caLIPSO

Protein sorting is a central process of eukaryotic cells that orchestrates secretory and endocytic pathways and is supported by the trans-Golgi Network (TGN) and early endosomes (EEs), respectively. While there is extensive crosstalk between these two dynamic sorting systems they have evolved different organelle structures and dynamics across eukaryotic kingdoms. Plants have evolved an original TGN which blurs the frontiers between secretion and endocytosis to the extreme. Our project is addressing how a single membrane compartment acts as both the TGN and EEs, therefore fusing two major sorting platforms of eukaryotic cells into a single entity of exquisite complexity. Plant TGN is differentiated in functional subdomains. TGN-associated Secretory vesicles (SVs) and TGN-associated Clathrin-Coated Vesicles (CCVs) represent two distinct sub-domains of TGN that likely regulate sorting toward the plasma membrane (PM) and the Late Endosomes (LEs)/vacuoles, respectively. However, while some protein markers of the different TGN subdomains have been uncover in recent years, lipids have received little attention, while at the same time it began evident that lipids are key determinants of membrane identity and sorting mechanisms. In plants, we previously showed partitioning of sphingolipids (SLs), sterols (partner 1) and phosphoinositides (PIPs) (partner 2) in TGN subdomains and our preliminary results suggest that SL and PIP patterning at TGN are interconnected specifically at SVs subdomain. By combining advanced approaches in lipidomics, cell and developmental biology, our project aims at describing the extent of lipid partitioning at the TGN, uncovering the underlying mechanisms and determining their importance in protein sorting, cell polarity and plant root development. To address these aims the project will be divided in three tasks. Task 1 (partner 1, Yohann Boutté) will map the precise subcellular localisation of PIPs at distinct subdomains of TGN by high resolution live imaging of fluorescent PIP sensors established in Partner2 lab and by performing in-depth lipidomic analyses on imnuno-purified TGN subdomains. Moreover, we will gain insights into SLs involvement in PIPs homeostasis by studying the impact of pharmacological and genetic perturbation of SL metabolism on PIP patterning and TGN morphodynamics. Task 2 (partner 2, Yvon Jaillais) will Set-up a new plant cell biology approach to acutely erase PI4P with subcellular resolution at distinct TGN subdomains by establishing the rapamycin-based chemically-inducible dimerisation system in Arabidopsis. Using this new targeted approach we will study the impact of PI4P on TGN morphology and dynamics, protein sorting and identify downstream proteins and lipids. Task 3 (partner 1 and partner 2) will determine the function of the SLs/PIPs connection in TGN subdomains maturation and protein sorting from TGN to PM using cutting edge tools in microscopy. In this task, Partner 1 and 2 will combine their expertise on SLs and PIPs in order to dissect some of the molecular mechanisms that pattern PIP within the plant endomembrane system. The identification of these mechanisms will provide insights into how lipids participate in TGN differentiation and thereby proteins trafficking, cell polarity and plant development. As a model system for plant development, we will investigate the trafficking and sorting of PIN2, an auxin efflux carrier polarly localized at the apical membrane of epidermal cells and which regulates the root response to gravity in Arabidopsis thaliana (i.e. bending of the root according to the gravity vector). Hence, this project will provide a unique and innovative package with complementary skills. Elucidation of function and dynamics of SLs/PIPs interplay during selective sorting of proteins at TGN represents a new entry to understand how lipid patterns may arise to differentiate TGN subcompartments and drive sorting events for both secretion and endocytosis.