EUROMembrane_LIPIDPROD_Detecting rafts in the live cell plsma membrane - from resting state to signaling

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 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. Another promoter of bilayer research was the introduction of the raft concept to subcompartmentalize cell membranes. 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 that are postulated to be functional in membrane trafficking and signaling. The stage is thus set to bring this research field to the next level of sophistication by coming to grips with how functional raft platforms form in cells. The vision of this CRP 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 as well as in vitro and in silico. In order to clarify the existence and to further characterize their properties, our group recently designed an experimental strategy for in vivo imaging of single membrane rafts by ultra-sensitive fluorescence microscopy, termed "Thinning Out Clusters while Conserving the Stoichiometry of Labeling" (TOCCSL). The methodology is based on advances to detect and characterize individual molecules in the live cell plasma membrane. In contrast to our efforts to track single molecules, we focus here on the brightness as a measure of the local stoichiometry. The overall idea is to detect rafts via their property to recruit specific membrane proteins. In this subproject, we focus on the direct visualization and analysis of lipid rafts in the live cell plasma membrane under various conditions. We will address the resting state of T cell rafts, and investigate the signaling induced formation of larger structures. Particular emphasis will be laid on studying raft heterogeneity: while there is increasing evidence for different types of lipid rafts, the origin still remains unclear. Using a two-color variant of TOCCSL, we will study the degree of colocalization of any two differently labeled membrane constituents in the same membrane rafts. Heterogeneous composition of membrane rafts will be investigated using differently labeled GPI-anchored proteins as markers. Finally, the recruitment of membrane proteins to lipid rafts will be analyzed.

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 lipid matrix itself was considered to be a solvent for membrane proteins. This changed with the increasing awareness of the complexity of the lipid composition of bilayers. One promoter of bilayer research was the introduction of the raft concept to subcompartmentalize cell membranes. This concept implies that cell membranes containing sphingolipids, saturated phosphatidylcholine and cholesterol are occupied by fluctuating nanoscale assemblies that are poised for coalescence into larger scale, more stable domains including some and excluding other proteins. The vision of our joint project was to provide the multidisciplinary support required to analyze how nanoscale protein-lipid assemblies interact to form functional platforms and how membrane proteins associate with lipids to modulate function. In our subproject we used two strategies to address this problem: i) Via single molecule microscopy. We have previously devised a method to analyze the co-diffusion of membrane proteins or lipids based on the brightness of the diffusing entities. We used this method here to study the co-diffusion of lipid-anchored proteins (so-called GPI-anchored proteins) in the live cell plasma membrane. We found out that about half of the GPI-anchored proteins diffuse as homodimers within the plasma membrane. The presence of dimers depends on the cholesterol concentration of the membrane, but also on temperature: slightly elevated fever-type temperatures completely abolished the formation of the dimers. In addition, we observed effects by activation of lipid-modifying enzymes. Together, these data indicated that lipids are important for the formation of membrane protein associates. ii) Via micropatterning of membrane proteins. In the second approach we used a method we had previously developed for studying the interaction of membrane proteins. Briefly, one of the membrane proteins of interest (termed bait) is spatially rearranged to micropatterns directly in the live cell plasma membrane by growing the cells on microstructured surfaces. The interaction with the second protein of interest (prey) is measured by determining the prey recruitment to the bait patterns. In this project, we were interested whether the enrichment of a GPI-anchored protein has any effect on the biophysical properties of the corresponding lipid bilayer region. Surprisingly, we did not find any measurable influence on the recruitment of other GPI-anchored proteins, or on the phase state of the lipids. We did find, though, a size-exclusion effect: due to the enrichment of the bait protein, the local density in the exoplasmic region of the plasma membrane increased so that the entry of other proteins was impeded. These data show that phase separation is less important for protein association than expected; it also shows that size exclusion phenomena in the immediate environment of the lipid bilayer are important for protein interactions.