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The dynamic behavior of plasma membrane proteins mediates various cellular processes such as cellular motility, communication, and signaling. It is widely accepted that the dynamics of the membrane proteins is determined either by the interactions of the transmembrane domain with the surrounding lipids or by the interactions of the intracellular domain with cytosolic components such as cortical actin. Although initiation of different cellular signaling events at the plasma membrane has been attributed to the extracellular domain (ECD) properties recently, the impact of ECDs on the dynamic behavior of membrane proteins is rather unexplored. Here, we investigate how ECD properties influence protein dynamics in the lipid bilayer by reconstituting ECDs of different sizes or glycosylation in model membrane systems and analyzing ECD-driven protein sorting in lipid domains as well as protein mobility. Our data show that increasing the ECD mass or glycosylation leads to a decrease in ordered domain partitioning and diffusivity. Our data reconcile different mechanisms proposed for the initiation of cellular signaling by linking the ECD size of membrane proteins with their localization and diffusion dynamics in the plasma membrane.

Suppressive regulatory T cell (Treg) differentiation is controlled by diverse immunometabolic signaling pathways. However, the impact of intracellular metabolites on Treg fate has not been well explored. Here we show that the α-ketoglutarate (αKG) tricarboxylic acid (TCA) cycle metabolite increases oxidative phosphorylation (OXPHOS) in naive CD4 T cells activated under Treg polarizing conditions, markedly attenuating Foxp3+ Treg differentiation and increasing inflammatory cytokine expression. Adoptive transfer of these T cells into tumor-bearing mice results in enhanced tumor infiltration and decreased Foxp3 expression. Mechanistically, αKG-induced OXPHOS is associated with lipidome remodelling, characterized by augmented mitochondrial lipids and triacylglyceride (TAG) stores. Inhibiting succinate dehydrogenase, the bridging enzyme between the TCA cycle and the ETC, enforces Treg differentiation. Strikingly, TAG storage directly promotes an inflammatory phenotype –– Treg differentiation is restored by inhibiting DGAT2-mediated TAG synthesis. Thus, we identify a novel crosstalk between αKG, mitochondrial metabolism and DGAT2-mediated TAG synthesis in controlling Treg fate.

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls. proof

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

The high-affinity IgE receptor, FcεR1, is responsible for the sensitization of mast cells and basophils to IgE-targeted anti- gens. The current model of FcεR1 triggering rests on the principle of aggregation-driven phosphorylation, yet several recent observations have indicated that this is incomplete. Here we re-examine the minimal requirements for FcεR1 triggering and observe that it is induced without receptor aggregation by surface-associated antigen in a size-dependent manner.

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

Nature of the cellular membrane heterogeneity has been a long-standing question in cell biology. One crucial measure for the membrane heterogeneity is the lipid packing. Measuring changes of lipid packing in the plasma membrane during cellular events has been an important part of elucidating the biological function of membrane physical chemistry. Here, we apply conventional and spectral imaging to figure out the nature of membrane heterogeneity and its role in immune signalling events.We observe bio-membranes sampling different lipid packing states that form coexisting domains (i.e., relatively ordered and disordered domains) with a variety of distinct compositions and functional characteristics. We first investigate the connection between membrane composition and lipid packing in synthetic liposomes and show that both the relatively ordered and disordered phase, and the difference between them, are tuned by lipid composition. We then give an example of live cells regulating the lipid packing phenotypes of their plasma membranes, accessing a variety of both ordered and disordered domains as a function of cellular activity. Finally, we find that in both synthetic and natural membranes, the tunable interdomain lipid packing disparity (i.e. the difference between coexisting domains) regulates component partitioning between domains and the functionality of membrane embedded lipidic receptors. By applying advanced image processing with spectral imaging, we show that even the marginal modulation of membrane heterogeneity is crucial for immune signalling events such as FCR and B-cell signalling.Membrane heterogeneity is an organizing principle for the membrane bio-activity by either concentrating certain molecules in transient domains or by physically adjusting the conformity of the molecules to favour or hinder their interactions in the membrane.

Proper supply of mammalian cells with cholesterol is crucial for membrane function and cellular survival and is regulated by several mechanisms, including cellular uptake, synthesis, storage and export. Transport of excess cholesterol from the periphery back to the liver for excretion into the bile is achieved by high-density lipoproteins (HDL). HDL particles are captured from the blood stream by the scavenger receptor class B type I (SR-BI), the so-called HDL receptor. The subsequent lipid transfer process, however, is highly speculative: it has been proposed to occur after HDL endocytosis or directly at the cell surface across an unstirred water layer or via a hydrophobic channel in the receptor. Here we show via direct imaging techniques that, upon contact with the membrane, HDL is incorporated into the hydrophobic core of the lipid bilayer and amphiphilic cargo is immediately released to the plasma membrane. Particle incorporation and cargo transfer is abolished at increased membrane cholesterol concentrations, as a consequence of the reduced membrane elasticity. Our observations reveal a mechanism for regulation of lipid uptake, which is based on sensing plasma membrane cholesterol levels: HDL-receptors tether particles close to the plasma membrane; once in proximity, elastic membrane properties regulate the particles’ fusion with the bilayer and subsequent cargo transfer. The data indicate that the plasma membrane itself signals a cell's cholesterol demand: high cholesterol levels act repulsive, low cholesterol levels fusogenic.

Selective uptake of high density lipoprotein (HDL)-associated cholesteryl-esters (CE) by hepatocytes is a crucial process for the removal of cholesterol from the circulation. Together with triglycerides, CE is transported in the hydrophobic core of HDL particles; free cholesterol, phospholipids, and apolipoproteins build up the particles' amphiphilic surface monolayer. The lipid transfer process itself is highly speculative: it is still unclear how lipids are removed from the circulation and transferred from HDL - a main carrier of cholesterol in the blood stream - to cells.In this study we provide a mechanistic understanding of the cargo exchange process between HDL and biomembranes. The interaction between HDL and synthetic lipid membranes was investigated with force spectroscopy and high speed atomic force microscopy; the transfer of single cargo molecules was directly visualized using a combined and simultaneously operating fluorescence and force microscope. Experimental evidence points to the fact that i) cargo transfer requires contact; ii) only amphiphilic cargo is transferred; iii) upon contact the particle incorporates into the hydrophobic core of the bilayer where it can diffuse. In particular, we compared the transfer of the fluorescently labelled lipids DiI, and Bodipy-labelled cholesterol and cholesteryl-ester. Live cell experiments confirmed the data obtained on the synthetic systems and on GPMV's (giant plasma membrane vesicles). Particle incorporation and cargo transfer was abolished at increased membrane cholesterol levels, as a consequence of the reduced membrane elasticity. These observations reveal a mechanism for regulation of lipid uptake based on sensing plasma membrane cholesterol levels. The function of the corresponding receptor SR-B1 is primarily an anchor to hold the particle close to the plasma membrane; once in proximity, elastic properties of the membrane regulate the fusion of the particle.