EUROMembrane_Dissecting lipid anchors to study lipid rafts heterogeneity for signal transduction

Membranes are central to understanding cellular organization and function. About one third of the genome encodes membrane proteins and many other proteins spend part of their lifetime bound to membranes. The major class of membrane proteins are transmembrane proteins, spanning the bilayer. The other class are the peripheral membrane proteins, which function by binding to the interfacial regions of the bilayer, either at the exoplasmic or at the cytoplasmic side. The third class of proteins is anchored in the membrane by covalently attached lipid moieties. The lipid bilayer, which constitutes the fluid matrix of the membrane was for years neglected. The lipid matrix itself was considered to be a mere solvent for membrane proteins. This changed with the increasing awareness of the complexity of the lipid composition of bilayers. Eukaryotic membrane lipids are glycerophospholipids, sphingolipids, and sterols and it is thought that more than 1000 different lipid species are present in mammalian cells. Why there are so many lipids in cell membranes is not understood. A promoter of bilayer research was the introduction of the lipid raft concept. This concept as it stands today implies that cell membranes containing sphingolipids, saturated phosphatidylcholine and cholesterol are occupied by fluctuating nanoscale assembles that are poised for coalescence into larger scale, more stable domains including some and excluding other proteins. In this scheme the biophysical propensity to phase separate is coupled to specific lateral association involving oligomerization, lipid-protein, and protein-protein interactions such that when amplified during raft coalescence specific liquid platforms form. The stage is thus set to bring this research field to the next level of sophistication by coming to grips with how raft platforms form in cells. The vision of this common research project is to provide the multidisciplinary support that will be required to analyze how nanoscale protein-lipid assemblies interact to form functional platforms and how membrane proteins associate with lipids to modulate function. Most importantly, we will apply the whole set of technologies to same cell and protein systems both in vitro and in silico. The lipid raft field was boostered by indications that rafts control signal transduction across the plasma membrane by associating only with transmembrane receptors when the latter are triggered by their ligands. This mechanism facilitates both to separate transmembrane receptors from submembrane signaling molecules (e.g. kinases) in the resting state, and to sequester them upon activation for signal transduction. One of the best studies transmembrane receptor and lipid raft-associated submembrane signaling component are the T cell antigen receptor and the Src protein tyrosine kinase (SFK) Lck, respectively. Besides Lck, other resident constituents of T cell lipid rafts are its sister SFK Fyn, numerous adaptor proteins such as LAT and PAG, and the GPI-anchored proteins CD48, CD55, CD59, known as regulatory molecules of T cell activation. In this subproject the group of Hannes Stockinger intends to analyze the contribution of the lipid/protein moieties of these major components of lipid rafts of T cells to their molecular organization in lipid rafts. In particular, the Stockinger lab will study the role of the exoplasmic and cytoplasmic lipid anchors of the GPI-anchored proteins and SFKs, respectively, for heterogeneity and oligomerisation in lipid rafts, and the consequence of these suborganizations in rafts to their function in signal transduction.

With the EUROMembrane programme LIPIDPROD we aimed to provide a deeper understanding of cell membranes. Cell membranes serve many essential biological functions, such as the build-up of important structures within the cell, the formation of boundaries between a cell and its exterior, and transport of cellular components. Another important function of cell membranes is the regulation of signalling pathways that cells use to transfer information from the environment or from other cells to pass into the cell to respond appropriately. In several types of experiments we have been able to show that the composition and organization of cell membranes have an influence on whether and how immune cells respond to stimulation from their environment. For example particular enzymes, which are essential for the transmission of signals in immune cells, gather at certain areas of the membrane where special lipids are present. These organized lipid structures in the membrane are called lipid rafts. In this way, lipids can influence the forwarding or receiving of information. In this project we discovered for instance also that lipid rafts play an important role in the interactions of lipids and proteins to resolve blood clots. In cooperation with the project partners from the biophysics we also explored the speed, dynamic of formation and dissolution as well as the size of these lipid rafts, and thereby provide an insight how cells use these lipid rafts as plasma membrane devices to regulate the information flow from the environment into the cell.