Protein misfolding is a constant threat to cellular health and is linked to pathologies. It has been recently demonstrated that misfolded nuclear proteins reversibly translocate into the nucleolus upon stress. This novel cell protective protein quality control pathway involving the nucleolus maintains protein homeostasis. The nucleolus is a paradigm example of biocondensate formed by phase separation of its constituents. Protein modifications have been reported to modulate biocondensate formation and properties. Here I propose to investigate how the widespread protein N-?-acetylation and lysine-?-acetylation regulate the capacity of the nucleolus to reversibly store misfolded proteins. The project will combine state of the art methodologies such as advanced proteomics, dynamic cellular imaging, optogenetics tools and in vitro reconstitution to shed light on the influence of protein acetylations on nuclear protein homeostasis, a fundamental question with disease relevance.

Sociality is classified as one of the major transitions in evolution, and the most advanced level of sociality in animals is found in eusocial insect societies, like honey bees. The success of social insect colonies relies on elaborate communication among the colony members, and in particular on the use of a high number of pheromones. But how does the social insect brain manage to encode such a plethora of highly?meaningful and ecologically?relevant signals? Does it encode social pheromones using dedicated pathways (labeled-line system), a relevant strategy when only a few pheromones are used by the animal (i.e. sexual pheromone), or does it use a combinatorial strategy (many weakly specific lines), in the manner of general odorants? To answer these questions, the PHEROBRAIN project aims to identify and characterize olfactory receptors of the honey bee tuned to pheromonal compounds using a phylogenetic approach and heterologous expression in the Drosophila empty-neuron system (Aim 1). We will then study the central circuits involved in pheromone processing using in vivo calcium imaging on transgenic bees expressing the calcium indicator GCaMP6, recently developed in the scientific coordinator’s team (Aim 2). Finally, we will study the effect of specific olfactory receptor knock-out on bee’s behavior (Aim 3), both in natural conditions and using associative conditioning. Overall, the PHEROBRAIN project will help to understand how evolution has shaped the social insect brain to cope with the increased need for accurate communication channels.

Long-term potentiation (LTP) of excitatory synapses is widely regarded as a cellular correlate of learning and memory. Among other events, it is accompanied by an increase in the size of the postsynaptic compartment known as dendritic spines. This structural change is driven by remodelling of the actin cytoskeleton. It is widely accepted that this remodelling results from signalling cascades initiated by calcium entry through NMDA receptors. Other lines of research however highlight the necesserty for LTP maintenance of another well-known actin regulator: integrin-beta1. Integrins are extracellular matrix (ECM)-binding transmembrane receptors with no intrinsic enzymatic activity. Studies in non-neuronal cells distinguish two ways by which they transmit information across the plasma membrane: biochemical signalling, notably via the activation of tyrosin kinases, and biophysical signalling mediated by protein-protein interactions between integrins and actin. In this context, integrins are recognized as central players in the molecular assemblies responsible for cellular adhesion and motility. While integrin activation and downstream signalling have been explored in the context of LTP at the mature hippocampal synapse, little is known about the existence and potential functional impact of a physical link between the ECM and the synaptic cytoskeleton. The IntSynCity project will therefore explore this overlooked aspect of integrin regulation in the context of LTP. It will combine advanced optical imaging, molecular biology and chemical biology to i) reveal the existence of an ECM-integrin-actin link at the synapse, ii) identify the adaptor protein(s) mediating it, iii) map the nanoscale co-organisation of proteins composing these adhesion sites, relatove to each other and to other synaptic components, and iv) investigate the functional impact of such linkage on various aspects of LTP, including the AMPA receptor mediated increase in synaptic strength and the actin regulatory cascades leading to increased spine size. Taken together, this project and its technological developments will shed new light on the molecular mechanisms via which integrin-b1 regulates LTP. By filling an important knowledge gap at the crossroads of several aspects of molecular neuroscience, the project will impact both the fields of synaptic adhesion and structural plasticity. Its ambition thus perfectly aligns with the scientific topics persued in the host team, while proposing a new line of research that will enable the coordinator to achieve scientific maturity, intellectual independence and visibility among her peers.

Investigations of the programmed DNA elimination (PDE) process of the unicellular ciliate Tetrahymena have provided important insights into how eukaryotes use small noncoding RNAs to regulate transposable elements (TEs), which impact genome organization and integrity. Despite the two decades of efforts to clarify the small RNA-directed PDE mechanism, previous studies using candidate-driven reverse genetics by gene knockout and biochemical protein complex purifications have failed to identify many of the molecular factors involved in this process. To further understand the molecular mechanism of small RNA-directed PDE in Tetrahymena, in this proposed project, we aim to optimize the CRISPR-Cas13-based gene knock-down method in Tetrahymena and establish sorting methods isolating cells and nuclei that are defective in different steps of PDE. Using these methods, we will perform genome-wide forward genetic screening to identify and characterize previously undescribed genes involved in PDE. In parallel, we also aim to use proximity labeling to identify and characterize novel proteins associated with the factors known to be involved in PDE. We expect that the proposed study will provide important insights into the evolution of small RNA-directed TE silencing as well as advanced understandings about the conserved molecular principles for small RNA-mediated epigenetic chromatin regulation. Moreover, we believe that the establishment of a cells sorting-based high-throughput forward genetics protocol will revolutionize Tetrahymena genetics to understand the various fundamental processes of eukaryotes.

Methionine oxidation (MO) converts Methionine (Met) into Methionine sulfoxide (MetO). In proteins, MO can be a double-edged sword. Surface-exposed Met scavenges oxidants, protecting key residues like catalytic sites from damage. This protective role is amplified by the Methionine Sulfoxide Reductase (Msr) enzymes, which reduce MetO, making Met prone to subsequent oxidations and thereby lower intracellular oxidant levels. Conversely, MO can lead to protein misfolding or aggregation. Here, the Msr system is necessary to prevent protein loss-of-function. Although pivotal in protein homeostasis (proteostasis), the Msr system's precise contribution to protein quality control remains elusive. Notably, questions such as (i) how these proteins find their substrates within the crowded intracellular environment, (ii) whether fluctuations of their regulation disrupt cell fate and (iii) if a cooperative mechanism with molecular chaperones exists remain unknown. Using Escherichia coli as a model, the AMORE project aims at investigating in what proportion the Msr system participates to proteostasis. The role of the Msr system will be studied in unperturbed and stressed conditions. We will focus on stresses mediated by reactive chlorine species (RCS) that are known to have high reactivity with Met. Specifically, we will study HOCl, a physiological RCS produced by neutrophils during host infection, and ClO2-, a chemical used in water treatment facilities, disinfectants and herbicides, known for its bactericidal activity through its MO effect. AMORE holds the promise of deciphering critical mechanisms underlying protein homeostasis and bacterial oxidative stress response, potentially heralding transformative insights in the field.