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The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10 -15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 m 2 ), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. membrane domains ͉ macrophages T he lipid raft hypothesis proposes that the lateral organization of cellular membranes is based on the presence of distinct, cholesterol-rich, rigid domains (rafts) (1), which are involved in signal transduction (2), protein sorting, and membrane transport (3, 4). Our understanding of lipid structure and the formation of specific lipid domains within membranes, however, is almost exclusively based on model membrane systems (5). Although phase separation of domains of liquid-ordered structure is predicted to exist in cellular membranes (6, 7), direct demonstration using methodologies such as fluorescence quenching has been difficult to apply to living cells (8). The evidence for the existence of lipid rafts in living cells is largely based on measurements of the clustering (9, 10) or diffusion (11, 12) of lipid raft proteins, which are secondary to the lipid organization.In the present study, we labeled living cells with the fluorescent probe 6-acyl-2-dimethylaminonapthalene (Laurdan), which has been previously used to characterize domain formation and phase separation in model membranes using phospholipid mixtures (13-15) or lipid extracted from cellular membranes (16)(17)(18). Laurdan is an environmentally sensitive fluorescence probe that exhibits a 50-nm red shift as membranes undergo phase transition from gel to fluid, due to altered water penetration into the lipid bilayer (19). Its dipole is aligned parallel to the hydrophobic lipid chains in membranes and is located in both bilayers (20). The probe's fluorescence in water is negligible, and it is not influenced spectroscopically by surface modifications such as lipoprotein binding (20,21). The environmentally induced red shift allows the translation of intensity measurements at different wavelengths into lipid packing orders within the membranes of intact and living cells (20,22,23). Generalized polarization (GP), with a correcting factor G for the experimental setup, is defined analogously to fluorescence polarization by measuring the in...

Based on LDL-C lowering and the absence of adverse signals, this EAS Consensus Panel concludes that functional foods with plant sterols/stanols may be considered 1) in individuals with high cholesterol levels at intermediate or low global cardiovascular risk who do not qualify for pharmacotherapy, 2) as an adjunct to pharmacologic therapy in high and very high risk patients who fail to achieve LDL-C targets on statins or are statin- intolerant, 3) and in adults and children (>6 years) with familial hypercholesterolaemia, in line with current guidance. However, it must be acknowledged that there are no randomised, controlled clinical trial data with hard end-points to establish clinical benefit from the use of plant sterols or plant stanols.

Objective-To study the acceptor specificity for human ABCG1 (hABCG1)-mediated cholesterol efflux. Methods and Results-Cells overexpressing hABCG1 were created in Chinese Hamster Ovary (CHO-K1) cells and characterized in terms of lipid composition. hABCG1 expressed in these cells formed homodimers and was mostly present intracellularly. Cholesterol efflux from hABCG1 cells to HDL 2 and HDL 3 was increased but not to lipid-free apolipoproteins. A range of phospholipid containing acceptors apart from high-density lipoprotein (HDL) subclasses were also efficient in mediating ABCG1-dependent export of cholesterol. Importantly, a buoyant phospholipidcontaining fraction generated from incubation of lipid-free apoA-I with macrophages was nearly as efficient as HDL 2 . The capacity of acceptors to induce ABCG1-mediated efflux was strongly correlated with their total phospholipid content, suggesting that acceptor phospholipids drive ABCG1-mediated efflux. Most importantly, acceptors for ABCG1-mediated cholesterol export could be generated from incubation of cells with lipid-free apoA-I through the action of ABCA1 alone. Conclusions-These

No abstract

After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)–signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.

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However, not all studies show a clear association between HDL-cholesterol and cardiovascular disease. 4,5 The observation that the capacity of serum to promote macrophage Molecular Medicine© 2015 American Heart Association, Inc. Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process. Methods and Results:We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions. Conclusions: