Microtubules are key components of the eukaryotic cytoskeleton involved in a multitude of essential cellular functions. In the past decades, thorough studies have elucidated the biophysical properties of microtubules, their interactions with multiple microtubule-associated proteins and molecular motors, as well as many specialized cellular functions of the microtubule network. While many of the basic functions and properties of microtubules have been studied in great detail, microtubules have generally been considered as homogeneous macromolecular assemblies. This is contrasted by the obvious existence of microtubule identities in cells, which allow microtubules to acquire specific properties for functional specialisation. Our project tackles an emerging regulatory mechanism that could play a key role in generating distinct microtubule identities, the posttranslational modifications of tubulin. To study how spatially and temporally controlled modification patterns determine the properties and the functional fate of microtubules, we will focus on two original tubulin modifications, polyglutamylation and polyglycylation. Having nearly completed the identification and characterization of the enzymes that catalyse these two modifications, our future project is focussed on their biological functions and the underlying molecular mechanisms. Applying an interdisciplinary approach ranging from in vitro systems over cell biology to mouse models, we will (1) develop new tools and methods for the observation of tubulin modifications at higher spatial and temporal resolution (e.g. recombinant antibodies, high-resolution imaging), (2) perform functional studies on mouse and cellular models to elucidate the functional roles of the modifications by focussing on microtubule assemblies with characteristic patterns of polyglutamylation (mitotic spindle and midbody, neuronal microtubules) and polyglycylation (axonemes in primary and motile cilia), (3) investigate the mechanisms that determine the specificity of modifying enzymes and control the generation of modification patterns on selected microtubules inside cells, and (4) analyze the molecular readout mechanisms of tubulin polyglutamylation and polyglycylation by in vitro studies. In summary, we will address how precise microtubule identity marks are generated by tubulin posttranslational modifications, and how these marks are translated into specialized microtubule functions. Our work might have a fundamental impact on the understanding of microtubule functions in cells and organisms.

Microtubules are essential components of the cytoskeleton involved in fundamental processes such as intracellular trafficking or cell division. A large part of these functions is underpinned by their dynamic properties, which allow microtubules to switch rapidly between polymerization and depolymerization phases upon cell needs. While microtubule dynamics is finely tuned in cells by accessory proteins, the ability to alternate between growing and shrinkage states is intrinsic to microtubules assembled from purified tubulin. The hydrolysis of the GTP bound to the exchangeable site of tubulin sustains this particular dynamic behavior, called 'dynamic instability'. According to the most widely spread model in the literature, a delay between polymerization and GTP-hydrolysis generates a 'GTP-cap' at microtubule growing ends. Stochastic loss of this GTP-cap would result in fast disassembly of the microtubule, while re-capping would allow the reverse event. Analysis of the GTP-cap has stimulated a large number of studies in the past. However, the absence of specific probes for the nucleotide state of tubulin in microtubules did not allow a detailed characterization of this structure at the molecular level. This project involves a collaboration between the Institute of Genetics and Development of Rennes (D. Chrétien, partner 1) and the Paul Scherrer Institute (M. Steinmetz, Villigen, Switzerland, partner 2). During a previous collaboration, we characterized the architecture of the GTP-cap by cryo-electron tomography (cryo-ET) using the plus-end binding protein 1 (EB1) conjugated to gold nanoparticles. EB1, which binds specifically to microtubule growing ends, displays a high affinity for microtubules assembled in the presence of some GTP-analogues, and thus is currently considered as a specific marker of the GTP-cap region. Our results suggest that the GTP-cap structure includes outwardly curved sheets present at microtubule growing ends, and extends onto closed parts of the microtubule lattice. In the present project, our objective is to extend this study by correlating tubulin conformational changes with its GTPase cycle. In this respect, our methodological approach will aim at characterizing at the highest possible resolution dynamic microtubules assembled in the presence of GTP and GDP-Pi analogues. This work will involve solving the high-resolution structure of tubulin in the presence of the analogues by X-ray crystallography (partner 2), analysis by cryo-ET of dynamic microtubules in the presence of the analogues (partner 2), and docking of the high-resolution structures of tubulin into the molecular envelopes determined by cryo-ET (partners 1 & 2). Malfunctions of microtubule dynamics are associated with a range of pathologies such as neuronal disorders or cell cycle anomalies. In this context, a better understanding of the molecular basis of microtubule dynamics is essential to develop new strategies against these pathologies. Our project will also bring to the scientific community new methodological tools to analyze the structure and dynamics of complex macromolecular assemblages. Methodological approaches will be disseminated though practical and theoretical workshops. Results will be published in high-impact journals and presented at international conferences. A particular effort will be devoted to presenting our work to the non-scientific public.

The sustainable supply and efficient conversion of energy is of major concern in today’s societies and economies. In particular, this is valid for Switzerland and France due to increasing scarcity of mineable fossil energy sources, hosting different industrial entities in the energy, transportation and power electronics sector. Metal oxide semiconductor field-effect transistors (MOSFETs) are of key importance for e.g. decentralized electrical supply grids, electrical drive trains and electric cars, core businesses for ABB, EDF and STmicroelectronics companies. To date, such power devices are mainly realized on silicon. However, Silicon Carbide (4H-SiC) MOSFETs allow higher switching speeds, lower losses and simpler system topologies, thus enabling a reduction in overall system and material costs as well as higher efficiencies and reliabilities. This project aims at analyzing and innovating a critical process step in the fabrication of silicon carbide (SiC) based MOSFET, namely the creation of the SiO2/SiC interface crucial for a high conductance inversion channel. When comparing to state-of-the-art silicon technology, the channel mobility in SiC MOSFETs is very low and strongly dependent on the crystallographic direction. Therefore, in SiC MOSFETs, the inversion channel significantly contributes to the total on-resistance and thus device losses. SiC is commercially available only with surface being tilted 4 degrees with respect to the [0001] crystallographic basal plane. Since the thermal oxidation process is strongly orientation-dependent, the surface morphology of 4H-SiC is expected to signi?cantly in?uence the formation of the SiO2/SiC interface potentially leading to non-ideal oxidations and nonstoichiometric near-interface regions. Yet, the question of how the surface, and especially agglomerated macrosteps, affects the performance of Metal-Oxide-Semiconductor Field-E?ect Transistors (MOSFETs) is still largely unsolved. Main reason for this lack of knowledge is that the commercially available SiC epi-wafers (wafers with an epitaxial layer) used for MOSFET fabrication have irreproducible surface steps to allow for a proper characterization. In this project, we propose to overcome these limitations by using different annealing techniques to induce controlled modifications of the surface morphology to generate either regions without steps (on-axis mesas) or regions with periodically distributed large steps and terraces (>10nm and >200nm, respectively). With such modifications, we aim at studying the effects of the local crystal structure on thermal oxidations and ultimately understand their impact on channel characteristics of 4H-SiC MOSFETs and present strategies for improvements. This study will be both theoretical, using hybrid DFT-force fields and TCAD simulations, and experimental, inducing surface reconstructions and characterizing thermal oxides by means of MOS capacitors and MOSFETs in such reconstructed surfaces. A full understanding of the factors limiting the channel mobility at the SiC/SiO2 interface will allow to design manufacturing processes and MOSFET structures which outperform today’s devices by a factor of three or more in terms of on-resistance for 1.2kV 4H-SiC MOSFETs. This will substantially boost the power electronic systems efficiency. This project proposal is set for four years. A PhD student at the University of Lyon (LMI) will develop processes to modify the surface of SiC samples. A PhD working both at PSI and at ETH Zurich will fabricate and characterize MOS capacitors and MOSFETs on such reconstructed samples. We will use a patented patterning technique developed at PSI to analyze the SiO2/SiC interface of terraces and steps separately. Finally, a PhD student working at the University of Basel will develop a novel atomistic simulation code to theoretically understand the role of the steps on the oxidation process and their impact on the device behavior.

The project entitled “Characterization of defects by Advanced Diffraction techniques to evaluate micro-crystals deformation stages” (CharADiff) aims at gaining a detailed understanding of the mechanics of small scale objects (micro and nano-crystals) by the implementation of an unprecedented combination of recent and specifically-developed in-situ cutting-edge x-ray diffraction techniques: 1) in-situ coherent x-ray diffraction that enables to detect and evaluate the number of lattice defects (dislocations) in small scale crystals during mechanical solicitation; 2) in-situ micro-Laue diffraction that allows for studying the nature and sequence of activated dislocation slip systems during plastic deformation of small objects; 3) post-mortem Bragg ptychography that provides a 3D image of the displacement field caused by lattice defects left in the deformed objects and 4) ptychographic topography, a novel technique developed in this project, to map individual lattice defects while remaining compatible with future in-situ deformation studies. These techniques are applied to a unique system: InSb semi-conductor micro-crystals of initial excellent crystalline quality whose mechanical properties have been already thoroughly studied, therefore being a robust benchmark. The outcomes of this original methodology are two-fold: - developing a tool box combining in-situ and post-mortem advanced diffraction techniques to characterize the deformation response of small scale objects (deformation stages and lattice defects storage): this will enrich the current knowledge on small scale mechanics, providing responses to some of the open questions still remaining in Materials Science on the effects of size reduction; - assessing the complementarity between some of the most recent diffraction techniques developed at synchrotrons with the perspective to push their applicability to the forefront of their current use. Moreover, a new non-destructive imaging technique will be developed, ptychographic topography, with the strong perspective to be implemented within an in-situ setup, as another step forward. The impact of the project is potentially far beyond the frontiers described above, in particular in the field of nanotechnology, where the x-ray diffraction techniques developed here could constitute non-destructive solutions to assess the crystalline quality of small-scale crystals that are the building blocks of miniaturized devices, therefore allowing for quantitative diagnostics and defect engineering. To guarantee the success of this 36 month-project, a well thought consortium of French and Swiss experts has been settled, with strong complementarity in the fields of experimental and simulation approaches, as well as Materials Science and diffraction. A total of 86.4 person-months will be deployed by the consortium, including 9 permanent staffs and 2 post-doctoral fellows.