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The missing link: how do membrane lipids interplay with ATG proteins to instruct plant autophagy

Periodic Reporting for period 2 - LIP-ATG (The missing link: how do membrane lipids interplay with ATG proteins to instruct plant autophagy)

Reporting period: 2021-08-01 to 2023-01-31

Autophagy is an intracellular degradation process critical to eukaryotic life and indispensable for plant survival to a wide range of environmental stresses including drought, nutrient scarcity or attacks by various pathogens. Autophagy relies on the formation of specialized vesicles which engulf and deliver cell components to the lytic vacuole. The formation of autophagy vesicles is carried out by a group of dedicated proteins and hinges on intense remodelling events and on the remarkable capacity of an initial membrane, the phagophore, to assemble de novo, shape like a cup, expand while maintaining structure and function and re-shape to a complete vesicle. At this time, the molecular mechanisms underlying these events remain elusive in plants. Research has focused on the role of autophagy proteins but, despite AP biogenesis being a membrane-based process, the fundamental contributions of lipids to AP membrane formation, identity and activities have been largely unexplored. In this project, we address the fundamental question of how autophagy vesicles form and shape by exploring how lipids’ nature, dynamics and lateral heterogeneity instruct the phagophore structure, its protein composition and its functions. We will tackle 3 complementary objectives: 1) Reveal the dynamic lipid signature of the phagophore, 2) Elucidate the implication of lipids nature and repartition in the phagophore ultrastructure, 3) Decrypt the molecular mechanisms by which lipids interplay with ATG proteins to control autophagy activity and plant physiology. Overall the project will articulate an integrated vision of the molecular processes controlling autophagy and provide fundamental knowledge in our understanding of plant responses and acclimation to environmental changes.
Despite extensive research on the topic, very little is known about the origin and the formation of autophagosome in plant cells. Using pharmacological and genetical approaches we uncovered the importance of the lipid phosphatidylinositol-4-phosphate (PI4P) in autophagy. Combining biochemical and live-microscopy analyses, we showed that PI4P is required for early stages of autophagosome formation. Further, our results show that the plasma membrane-localized PI4Kα1 is involved in autophagy and that a substantial portion of autophagy structures are found in proximity to the PI4P-enriched plasma membrane. Together, our study unravels critical insights into the molecular determinants of autophagy, proposing a model whereby the plasma membrane provides PI4P to support the proper assembly and expansion of the phagophore thus governing autophagosome formation in Arabidopsis (Gomez et al., Nature Communications, 2022).

We purified autophagy structures and we are now establishing their molecular profile. We already characterized their glycerophospholipid and sterol composition and further analyses are ongoing to provide an exhaustive map of the phagophore lipid composition, compare it to that of other cell compartments and identify lipids enriched in the phagophore as candidates for further functional analyses. Additionally, we established the proteome of autophagy compartments thus unraveling the presence of several lipid-related proteins including lipid-remodeling enzymes which characterization (localization, function) is in progress with the aim to explore their contribution in lipid dynamics during autophagosome formation.

We started to characterize the architectural dynamics of autophagosome formation in wild-type plants using Correlative Light Electron Microscopy combined to Electronic Tomography, including quantitative analyses of the phagophore morphology and contacts with additional subcellular compartments (see Gomez et al., Nature Communications, 2022 and unpublished). We further generated the transgenic lines and initial experiments to develop a novel technology in plant cells, aiming at exploring the repartition of key lipids and proteins within the phagophore membrane at nanometric scale.
Autophagy has been extensively studied but little focus has been given to the potential of lipids as critical functional actors behind the series of membrane remodeling events that instruct the remarkable dynamic structure of the phagophore. Our research aims at addressing the functions of lipids in plant autophagy in an integrated fashion. This project represents a change of perspective by 1) considering and demonstrating that lipids are critical functional components of the autophagy machinery, 2) considering that the nature and spatiotemporal distribution of lipids specify functional territories in the phagophore, 3) considering protein/lipid interactions and their dynamics during AP formation, 4) incorporating a portfolio of highly complementary and cutting-edge experimental approaches to explore the nature and function of lipids in the regulation of AP formation, autophagy flux and plant plasticity. Unraveling the molecular composition of the phagophore and the role of lipids in the phagophore structure, identity and functions in plants will provide critical novel information, opening new research directions and will set foundation for future studies. Autophagy is conserved in eukaryotes, hence concepts and strategies established by our work will be relevant and influential to autophagy research at large with the potential to unlock AP biogenesis in other models. If successful, our project will provide a first hint on the molecular mechanisms by which lipids instruct functional modeling of the phagophore to control autophagy and plant adaptive responses. Consequently, this project has potential to lead to fundamental breakthroughs in our understanding of how molecular plasticity translates to phenotypic plasticity and how this is established and regulated in plants. In that context, we sincerely hope that our work will provide keys to predict and manage the effects of environmental changes on native species as well as crop plants.
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