Starch is a storage form of carbohydrates in plants and the main source of calories in human and animal diets. Moreover, this polymer is used in many industrial applications for food and non-food purpose. During the last decades, it became a major renewable raw material for manufacturing products such as plastics and paints. These materials are agrobased, potentially biodegradable and thus are of great interest in a context of ecological transition. Consequently, new industrial sectors are arising that rely on starch as a raw material. In the “Hauts de France” region, starch-rich crops like potato are cultivated locally and represent a valuable resource to supply these sectors, without the need for long transportation of the raw material thus limiting greenhouse gas emissions. However, starch quality determines downstream applications that often require chemical modifications. The latter are costly and will impact the carbon balance and ecological footprint of the sector. Improving starch quality in vivo (i.e. before extraction) requires the development of new plant breeding programs that take the corresponding traits into account. Phosphate groups are naturally present in starch at C3- or C6-position of the glucose residues and impact the structure of starch granules. Other locks also impact post-extraction processing, namely the size of starch granules and the structure of starch polymers. This project aims at upgrading the knowledge regarding starch properties in vivo that are of interest in emerging starch-based industrial sectors. Moreover, the project intents to offer a phenotyping support to breeding programs targeting the new potato flour markets. In a previous work funded by ANR (PoStaTic project: Structural and morphological variability of potato starch and associated biological processes towards bio-plastic production), we have developed a semi high-throughput platform (including a new a capillary electrophoresis method to determine both C3- and C6-phosphoesters in starch) for characterizing starch structure and morphology and estimated their variability in numerous potato cultivars. Moreover, we deciphered the complete potato starch-associated proteome. In addition to the already known starch-bound proteins, we identified proteins with a high targeting potential. Interestingly, there is growing evidence that the amount of starch-bound proteins follows specific stoichiometric requirements related to starch initiation, synthesis, phosphorylation, and degradation as well as the control of granule size and number. With this project, we ambition to: i. decipher the stoichiometry of starch-bound proteins and its impact on starch structure and morphology by quantitative proteomics of single-starch granules; ii. understand the function of new target genes in potato by reverse genetics using the CRISPR/Cas9 system; iii. determine the exact repartition of the phosphoesters within the polymers and understand how they impact the molecular organization of starch; iv. characterize the variability of starch phosphorylation and morphology in wild-type potato-related species to open new perspectives in trait introgression approaches. This project will, for the first time, lead to single starch granule characterization and allow to understand how starch-bound proteins participate to the establishment and maintenance of the granule architecture. Furthermore, it will report on the natural variability of starch in wild-type potato species, allowing for instance to provide solutions to breeding programs targeting emerging sectors. Finally, this project will result in the characterization of new gene functions related to starch metabolism. The latter will be edited in potato by CRISPR/Cas9 to provide new functional traits in one of the most cultivated crops. Future legislation regarding gene-edited plants may facilitate bioeconomical applications of these plants.

Voltage-gated ion channels (VGICs) mediate electical signaling in cells. Several decades of research into VGICs has yielded much insight into their structure and function; however, the resting state of their voltage-sensing domains (VSDs) has remained elusive, since it is adopted in the presence of the cell's resting membrane potential which so far could not be reproduced in structural studies. I propose a novel approach to this problem by establishing a Donnan transmembrane potential in liposomes via the asymmetric presence of impermeable polyelectrolytes and by applying solid-state NMR spectroscopy to VGICs reconstituted into such liposomes. This will allow us to delineate the resting state of VGIC voltage sensors and their motion during gating. The method proposed will for the first time allow for direct structural investigation of a defined functional VGIC state in the presence of a transmembrane potential and resolve a long-standing problem in ion channel structural biology.

Sialic acids (SA) are major players in many biological functions such as host-pathogen recognition. Whereas the biosynthesis pathways of SA and the associated enzymes are quite well described in eukaryotes, this is not the case in bacteria. The objectives of the NEURAPROBE project are to develop new labeling tools and study incorporation of sialic acids using the bioorthogonal chemical reporter strategy, in pathogenic bacteria compared to human cells, as well as well as in cellular infection models, in order to detect key differences and characterize phenotypic variations that will be used to screen chemical libraries. While the majority of published articles focus on cell-level bioimaging using a fluorescent probe, we will attempt to gain in sensibility and resolution by using metal probes detectable by modern electronic microscopy methods. This engineering has never been used on such biological systems yet.

Starch is a main storage form of carbon for plants. It is composed by glucose polymers forming water insoluble granules that harbour a wide range of sizes and morphologies regarding to their origin (e.g., plant species, organs, tissues, cells). In addition, starch granules contain minor fractions of proteins, lipids and phospholipids as well as phosphate groups covalently bound to glucose moieties. Starch is an important raw material for food and non-food industries and represents a promising way for atmospheric carbon capture. In 2021, starch-like polymers were successfully synthesized chemoenzymatically from atmospheric carbon dioxide and hydrogen in vitro. However, no evidence for a granular, semi-crystalline packed organization neither for mimicking starch physicochemical properties were presented. This raises the questions about which additional natural components (e.g., structural proteins, phosphoesters) are necessary for native starch synthesis and how to normalize synthesis steps to produce homogenous starch granules. In previous projects, we developed methods for studying miniaturized starch samples and, to date, could characterize glucan length distribution and the proteome of single potato starch granules. We also established methods for the structural analysis of phosphoglucans and miniaturized lipidomics. With this project these developments will be finalized and used to characterize single starch granules and investigate their heterogeneity as well as the contribution of minor compounds to granule architecture. Establishing the relationships between granule composition, structure and morphology at the level of single granules will help feeding mathematical models contribute in designing homogenous artificial synthesis workflows.

The aggregation of neuronal Tau protein to form fibrilar structures inside neurons is associated with tauopathies, notably Alzheimer’s disease (AD). A trans-synaptic transfer of Tau may participate in the spatio-temporal progression of the disease and this supposes that Tau is secreted into the interstitial fluids. Molecular and cellular mechanisms behind these processes, aggregation and transfer, are still unknown. Additionally, although Tau immunotherapy is a potential therapeutic strategy in AD, fundamental knowledge is still lacking to implement this strategy. The objective of ToNIC (Tau Nanobodies Interaction Characterization) is to identify, optimize and evaluate potent and selective VHHs (nanobodies) targeting Tau, as well as to generate novel basic knowledge on the pathophysiology of tauopathies to validate, in the long term, immune-targets in the treatment of tauopathies. Nanobodies correspond to the variable domains of camelid heavy-chain antibodies (VHHs). The antigen binding capacity of VHHs relies on a single domain and therefore VHHs correspond to the smallest antigen-binding fragment of an antibody. A unique and original multi-disciplinary strategy will be implemented to reach our objectives thanks to the year-long expertise in Tau biology of both partners, Partner 1 I. Landrieu UMR8576 and Partner 2 L. Buée UMR- S 1172. Complementary approaches will be used to transfer the in vitro experimental biophysical data into cellular contexts. VHHs directed against soluble, phosphorylated or aggregated Tau protein will be selected from a validated synthetic library (sub-contracting Hybrigenics services, Paris) by phage-display. These VHHs will be fully characterized for their specificity, affinity and capacity to recognize their target in a cellular context. Selected VHHs, based on these criteria, will be further assayed to evaluate their capacity to inhibit Tau aggregation, both in vitro and in a cellular model of aggregation, and/or their capacity to block Tau transfer using microfluidic devices. These experiments should provide novel knowledge on which pathway(s) might preferentially be altered in Tau immune strategy. Preliminary data show the feasibility of our approaches which combine high resolution Nuclear Magnetic Resonance spectroscopy, with other biophysical methods (partner 1) and in cell studies (partner 2) as one of our VHH, directed against soluble Tau, was found to inhibit Tau aggregation, both in vitro and in a cellular context. This project of fundamental interest, aiming at answering key questions in Tau biology, has potential exciting perspectives in human health.