Mechanisms of lipid patterning at the trans-Golgi Network and roles in protein sorting, cell polarity and plant development – caLIPSO

As we know that TGN is subdivided in distinct populations that can overlap in conventional confocal microscopy, we use high resolution airyscan confocal microscope Zeiss 880, equipped with the latest cutting edge tool, the fast airy scan module, to combine 100 nm X-Y resolution and fast acquisition for live imaging (100 ms scale). This gives us access to the dynamics of PIPs-labelled compartments and study their interaction with other compartments. To identify and quantify lipids at TGN, we use immuno-isolation of intact and entire TGN by GFP-trap approach. Partner1 proved that lipid identification and quantification is possible in distinct sub-populations of TGN and in Golgi apparatus. To isolate entire and intact compartments, the purification has to be detergent-free. We use, in the first steps of immuno-isolation, density gradient centrifugation to coarsely separate endomembrane compartments and then incubate this pre-purified fraction with magnetic beads coupled to anti-GFP antibodies. These beads allow for very low background and efficient immuno-isolation of entire and intact compartments. Using LC-ESI-MS we attempt to identify and quantify PIP species but also all the other phospholipid species. LC-ESI-MS combined with HPTLC/GC-MS analyses will give important and detailed information on the head group, length of the acyl-chain, saturation, hydroxylation of each lipid classes. We additionally use quantitative label-free proteomics on immuno-isolated TGN. To acutely deplete PI4P at distinct TGN subdomains, we will use the rapamycin-based chemically-inducible dimerisation system to recruit a PI4P-specific phosphatase in an inducible manner at TGN subdomains. In order to do that, we will fuse FKBP to the catalytic domains of the Saccharomyces cerevisiae a PI4P phosphatase Sac1 protein. As compartment specific anchors, we will use the same markers than for compartment immuno-isolation of TGN since their localization specificity were extensively validated.

Results from partner 1 allowed to identify the role of the acyl chain length of SLs in the homeostatic regulation of PIPs at TGN. By label-free proteomics on immuno-purified TGN fraction, we identified a phospholipase C (PLC) specific of the PIPs (PI-PLC). PI-PLC consume PI4P by cleaving the molecule to produce on one side a diacylglycerol molecule (DAG), which is rapidly converted into phosphatidic acid (PA) in plants, and a polyphosphorylated inositol molecule on another side. Using pharmacological and genetic approaches coupled to biochemical approach we have shown that PI-PLC function is required to modulate PI4P quantity at TGN downstream of SLs. Moreover, we revealed that the homeostatic relationship between SLs and PI4P is instrumental during polar secretory trafficking of the PIN2 auxin carrier from the TGN to the polar apical domain of the plasma membrane of root epidermal cells. As PIN2 is involved in auxin distribution and modification of the orientation of root growth after a change of the axis of gravity (also called gravitropism), we used this as a phenotypic readout. Our results show that a double mutant impacted in PI4P production at TGN is insensitive to the effect triggered by a modification of SLs composition on root gravitropism. These results show that PI4P is acting downstream of SLs during a functional phenotypic response. Partner 2 focused on the enzymes that consume PI4P by dephosphorylating it and producing phosphatidylinositol (PI), unlike PI-PLC that cleaves PI4P to produce DAG. Enzymes dephosphorylating PI4P are SAC6, SAC7 et SAC8. The team is studying the subcellular localization of SAC6-8 enzymes and their function in PI4P homeostasis, endomembrane trafficking, protein sorting and plant development.

The results produced by the two teams are complementary and allowed the identification of two mechanisms that regulate the quantity of PI4P at TGN. One through the function of SLs on PI-PLC amount at TGN and another one through the function of SACs phosphatases. In animal cells, the polar head of SLs modulates PI4P/sterols exchange mechanisms between the endoplasmic reticulum and the TGN, a process which regulates the quantity of PI4P at TGN. Data obtained by partner 1 show that the length of the acyl chain of SLs modulates the quantity of PI4P at TGN through PI-PLCs. Thus it would be interesting in this project to test whether the polar head of SLs is involved in the regulation of PI4P quantity at TGN through the SACs and potentially in lipid exchange mechanisms at membrane contact sites. Interestingly, the grafting of the polar head of SLs happens at TGN and partner 1 has developed genetic tools to test the hypothesis described above. In the future, we will test the function of the polar head of SLs in SAC-mediated PI4P homeostasis. More generally, we will address the function of the SLs/PI4P and SAC/PI4P pathways in protein sorting, especially during secretory and endocytic sorting of auxin carriers. This will allow us to shed new lights on how TGN sorts secretory and endocytic materials within a same endomembrane compartment.

1. Ito Y#, Esnay N#, Platre M, Noack L, Menzel W, Claverol S, Moreau P, Jaillais Y, Boutté Y*. Sphingolipids mediate polar sorting of PIN2 through phosphoinositide consumption at the trans-Golgi Network. Nature Communications (revision)

2. Ito Y#, Grison M#,*, Esnay N, Fouillen L, Boutté Y*. (2020). Immuno-purification of intact endosomal compartments for lipid analyses in Arabidopsis. Methods in Mol Biol., 2177:119-141.

3. Boutté Y*, Jaillais Y*. (2020). Metabolic cellular communications: feeback mechanisms between membrane lipid homeostasis and plant development. Developmental Cell, 2:S1534-5807(20)30394-4.

4. Noack LC, Jaillais Y*. (2020). Functions of Anionic Lipids in Plants. Annu Rev Plant Biol. 71:71-102.

5. Jaillais Y*, Ott T*. (2020). The Nanoscale Organization of the Plasma Membrane and Its Importance in Signaling: A Proteolipid Perspective. Plant Physiol. 182(4):1682-1696.

6. Colin LA, Jaillais Y*. (2020). Phospholipids across scales: lipid patterns and plant development. Curr Opin Plant Biol. 53:1-9.

7. Trinh DC, Lavenus J, Goh T, Boutté Y, Drogue Q, Vaissayre V, Tellier F, Lucas M, Voß U, Gantet P, Faure JD, Dussert S, Fukaki H, Bennett MJ, Laplaze L, Guyomarc'h S. (2019). PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation. Proc. Natl. Acad. Sci. U S A., 116(28):14325-14330.

8. Gendre D, Baral A, Dang X, Esnay N, Boutté Y, Stanislas T, Vain T, Claverol S, Gustavsson A, Lin D, Grebe M, Bhalerao RP. (2019). Rho-of-plant activated root hair formation requires Arabidopsis YIP4a/b gene function. Development, 146(5). pii: dev168559.

9. Mamode Cassim A, Gouguet P, Gronnier J, Laurent N, Germain V, Grison M, Boutté Y, Gerbeau-Pissot P, Simon-Plas F, Mongrand S. (2019). Plant lipids: Key players of plasma membrane organization and function. Prog. Lipid Res., 73:1-27.

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.