Understanding how asymmetry emerges from an initial symmetrical condition is a major scientific goal. Our project will focus on the emergence of LR asymmetry or Chirality in biological systems, using a complementary, multiscale approach. Our previous work identified Myosin 1D (Myo1D) as a unique chiral factor essential for LR asymmetry and capable of breaking symmetry at all biological scales in Drosophila. While the actin cytoskeleton emerges as a pivotal and conserved pathway for chirality formation, the exact cytoskeletal factors involved in this process, their interplay and the molecular mechanisms involved remain to be explored. Collaborative work between Partners will study molecular-to-organismal chirality through the following tasks: 1. New regulators of Myo1D chiral function and development of the tracheal model system 2. Role of the Myo1D-DAAM interaction to induce cytoskeleton chirality in vitro 3. Role of Myo1D and DAAM in actin organization in cells, minimal tissues and organs in vivo.

Size scaling of biological systems is a general property found throughout nature by which individual cells can adapt essential elements according to their size. The size and shape of cells depend largely on the actin cytoskeleton to build both stable and dynamic intracellular organizations. Due to their complexity, a complete understanding of the molecular mechanisms underlying the self-organization of actin cytoskeletal architectures is still missing. Furthermore, the relationship between the self-assembling properties of actin cytoskeletal architectures and their ability to scale with cell size remains poorly understood. The goal of this project is to study the scaling properties of dynamic actin architectures reconstituted in cell-sized compartments. Subsequently, a comparative analysis will be conducted between the scaling properties observed in the reconstituted systems and those present in corresponding cellular architectures. I will address the following questions: (1) What defines the size of a dynamic actin architecture? (2) How do competing actin architectures for the same resources define and maintain their size and their dynamic? (3) How do actin architectures adapt their size and dynamic when faced with perturbations? (4) What provides robustness to the intracellular scaling mechanisms? Overall, this project will provide a comprehensive understanding of how the size and dynamics of intracellular architectures are linked and how they scale with cell size.

Flowers ensure angiosperm reproduction and are the basis for fruits and seeds. The LEAFY transcription factor orchestrates flower development in angiosperms. Its activity highly depends on its physical interaction with the UFO ubiquitin ligase but the reason for this has remained obscure. Our project aims at understanding how LFY and ubiquitination pathways interact to make flowers. We will use a combination of genomics, imaging, structural biology and proteomics to understand i) where this interaction occurs at the tissue, subcellular and genomics levels ii) the biochemical and structural properties of the UFO-LFY complex iii) how it controls gene expression iv) the function of ubiquitination. Moreover, we propose to create a tool combining LFY and UFO able to trigger the development of flowers from any tissue allowing to modify inflorescence structures at will, bypassing species or environmental constraints.

Phosphate (Pi) starvation is a frequent nutrient stress impacting crop yields. To adapt to this stress, plants exert different mechanisms to increase the uptake of extracellular Pi and to remobilize intracellular reserves. In cells, one third of the Pi is retained in phospholipids, the main components of extra-plastidial membranes. During Pi starvation, phospholipids are partially degraded to release Pi and are replaced by a non-phosphorous lipid synthesized in plastids: the digalactosyldiacylglycerol (DGDG). Thus, the synthesis and transfer of DGDG from plastids to other organelles is highly stimulated in this situation. However, the mechanisms involved in lipid remodeling remain poorly understood. DGDG transfer to mitochondria is thought to occur by non-vesicular pathways at contact sites between mitochondria and plastids. Recently, we have identified in Arabidopsis thaliana a super-complex, the MTL (Mitochondrial Transmembrane Lipoprotein) complex, involved in DGDG transfer to mitochondria during Pi starvation. Among this complex, AtMic60, a protein located in the inner membrane of mitochondria, plays an indirect role in DGDG transfer 1) by regulating contact sites formation between both mitochondrial membranes and 2) by destabilizing membranes. Only a partial decrease of DGDG transport is observed in the absence of AtMic60, suggesting that other pathways are also involved. In addition, we currently do not know how plastid-mitochondria contact sites, an important structure for lipid transport, are formed. The goal of the MiCoSLiT project is to identify keys actors involved in DGDG transport to mitochondria and/or in the formation of plastid-mitochondria contact sites in response to Pi deprivation. Our working hypothesis is that the MTL complex and other actors, which remain to be identified, are involved. First of all, a combination of three biochemical approaches will be optimized to identify new candidate proteins potentially involved in such processes. A first approach corresponds to a deep analysis of the composition and organization of the MTL complex in order 1) to better understand its functions and 2) to highlight new components putatively involved in lipid transport and/or membrane contact sites formation. Two non-targeted approaches, corresponding to the analysis of the mitochondrial proteome in response to Pi starvation and the optimization of a method to isolate plastid-mitochondria contact sites, will be performed in parallel to identify new pathways. All these approaches will be performed from A. thaliana cell cultures grown in presence and in absence of Pi to highlight candidates specifically involved in Pi-starvation response. Then, a functional analysis of a small subset of candidates will be undertaken to decipher their role(s) in the transport of lipids to mitochondria and/or in the formation of plastid-mitochondria contact sites. Finally, the involvement of these candidates in the global response of plant to Pi starvation will be studied in order to demonstrate the important role of lipid remodeling in the adaptation of plant to this situation. Impacts of the MiCoSLiT project are expected in both basic research and agricultural sciences. Indeed, the project will increase our understanding of the mechanisms involved in mitochondrial lipid transport and in the formation and regulation of contact sites between organelles, processes which remains poorly characterized, particularly in plants. In addition, by the investigation of the cellular mechanisms involved in plant response to Pi starvation, the project will open important perspectives in the development of crops presenting higher yield when grown in low-Pi soils.

Pollution of terrestrial and aquatic ecosystems by trace metal elements, also referred to as heavy metals, is a major and ever-growing threat to environmental and human health. A better understanding of the effects of toxic elements on land plants and microalgae is critical to develop approaches for treating contaminated environments using phyto- and phycoremediation processes The identification and characterization of organisms that tolerate and accumulate metals are essential to reach these objectives. Indeed, these organisms evolved sophisticated molecular mechanisms to cope with toxic elements. Deciphering these strategies may indicate how plants/algae might behave in contamination scenario and could provide clues for new biotechnological applications for the capture of metals. We isolated a metal-hypertolerant unicellular photosynthetic microalga from an environment contaminated with uranium, a chemotoxic radionuclide. This green microalga was identified by 18S rDNA sequencing as a Coelastrella species, hereafter designated Cos. Because it is able to live in culture media contaminated with high concentrations of uranium or silver, we assume that Cos has evolved unique molecular mechanisms to survive in environments polluted by toxic elements. In support of our hypothesis, it is known that some metal-tolerant land plants and microalgae have established efficient strategies to cope with metals (e.g. cellular uptake and efflux, compartmentalization, detoxification by chelators). The key genetic loci that explain the unique metal-tolerance and accumulation properties have been identified only in some land plants, for example in the zinc and cadmium hypertolerant Arabidopsis halleri species. These findings were essential to better understand metal homeostasis in both tolerant and non-tolerant species. In addition, they were the basis for new strategies to improve the phytoextraction properties of fast-growing, high-biomass but non-tolerant plant species. In green microalgae, however, the genes involved in metal tolerance and accumulation have never been identified. The objective of the DemoniaCo project is to fill this gap and unravel the molecular mechanisms involved in the tolerance and accumulation of toxic metals in Coelastrella sp. To this aim, we will use a combination of cell physiology and systems-based approaches, including a thorough analysis of the toxicological outcomes of metals on the transcriptome, proteome, ionome, and metabolome of the alga. This unprecedented multiscale and integrative strategy will provide new insights into the fundamental and applied biology of a metal-hypertolerant green microalga. Besides the identification of the gene network enabling Cos to tolerate uranium and silver, the expected results of the project include the characterization of Cos tolerance to a variety of toxic elements, the characterization of metal uptake and subcellular distribution in algal cells, the behaviour of the microalga in natural metal-contaminated waters to estimate its performance for phycoremediation, and the investigation of the potential of Cos as an oleaginous model species for the production of lipids for biofuel applications.