ROP6-mediated auxin signaling relies on plasma membrane lipid interleaflet coupling

Abstract

International audienceIn cell signaling, extracellular signals are transmitted across the plasma membrane to activate downstream elements inside the cell. These signaling processes are based on protein complexes e.g. activation of receptor-like kinases (RLKs), transmembrane kinases and/or membrane associated kinase regulators. However, it now appears that lipids play a major role for signal transduction across membranes. Indeed, they could act on protein activity or activation, mobility, clustering or interaction with other proteins. More particularly, at the inner leaflet of the PM, phosphatidylserine (PS) is a crucial lipid that acts in the auxin- induced nano-clustering of the Rho-GTPase ROP6, thereby triggering auxin signaling1. However, PS nanodomains exist prior to any cell stimulation and a mechanism is lacking to explain how PS nanodomains are primarily formed within the lipid matrix of the PM. In this work, we used a combination of Fluorescence Recovery After Photobleaching (FRAP), Total Internal Reflection Fluorescence (TIRF) microscopy, super- resolution PhotoActivated Localization Microscopy (PALM) and molecular dynamics modeling to address the role of lipids in the lateral mobility and nano-clustering of PS and ROP6 at the PM. Our work reveals that the very long chain fatty acids (VLCFAs) of lipids are crucial in this process. In plants, VLCFAs are found in two main pools of membrane lipids, i.e the sphingolipids and PS. We show that VLCFAs of both sphingolipids and PS are involved in the lateral mobility and nano-clustering of PS and ROP6. Molecular dynamics simulations confirmed these experimental observations and further revealed that sphingolipids, that are located in the outer leaflet of the PM, interact with PS that are located in the inner leaflet of the PM. Computational simulations show that sphingolipid-PS interactions are dependent on their acyl-chain length and create a coupling between the two leaflets of the PM through lipid-lipid interdigitation. Altogether, our results identified an interleaflet lipid coupling mechanism that creates an orthogonal membrane organization that stabilizes or anchors PS and ROP6 signaling nanodomains.1. Platre, M. P. et al. Developmental control of plant Rho GTPase nano-organization by the lipid phosphatidylserine. Science 364, 57–62 (2019)