of chromosomes. Cohesion is mediated by ring-shaped cohesin complexes, which are thought to entrap sister DNA molecules topologically. In mammalian cells, cohesin is loaded onto DNA at the end of mitosis by the Scc2-Scc4 complex, becomes acetylated during S-phase, and is stably “locked” on DNA during S- and G2-phase by sororin. Sororin stabilizes cohesin on DNA by inhibiting Wapl, which can otherwise release cohesin from DNA again. In addition to mediating cohesion, cohesin also has important roles in organizing higher-order chromatin structures and in gene regulation. Cohesin performs the latter functions in both proliferating and post-mitotic cells and mediates at least some of these together with the sequence-specific DNA-binding protein CTCF, which co-localizes with cohesin at many genomic sites. Although cohesin and CTCF perform essential functions in mammalian cells, it is poorly understood how cohesin is loaded onto DNA by Scc2-Scc4, how cohesin is positioned in the genome, how cohesin is released from DNA again by Wapl, and how Wapl is inhibited by sororin. Likewise, it is not known how cohesin establishes cohesion during DNA replication and how cohesin cooperates with CTCF to organize chromatin structure. Here we propose to address these questions by combining biochemical reconstitution, single-molecule TIRF microscopy, genetic and cell biological approaches. We expect that the results of these studies will advance our understanding of cell division, chromatin structure and gene regulation, and may also provide insight into the etiology of disorders that are caused by cohesin dysfunction, such as Down syndrome and “cohesinopathies” or cancers, in which cohesin mutations have been found to occur frequently.

2 500 000 €

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

Chromosomes undergo dramatic changes in their three-dimensional organisation during all aspects of genome function, ranging from the regulation of gene expression during cellular differentiation to chromosome duplication and partitioning over the course of a cell division cycle. The multi-subunit condensin protein complex plays major roles for these changes in DNA topology. Despite its fundamental importance, the mechanisms of condensin’s action are not understood. Here, I propose a comprehensive research program that aims to reveal the elusive mechanisms behind the functions of the condensin complex. We intend to unravel how the condensin complex engages DNA, how this interaction activates large-scale ATPase-dependent conformational rearrangements within the complex, and how condensin eventually encircles chromatin fibres within its ring-shaped architecture. Insights from these mechanistic studies will be invaluable for understanding how networks of condensin-mediated linkages can shape linear DNA helices into higher-order chromosome structures. To achieve this ambitious and timely goal, we will combine an integrative structural biology approach with biochemical and cell biological methods. By applying complementary technologies, including X-ray protein crystallography, electron microscopy, cross-linking mass spectrometry, single molecule fluorescence microscopy and reconstitution experiments, we anticipate to build the first model of the entire condensin complex at near-atomic resolution and explain how dynamic conformational changes confer function. The insights gained from this research program will provide an in-depth mechanistic comprehension of the core molecular machinery that determines the architecture of our genomes and will have major implications for understanding how genomic integrity is affected in various disease conditions.

1 982 479 €

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

Structural basis of controlling the membrane attack complex Complement is a fundamental component of the human immune system; central to the battle between hosts and pathogens. The membrane attack complex (MAC) is the direct killing arm of complement that acts by forming large pores in target cell membranes. Uncontrolled activation results in by-stander damage, which can have devastating consequences for host cells and impact inflammatory pathologies, thrombosis and cancer. Understanding how MAC activity is controlled on human cells during an immune response is a major unresolved question. My lab has pioneered the use of cryo electron microscopy (cryoEM) to investigate the molecular mechanism underpinning MAC assembly. We have defined the stoichiometry of the complex and identified interaction interfaces that determine its sequential assembly mechanism. Recent data from my lab has now revealed atomic resolution information for the complete transmembrane pore. Results from my lab have provided a molecular and biophysical basis for MAC pore formation, which has led to a general mechanism for how proteins cross lipid bilayers. Here, the goal is to understand the structural basis for how MAC activity is controlled by (i) cell surface receptor CD59, (ii) removal of pores from the plasma membrane, and (iii) clearance of assembly by-products from the plasma. MAC interacts with a defined set of cellular proteins through these three pathways. In this proposal, we will integrate structural information that spans cellular to molecular length scales. Recent technical advances in cryoEM, cryo soft X-ray tomography (cryoSXT) and correlated fluorescence imaging make it now possible to address how MAC activity is controlled in and around the plasma membrane. In doing so, we will answer a longstanding question in immunology and open new research directions exploring fundamental cellular processes. These results will provide a foundation for the development of novel therapeutics.

1 999 990 €

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

The human genome is highly transcribed, with over 90% of sequences contributing to the production of RNA. The function of the vast majority of these RNAs is unknown. Evidence over many years has revealed that transcription factors and chromatin regulators are associated with a variety of non-coding (nc)RNAs, but their function remains largely unknown. There are a few cases where a role has been ascribed for ncRNAs in transcription, but no clear mechanistic insight has been defined yet. We predict that many of the newly identified ncRNAs emanating from the genome will play a role in transcriptional processes. We intend to identify and characterise such ncRNAs. This will take place in two phases. In the first phase we will use biochemical approaches to identify ncRNAs involved in the regulation of chromatin and transcription. Our investigations will focus on proteins leading to the induction of pluripotency and oncogenesis. ncRNAs associated with such proteins will be identified using targeted screens. In the second phase, the importance of these RNAs in determining pluripotency and oncogenesis will be analysed. In addition, a variety of molecular approaches will be used to investigate the mechanism by which these ncRNAs regulate the function of the proteins or complexes they associate with. One particular hypothesis we will explore is that such ncRNAs play a role in guiding proteins to DNA sequences, via the formation of RNA/DNA triplexes. This concerted and focused analysis will provide mechanistic insights into the functions of ncRNAs in transcriptional regulation and validate their role in key biological processes. The identification of such new ncRNA-regulated pathways may open up new avenues for therapeutic intervention.

2 141 470 €

Start date: 2011-05-01, End date: 2017-04-30

The constant arms race between prokaryotic microbes and their molecular parasites such as viruses, plasmids and transposons has driven the evolution of complex genome defence mechanisms. The CRISPR-Cas defence systems provide adaptive RNA-guided immunity against invasive nucleic acid elements. CRISPR-associated effector nucleases such as Cas9, Cas12a and Cas13 have emerged as powerful tools for precision genome editing, gene expression control and nucleic acid detection. However, these technologies suffer from drawbacks that limit their efficacy and versatility, necessitating the search for additional exploitable molecular activities. Building on our recent structural and biochemical studies, the goal of this project is to investigate the molecular architectures and mechanisms of CRISPR-associated systems and other genome defence mechanisms, aiming not only to shed light on their biological roles but also inform their technological development. Specifically, the proposed studies will examine (i) the molecular basis of cyclic oligoadenylate signalling in type III CRISPR-Cas systems, (ii) the mechanism of transposon-associated type I CRISPR-Cas systems and their putative function in RNA-guided DNA transposition, and (iii) molecular activities associated with recently described non-CRISPR defence systems. Collectively, the proposed studies will advance our understanding of the molecular functions of genome defence mechanisms in shaping the evolution of prokaryotic genomes and make critical contributions to their development as novel genetic engineering tools.

1 996 525 €

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

"Translation of the genetically encoded information into polypeptides, protein biosynthesis, is a central function executed by ribosomes in all cells. In the case of membrane protein synthesis, integration into the membrane usually occurs co-translationally and requires a ribosome-associated translocon (SecYEG/Sec61). This highly coordinated process is poorly understood, since high-resolution structural information is lacking. Although single particle cryo-electron microscopy (cryo-EM) has given invaluable structural insights for such dynamic ribosomal complexes, the resolution is so far limited to 5-10 Å for asymmetrical particles. Thus, the mechanistic depth and reliability of interpretation has accordingly been limited. Here, I propose to use single particle cryo-EM at improved, molecular resolution of 3-4 Å to study two fundamental ribosome-associated processes: (i) co-translational integration of polytopic membrane proteins and (ii) recycling of the eukaryotic ribosome. First, we will visualize nascent polytopic membrane proteins inserting into the lipid bilayer via the bacterial ribosome-bound SecYEG translocon. Notably, the translocon will be embedded in a lipid environment provided by so-called nanodiscs. Second, we will visualize in a similar approach membrane protein insertion via the YidC insertase, the main alternative translocon. Third, as a novel research direction, we will determine the structure and function of eukaryotic ribosome recycling complexes involving the ABC-ATPase RLI. The results will allow, together with functional biochemical data, an in-depth molecular structure-function analysis of these fundamental ribosome-associated processes. Moreover, reaching molecular resolution for asymmetrical particles by single particle cryo-EM will lift this technology to a level of analytical power approaching X-ray and NMR methods. ERC funding would allow for this highly challenging research to be conducted in an internationally competitive way in Europe."

2 995 640 €

Start date: 2012-05-01, End date: 2017-04-30

Specificity in the ubiquitin-proteasome system is largely conferred by ubiquitin E3 ligases (E3s). Cullin-RING ligases (CRLs), constituting ~30% of all E3s in humans, mediate the ubiquitination of ~20% of the proteins degraded by the proteasome. CRLs are divided into seven families based on their cullin constituent. Each cullin binds a RING domain protein, and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 200 distinct CRLs. All CRLs are regulated by the COP9 signalosome (CSN), an eight-protein isopeptidase that removes the covalently attached activator, NEDD8, from the cullin. Independent of NEDD8 cleavage, CSN forms protective complexes with CRLs, which prevents destructive auto-ubiquitination. The integrity of the CSN-CRL system is crucially important for the normal cell physiology. Based on our previous work on CRL structures (Fischer, et al., Nature 2014; Fischer, et al., Cell 2011) and that of isolated CSN (Lingaraju et al., Nature 2014), We now intend to provide the underlying molecular mechanism of CRL regulation by CSN. Structural insights at atomic resolution, combined with in vitro and in vivo functional studies are designed to reveal (i) how the signalosome deneddylates and maintains the bound ligases in an inactive state, (ii) how the multiple CSN subunits bind to structurally diverse CRLs, and (iii) how CSN is itself subject to regulation by post-translational modifications or additional further factors. The ERC funding would allow my lab to pursue an ambitious interdisciplinary approach combining X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology. This is expected to provide a unique molecular understanding of CSN action. Beyond ubiquitination, insight into this >13- subunit CSN-CRL assembly will allow examining general principles of multi-subunit complex action and reveal how the numerous, often essential, subunits contribute to complex function.

2 200 677 €

Start date: 2016-01-01, End date: 2020-12-31

"Cytokine receptor signaling is an essential part of the intercellular communication networks that govern key physiological processes in the body. Cytokine dysfunction is associated with numerous pathologies including autoimmune disorders and cancer, and both cytokines and cytokine antagonists have found their way into the clinic. Yet, there are still many unfulfilled promises and opportunities. In this project we will reinvestigate key aspects of cytokine receptor activation and signaling using novel insights and techniques recently developed in our laboratory. This will include the AcTakine concept for cell-specific targeting of cytokine activity, and applications of our MAPPIT, KISS and Virotrap toolboxes to systematically map protein interactions involved in cytokine signaling. We expect to obtain important new insights, both in fundamental and in applied medical sciences."

2 487 728 €

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

We pioneered an initially highly controversial connection between DNA damage and (accelerated) aging. In the previous ERC grant ‘DamAge’ we reached the stage that (segmental) aging in DNA repair-deficient mice can be largely controlled. The severity of the repair defect determines the rate of segmental aging; the repair pathways affected influence which organs age fast; conditional repair mutants allow targeting accelerated aging to any organ. Importantly, we found that dietary restriction (DR), the only universal intervention known to delay aging, extends remaining life- and healthspan in progeroid Ercc1Δ/- mutants by 200% (see Vermeij et al., Nature 2016 and fig.2). Also Xpg-/- progeroid repair mice strongly benefit from DR, generalizing this finding. The prominent Alzheimer- and Parkinson-like neurodegeneration is even retarded up to 30-fold(!) disclosing powerful untapped reserves unleashed by 30% less food, with enormous clinical potential. Also we discovered that in accelerated and normal aging gene expression declines due to accumulating stochastic transcription-blocking lesions and that DR counteracts genomic dysfunction. In Dam2Age we will focus on the cross-talk between DNA damage, aging and DR with emphasis on the relevance for normal aging, elucidate underlying mechanisms and use our unique -for DR research superior- mouse models and a variety of novel assays to search for effective nutritional-pharmacological DR mimetics. This serves as a stepping stone towards potent universal therapy for a range of repair-deficient progeroid syndromes and prevention of many aging-related diseases, most urgently dementia’s, to promote sustained health.

2 251 719 €

Start date: 2017-09-01, End date: 2022-08-31

During its duplication, DNA, the carrier of our genetic information, is particularly vulnerable to decay, and the capacity of cells to deal with replication stress has been recognised as a major factor protecting us from genome instability and cancer. A major pathway that allows cells to overcome or bypass DNA lesions during replication is activated by posttranslational modifications of the sliding clamp protein PCNA. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required for an error-free pathway that involves template switching to the undamaged sister chromatid, involving a recombination-like mechanism. Hence, damage bypass contributes to genome maintenance, but can itself be a source of genomic instability. It is therefore not surprising that PRR is a highly regulated process whose activity is limited to the appropriate situations by stringent control mechanisms. The proposed project aims at understanding DNA damage bypass in its cellular context. Using a combination of new and established technology, we will address the role of chromatin dynamics in the reaction, its spatial and temporal control in relation to genome replication, and its coordination with other PCNA-dependent processes in the cell. To this end, we will establish technology to isolate and analyse the composition of damage bypass tracts, develop and implement novel methods to induce lesions and image damage processing in live cells, and exploit a spectrum of biochemical and biophysical techniques to investigate the role of PCNA as a molecular tool-belt in the coordination of its interaction partners. In combination, these approaches will give important insight into how the replication of damaged DNA is managed with high efficiency and accuracy within the cell.

2 476 388 €

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

DNA, if damaged, cannot be replaced. If not replaceable, it must be repaired. The so-called “DNA damage response” (DDR) is a coordinate set of evolutionary conserved events that arrest the cell-cycle (DNA damage checkpoint function) in proliferating cells and attempts DNA repair. Until DNA damage has not been repaired in full, cell proliferation is not resumed in normal cells. DNA damage is a physiological event. Ageing and cancer are both associated with DNA damage accumulation. In the past, we contribute to better understand the mechanisms and the consequences of DNA damage generation and DDR activation in both settings. We have recently identified a completely hitherto undiscovered level of control of DDR activation, so far considered a proteinaceous only signaling cascade. We have discovered that short RNA species are detectable at DNA damage sites and are necessary for DDR activation at DNA lesions. These RNA species are generated by an evolutionary-conserved RNA processing machinery. However, they serve purposes never reported before. We believe that our findings change radically our understanding of DDR modulation in mammals and disclose a fertile unspoilt ground for exciting investigations.

2 329 200 €

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

"The degradation of mature mRNAs has emerged as a key step in the regulation of eukaryotic gene expression. Modulation of the half-life of mRNAs via their degradation is a powerful and versatile mechanism to swiftly alter the expression of proteins in response to changes in physiological conditions. The decay of mRNAs is performed by a set of macromolecular complexes that act in a sequential and coordinated manner, progressively eroding the ends of the transcript until its degradation is complete. These macromolecular assemblies contain only a few catalytically active subunits and a large number of regulatory components. How and why the activities are regulated within the architecture of the complexes is largely unknown. Also unclear are the mechanisms with which the complexes communicate with each other and/or with the changing composition of the nucleic acid. In this project, we will reconstitute the key protein complexes in mRNA decay from recombinant proteins in vitro. Specifically, we will focus on the evolutionary conserved deadenylation, decapping and exosome-Ski complexes. The reconstituted complexes will be used for structural studies to derive atomic models of the holoenzymes using a combination of X-ray crystallography and cryoelectron microscopy. In parallel to obtaining static views of the individual steps in the pathway, we will establish the assays to study how information from one processing step is passed on to the next in a dynamic manner. We will address the basis for the timing and interrelationship of the conserved enzymatic machineries and the influence of the mRNP composition on their activity. Our final goal is to recapitulate the complex behavior of the mRNA decay pathway in vitro. Our lab has extensive experience in biochemical reconstitution of protein complexes, in vitro biochemical assays and X-ray crystallography. In the next five years, we plan to implement cryoelectron microscopy within the scope of this proposal."

2 499 344 €

Start date: 2012-04-01, End date: 2017-03-31

The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) largest subunit coordinates co-transcriptional processing and it is decorated by many processing factors throughout the transcription cycle. The composition of this supramolecular assembly is diverse and highly dynamic. Many of the factors associate with RNAPII weakly and transiently, and the association is dictated by different post-translational modification patterns and conformational changes of the CTD. To determine how these accessory factors assemble and exchange on the CTD of RNAPII has remained a major challenge. Here, we aim to unravel the structural and mechanistic bases for the dynamic assembly of RNAPII CTD with its processing factors. Using NMR, we will determine high-resolution structures of several protein factors bound to the CTD carrying specific modifications. This will enable to decode how CTD modification patterns stimulate or prevent binding of a given processing factor. We will also establish the structural and mechanistic bases of proline isomerisation in the CTD that control the timing of isomer-specific protein-protein interactions. Next, we will combine NMR and SAXS approaches to unravel how the overall CTD structure is remodelled by binding of multiple copies of processing factors and how these factors cross-talk with each other. Finally, we will elucidate a mechanistic basis for the exchange of processing factors on the CTD. Our study will answer the long-standing questions of how the overall CTD structure is modulated on binding to processing factors, and whether these factors cross-talk and compete with each other. The level of detail that we aim to achieve is currently not available for any transient molecular assemblies of such complexity. In this respect, the project will also provide knowledge and methodology for further studies of large and highly flexible molecular assemblies that still remain poorly understood.

1 844 604 €

Start date: 2015-08-01, End date: 2020-07-31

Bacterial dehalorespiration is a microbial respiratory process in which halogenated hydrocarbons, from natural or anthropogenic origin, act as terminal electron acceptors. This leads to effective dehalogenation of these compounds, and as such their degradation and detoxification. The bacterial species, their enzymes and other components responsible for this unusual metabolism have only recently been identified. Unlocking the full potential of this process for bioremediation of persistent organohalides, such as polychlorinated biphenyls (PCBs) and tetrachloroethene, requires detailed understanding of the underpinning biochemistry. However, the regulation, mechanism and structure of the reductive dehalogenase (the enzyme responsible for delivering electrons to the halogenated substrates) are poorly understood. This ambitious proposal seeks to study representatives of the distinct reductive dehalogenase classes as well as key elements of the associated regulatory systems. Our group has been at the forefront of studying the biochemistry underpinning transcriptional regulation of dehalorespiration, providing detailed insights in the protein CprK at the atomic level. However, it is now apparent that only a subset of dehalogenases are regulated by CprK homologues with little known about the other regulators. In addition, studies on the reductive dehalogenases have been hampered by the inability to purify sufficient quantities. Using an interdisciplinary, biophysical approach focused around X-ray crystallography, enzymology and molecular biology, combined with novel reductive dehalogenase production methods, we aim to provide a detailed understanding and identification of the structural elements crucial to reductive dehalogenase mechanism and regulation. At the same time, we aim to apply the knowledge gathered and study the feasibility of generating improved dehalorespiratory components for biosensing or bioremediation applications through laboratory assisted evolution.

1 148 522 €

Start date: 2008-09-01, End date: 2013-08-31

A prerequisite for chromosome segregation in all living organisms is the topological unlinking of sister DNA molecules, called DNA decatenation. Decatenation is performed by DNA topoisomerases that work by transiently breaking strand(s) in one DNA double helix and passing another double helix through the temporarily created gate. How DNA topoisomerases manage to recognize linkages between sister DNA molecules and how they promote decatenation of sister chromatids (but not catenation) is still largely unknown. The driving hypothesis of this project is that condensin promotes chromosome decatenation by guiding the unlinking activity of DNA topoisomerases. Condensin is a member of the family of SMC (Structural Maintenance of Chromosomes) protein complexes that is conserved from bacteria to humans. It forms large, ring-like structures that bind to and organize chromosomes. Efficient separation of sister chromosomes in the bacterium B. subtilis depends on the condensin complex. However, so far the precise role of condensin in chromosome segregation and its mechanisms are unclear. To test our hypothesis, we will establish a minichromosome in bacteria that segregates in a condensin-dependent manner and measure its decatenation in vivo in the presence and absence of condensin. We will investigate the mechanism by which condensin organizes DNA within the (mini-)chromosome using techniques like chromosome conformation capture (3C) and electron microscopy. Finally, we will attempt to reconstitute for the first time the entrapment of DNA double helices by ring-like SMC protein complexes using purified components. Our results will be pivotal for understanding the action of SMC proteins in general with important implications in the separation and segregation of chromosomes in bacteria, the shaping of mitotic chromosomes and the resolution of sister chromatids during mitosis in eukaryotes.

1 376 734 €

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

Beta-thalassemia and sickle cell disease (SCD) are caused by mutations affecting the synthesis or the structure of the adult hemoglobin (Hb) beta-chain. The only definitive cure is transplantation of allogeneic hematopoietic stem cells (HSCs) from an HLA-matched donor, an option available to <30% of the patients. The clinical severity of beta-hemoglobinopathies is alleviated by the co-inheritance of mutations causing expression of fetal gamma-globin in adult life - a condition termed hereditary persistence of fetal hemoglobin (HPFH). Transplantation of autologous, genetically modified HSCs is an attractive therapeutic option for patients lacking a suitable donor. To this aim, genome editing approaches based on the use of site-specific nucleases have been explored by many groups, including ours. These approaches may either revert the single point mutation causing SCD or reactivate fetal globin expression, by mimicking HPFH mutations or by decreasing the level of BCL11A, a master repressor of fetal Hb synthesis. Site-specific nucleases, however, generate double-strand breaks (DSBs) in the genome and raise safety concerns for clinical applications, particularly when used in DSB-sensitive HSCs. In this proposal, we aim at exploiting targeted base-editing to develop novel, efficacious and safe strategies for beta-hemoglobinopathies without generating DSBs. This will be attempted by (i) correcting the SCD-causing mutation, (ii) mimicking HPFH mutations in the gamma-globin promoters, or (iii) modulating the activity of a BCL11A erythroid-specific enhancer. These approaches will be tested in human adult erythroid cell lines and patient HSCs, differentiated in vitro and in vivo into mature red cells to evaluate editing efficiency, fetal Hb expression, phenotypic cell correction and biosafety. The ultimate goal of the project is to provide sufficient proof of efficacy and safety to enable the clinical development of base-edited HSCs for the therapy of beta-hemoglobinopathies.

2 000 000 €

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

Cells must ensure the integrity of their genome, and failure to do so can lead to mutations and cause disease. A sophisticated molecular network senses genomic lesions and coordinates their faithful repair with other DNA transactions, including transcription and DNA replication. Research over the last years has significantly advanced our understanding of the DNA damage response and continues to provide crucial insights that explain how cells deal with genotoxic stress to avoid malignant transformation. More recently, the intriguing phenomenon of cellular heterogeneity reached into the limelight as it became increasingly clear that significant variability exists between individual cells, even of the same genetic background and cell type. Single cells matter, for instance during cellular transformation or tumor relapse, and cellular variability thus impacts disease development and therapeutic outcome. Its determinants are surprisingly unexplored, however, and have not been studied in context of genome integrity maintenance. The main objective of this project is to systematically assess cellular heterogeneity in genome integrity maintenance and characterize its causes and consequences. Quantitative automated high-content imaging of large cell cohorts will be used to identify hitherto unknown determinants of variability in the cellular responses to genotoxic stress and dissect at the single cell level the variability in (1) the chromatin response to DNA double-strand breaks, (2) the cellular response to replication stress, and (3) the cellular capacity to trigger phase transitions, a newly emerging mechanism of dynamic compartmentalization, at sites of genomic lesions. This project will bridge two thus far independently developed research fields (genome stability and cellular heterogeneity), reveal how cell-to-cell variation impacts cell fate and survival in response to genotoxic stress, and may uncover ways to homogenize this response for improved cancer therapies.

1 500 000 €

Start date: 2017-04-01, End date: 2022-09-30

For cells and organisms to survive and propagate, they must accurately pass on their genetic information to the next generation. Errors in this process may arise from spontaneous mistakes in normal cellular metabolism, or from exposure to external agents, such as chemical mutagens and radiation. To protect themselves from the consequences of DNA damage, cells have evolved a vast array of pathways DNA repair mechanisms, each optimised for the resolution of a particular problem. One method of DNA repair, called homologous recombination (HR), involves using intact undamaged DNA sequences as a template to repair the damaged copy. HR is used extensively in meiotic cells to repair DNA breaks that are purposely created by the cell. In this context, HR is not just a repair mechanism, but also a method to drive interaction and genetic exchange between maternally and paternally inherited chromosomes, creating haploid genomes which are chimeras of the parental genetic information. Thus, the study of DNA repair and recombination informs our understanding of mechanisms that maintain genome stability, but which also generate genetic diversity, topics that are as critical to the survival of an individual cell as they are for the evolution and survival of an entire ecosystem. In recent decades a great deal has been learned of the genetic and biochemical control of the DNA repair and recombination mechanism. In general we infer gene function from what happens (or doesn’t happen) when we mutate a pathway of interest, and use biochemistry to test function using surrogate, simplified in vitro assays. Here, to bridge the divide between these classic approaches, I propose to develop biochemical methods using intact chromatin prepared from living cells. I believe that integrating chromatin biochemistry, with cell biology and genome-wide analysis will enable a new mode of scientific investigation, detailing how molecular reactions occur on biologically-relevant chromosomal substrates.

1 747 823 €

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