ERC FUNDED PROJECTS

The three-dimensional organization of chromosomes is necessary for hereditary fidelity and gene regulation. Recent studies have found that eukaryotic interphase chromosomes are spatially organized in compartments, chiefly topologically associated domains (TADs), in a hierarchical order of nested chromatin loops, coining the term “chromosome folding”. TADs are clusters of genes and regulatory elements that are confined to their genomic compartment by spatially constricting their accessible range of action. The folded structure of chromosomes through long-range loops enables mutual interactions of distant genomic loci that otherwise would not be in contact. While crosslinking-based chromosome conformation capture (3C) techniques have revealed the underlying structure of interphase chromosomes, the molecular mechanism of how chromosome-organizing proteins, such as the insulator CTCF or the structural maintenance of chromosomes (SMC) complex cohesin build the chromosomal scaffold and contribute to genomic organization, is not understood. Due to the complexity of the processes involved, biochemical information on how chromosomal proteins contribute to the establishment of TADs is scarce. I have previously demonstrated that single molecule techniques can be used to study the interactions of single cohesin complexes with DNA, chromatin and DNA-bound proteins and to resolve processes that are inaccessible in bulk biochemical experiments. In this project, I will use and expand the high-throughput single molecule technique of DNA curtains to study the molecular details of how chromosomal scaffolding proteins and genetic insulators form the basis for the three-dimensional folding of chromosomes. My experiments will build a novel experimental platform to study the dynamics of chromosomal configuration and maintenance in a reconstituted single molecule assay and will reveal the molecular details that drive the organization of chromosomes into hierarchically organized structures.

1 499 350 €

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

Our body constantly encounters pathogens or malignant transformation. Consequently, the adaptive immune system is in place to eliminate infected or cancerous cells. Specific immune reactions are triggered by selected peptide epitopes presented on major histocompatibility complex class I (MHC-I) molecules, which are scanned by cytotoxic T lymphocytes. Intracellular transport, loading, and editing of antigenic peptides onto MHC-I are coordinated by a highly dynamic multisubunit peptide-loading complex (PLC) in the ER membrane. This multitasking machinery orchestrates the translocation of proteasomal degradation products into the ER as well as the loading and proofreading of MHC-I molecules. Sampling of myriads of different peptide/MHC-I allomorphs requires a precisely coordinated quality control network in a single macromolecular assembly, including the transporter associated with antigen processing TAP1/2, the MHC-I heterodimer, the oxidoreductase ERp57, and the ER chaperones tapasin and calreticulin. Proofreading by MHC-I editing complexes guarantees that only very stable peptide/MHC-I complexes are released to the cell surface. This proposal aims to gain a holistic understanding of the PLC and MHC-I proofreading complexes, which are essential for cellular immunity. We strive to elucidate the mechanistic basis of the antigen translocation complex TAP as well as the MHC-I chaperone complexes within the PLC. This high-risk/high-gain project will define the inner working of the PLC, which constitutes the central machinery of immune surveillance in health and diseases. The results will provide detailed insights into the architecture and dynamics of the PLC and will ultimately pave the way for unraveling general principles of intracellular membrane-embedded multiprotein assemblies in the human body. Furthermore, we will deliver a detailed understanding of mechanisms at work in viral immune evasion.

2 181 250 €

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

Acoustic waves exert forces when they interact with matter. Sound, and in particular ultrasound, which has a wavelength of a few hundred microns in water, is a benign and versatile tool, that has been successfully used to manipulate, trap and levitate microparticles and cells. The acoustic contrast between the material and the medium, and the spatial variation of the ultrasound field determine the interaction. Resonators and arrays of a few hundred transducers have thus far been used to generate the sound fields, but the former only yields highly symmetrical pressure patterns, and the latter cannot be scaled to achieve complex fields. Our radically new approach uses a finely contoured 3D printed acoustic hologram to generate pressure fields with orders of magnitude higher complexity than what has been possible to date. The acoustic hologram technology is a route towards truly sophisticated and 3D sound fields. This project will research the necessary computational and experimental tools to generate designed 3D ultrasound fields. We will investigate ways to use acoustic holograms for rapid manufacturing, the controlled manipulation of microrobots, and the assembly of cells. The 3D pressure fields promise the assembly and fabrication of an entire 3D object in “one shot”, something that has not been realized to date. We will also study the formation of 3D cellular assemblies, and more realistic 3D tumour models. This project will develop the technology, materials, processes, and understanding needed for the generation and use of sophisticated 3D ultrasound fields, which opens up entirely new possibilities in physical acoustics and the manipulation of matter with sound.

2 420 125 €

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

Protein quality control systems maintain a functional proteome through detection and removal of abnormal proteins. While typically only misfolded or damaged molecules are thought of as abnormal, recent work has revealed that also mislocalized proteins are subject to quality control. Mislocalized proteins are defined as proteins that fail to reach their native compartment or fail to assemble into their native complex, and thus cannot function normally. Protein mislocalization is a constitutive problem caused by inefficiencies of cellular processes and increases with aging. Proteins can also mislocalize due to mutations, as seen in various metabolic, cardiovascular and neurodegenerative diseases, and some types of cancer. Despite the ubiquity of protein mislocalization, the systems performing quality control of mislocalized proteins are unknown for most of the proteome because quality control substrates are usually rare, thus difficult to identify, and there is considerable redundancy built into quality control systems. Here, I propose to systematically dissect quality control mechanisms of mislocalized proteins through a combination of molecular biology, genetics, biochemistry and computational biology in yeast and human cells. We will establish a platform for conditional protein mislocalization and apply it (i) to identify quality control substrates proteome-wide, (ii) to dissect redundancies in quality control systems, (iii) to identify the machinery responsible for quality control of mislocalized proteins and (iv) to map the features involved in substrate recognition by the quality control machinery. Finally, we will exploit our findings to selectively target aneuploid cancer cells, which exhibit a high burden of mislocalized proteins. This work will provide a comprehensive picture of quality control systems for mislocalized proteins and shed light on their roles under both normal and perturbed conditions.

1 497 750 €

Start date: 2018-03-01, End date: 2023-02-28

Crystalline defects in metals and semiconductors are responsible for a wide range of mechanical, optical and electronic properties. Controlling the evolution of dislocations, i.e. line-like defects and the carrier of plastic deformation, interacting both among themselves and with other microstructure elements allows tailoring material behaviors on the micro and nanoscale. This is essential for rational design approaches towards next generation materials with superior mechanical properties. For nearly a century, materials scientists have been seeking to understand how dislocation systems evolve. In-situ microscopy now reveals complex dislocation networks in great detail. However, without a sufficiently versatile and general methodology for extracting, assembling and compressing dislocation-related information the analysis of such data often stays at the level of “looking at images” to identify mechanisms or structures. Simulations are increasingly capable of predicting the evolution of dislocations in full detail. Yet, direct comparison, automated analysis or even data transfer between small scale plasticity experiments and simulations is impossible, and a large amount of data cannot be reused. The vision of MuDiLingo is to develop and establish for the first time a Unifying Multiscale Language of Dislocation Microstructures. Bearing analogy to audio data conversion into MP3, this description of dislocations uses statistical methods to determine data compression while preserving the relevant physics. It allows for a completely new type of high-throughput data mining and analysis, tailored to the specifics of dislocation systems. This revolutionary data-driven approach links models and experiments on different length scales thereby guaranteeing true interoperability of simulation and experiment. The application to technologically relevant materials will answer fundamental scientific questions and guide towards design of superior structural and functional materials.

1 499 145 €

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

Post-translational modification by ubiquitin and ubiquitin-like proteins (UBLs) is a major eukaryotic regulatory mechanism. Nonetheless, we have little understanding of the detailed mechanisms by which most E3 ligases mark specific targets with monoubiquitin, multiple ubiquitins or specific polyubiquitin chains, or of how UBL modifications transform the functions of their targets. This proposal addresses both problems. First, we will discover the structural mechanisms by which the UBL NEDD8 (58% identical to ubiquitin) activates numerous distinct functions of its targets, which are cullin subunits of cullin-RING E3 ubiquitin ligases (CRLs). Second, we will take a tour-de-force structural, biochemical, and molecular cell biological approach to determine how NEDD8-activated E3 ligases regulate their substrates. Because CRLs form nearly half of all E3 ligases, and as we recently discovered, neddylated CRLs act in part by activating monoubiquitylation by another family of E3 ligases (Ariadne-family RBR E3s), the proposed studies will establish paradigms for a major fraction of ubiquitylating enzymes. To achieve these goals, we will devise novel chemical biology tools to capture fleeting assemblies that typically only occur during chemical reactions, and visualize structures of neddylated CRLs “in action” by cryo EM. We will generate a resource of novel reagents that detect, label, and affinity purify activated forms of E3 ligases to temporally track their interactions during pathways they regulate in cells. And we will define the mechanisms and structures of a class of atypical, disease-associated giant E3 ligases whose domains and interacting partners are so peculiar that their activities remain elusive. Overall, we will comprehensively define how a UBL directly regulates its targets, and how two major E3 ligase families mediate regulation.

2 193 871 €

Start date: 2018-10-01, End date: 2023-09-30

Dynamic fragmentation of metals is typically addressed within a statistical framework in which material and geometric flaws limit the energy absorption capacity of protective structures. This project is devised to challenge this idea and establish a new framework which incorporates a deterministic component within the fragmentation mechanisms. In order to check the correctness of this new theory, I will develop a comprehensive experimental, analytical and numerical methodology to address 4 canonical fragmentation problems which respond to distinct geometric and loading conditions which make easily identifiable from a mechanical standpoint. For each canonical problem, I will investigate traditionally-machined and 3D-printed specimens manufactured with 4 different engineering metals widely used in aerospace and civilian-security applications. The goal is to elucidate whether at sufficiently high strain rates there may be a transition in the fragmentation mechanisms from defects–controlled to inertia–controlled. If the new statistical-deterministic framework is proven to be valid, defects may not play the major role in the fragmentation at high strain rates. This would bring down the entry barriers that the 3D-printing technology has found in energy absorption applications, thus reducing production transportation and repairing, energetic and economic costs of protective structures without impairing their energy absorption capacity. It is anticipated that leading this cutting-edge research project will enable me to establish my own research team and help me to achieve career independence in the field of dynamic behaviour of ductile solids.

1 497 507 €

Start date: 2018-03-01, End date: 2023-02-28

Protein domains start to fold co-translationally while they are being synthesized on the ribosome. Co-translational folding starts in the confined space of the ribosomal polypeptide exit tunnel and is modulated by the speed of translation. Although defects in protein folding cause many human diseases, the mechanisms of co-translational folding and the link between the speed of translation and the quality of protein folding is poorly understood. Here I propose to study when, where and how proteins emerging from the ribosome start to fold, how the ribosome and auxiliary proteins bound at the polypeptide exit affect nascent peptide folding, what causes ribosome pausing during translation, and how pausing affects nascent peptide folding. Our recent results (Holtkamp et al., Science 2015; Buhr et al., Mol Cell 2016) provide the proof of principle for monitoring translation and protein folding simultaneously at high temporal resolution. First, we will follow translation processivity and folding trajectories for proteins of different domain structure types using time-resolved ensemble kinetics and single-molecule setups. The structures of complexes with stalled folding intermediates will be solved by cryo-electron microscopy. Second, we will investigate the effects of the chaperone trigger factor, the signal recognition particle, and other protein biogenesis factors on the folding landscape. Third, we will analyze transient ribosome pauses in vivo (based on ribosome profiling data) and in vitro (based on time-resolved translation assays and mathematical modeling) and identify the events that cause pausing. Finally, we will probe how changes in translational processivity affect the conformational landscape of a protein. We expect that these results will open new horizons in understanding co-translational protein folding and will help to understand the molecular basis of many diseases.

2 482 600 €

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

Roughness on most natural and man-made surfaces shares a common fractal character from the atomic to the kilometer scale, but there is no agreed-upon understanding of its physical origin. Yet, roughness controls many aspects of engineered devices, such as friction, adhesion, wear and fatigue. Engineering roughness in surface finishing processes is costly and resource intensive. Eliminating finishing steps by controlling roughness in primary shaping or in subsequent wear processes could therefore revolutionize the way we manufacture, but this requires a deep understanding of the relevant processes that is presently lacking. Roughness emerges during mechanical deformation in processes such as folding, scratching or chipping that shape surfaces. Deformation occurs in the form of avalanches, individual bursts of irreversible motion of atoms. The central hypothesis of this project is that roughness is intrinsically linked to these deformation avalanches, which themselves are well-documented to be fractal objects. This hypothesis will be tested in large-scale atomic- and mesoscale simulations of plastic forming and fracture on state of the art high performance computing platforms. Results of these calculations will be used to develop process models for evolving the topography of large surface areas under the action of an external mechanical force, such as experienced in shaping, finishing or wear. In addition to these simulations, a public repository for sharing topography data will be build. This repository is the connection to experiments: It is a database of experimental topographies whose contents will be mined for features identified in simulations. Beyond the present project, this web-repository will advance sharing, visualization and analysis of topography data, and aid researchers to correlate surface topography with surface functionality and processing. Simulations and database lay the foundation for a rational design of surface functionality in manufacturing.

1 499 101 €

Start date: 2018-02-01, End date: 2023-01-31

The main question to be addressed by SPOCk’S MS is how protein complex conformation adapts to local changes, such as processing of polyproteins, protein phosphorylation or conversion of substrates. While labelling strategies combined with mass spectrometry (MS), such as hydrogen deuterium exchange and hydroxyl footprinting, are very versatile in studying protein structure, these techniques are employed on bulk samples averaging over all species present. SPOCk’S MS will remedy these by studying the footprinting and therefore exposed surface area on conformation and mass selected species. Labelling still happens in solution avoiding gas phase associated artefacts. The labelling positions are then read out using newly developed top-down MS technology. Ultra-violet and free-electron lasers will be employed to fragment the protein complexes in the gas phase. In order to achieve the highest possible sequence and thus structural coverage, lasers will be complemented by additional dissociation and separation stages to allow MS^N. SPOCk’S MS will allow sampling conformational space of proteins and protein complexes and especially report about the transient nature of protein interfaces. Constraints derived in MS will be fed into a dedicated software pipeline to derive atomistic models. SPOCk’S MS will be used to study intracellular viral protein complexes, especially coronaviral replication/transcription complexes, which are highly flexible and often resist crystallisation and are barely accessible by conventional structural biology techniques. Objectives: - Integrate labelling with complex species selective native MS for time-resolved structural studies - Combine fragmentation techniques to maximise information content from MS - Develop software suite to analyse data and model protein complex structures based on MS constraints - Apply SPOCk’S MS to protein complexes of human pathogenic viruses

1 999 000 €

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