Size Of Lipid Rafts Research Articles

Docosahexaenoic Acid Controls Pulmonary Macrophage Lipid Raft Size and Inflammation

BackgroundDocosahexaenoic acid (DHA) controls the biophysical organization of plasma membrane sphingolipid/cholesterol-enriched lipid rafts to exert anti-inflammatory effects, particularly in lymphocytes. However, the impact of DHA on the spatial arrangement of alveolar macrophage lipid rafts and inflammation is unknown. ObjectivesThe primary objective was to determine how DHA controls lipid raft organization and function of alveolar macrophages. As proof-of-concept, we also investigated DHA’s anti-inflammatory effects on select pulmonary inflammatory markers with a murine influenza model. MethodsMH-S cells, an alveolar macrophage line, were treated with 50 μM DHA or vehicle control and were used to study plasma membrane molecular organization with fluorescence-based methods. Biomimetic membranes and coarse grain molecular dynamic (MD) simulations were employed to investigate how DHA mechanistically controls lipid raft size. qRT-PCR, mass spectrometry, and ELISAs were used to quantify downstream inflammatory signaling transcripts, oxylipins, and cytokines, respectively. Lungs from DHA-fed influenza-infected mice were analyzed for specific inflammatory markers. ResultsDHA increased the size of lipid rafts while decreasing the molecular packing of the MH-S plasma membrane. Adding a DHA-containing phospholipid to a biomimetic lipid raft-containing membrane led to condensing, which was reversed with the removal of cholesterol. MD simulations revealed DHA nucleated lipid rafts by driving cholesterol and sphingomyelin into rafts. Downstream of the plasma membrane, DHA lowered the concentration of select inflammatory transcripts, oxylipins, and IL-6 secretion. DHA lowered pulmonary Il6 and Tnf-α mRNA expression and increased anti-inflammatory oxylipins of influenza-infected mice. ConclusionsThe data suggest a model in which the localization of DHA acyl chains to nonrafts is driving sphingomyelin and cholesterol molecules into larger lipid rafts, which may serve as a trigger to impede signaling and lower inflammation. These findings also identify alveolar macrophages as a target of DHA and underscore the anti-inflammatory properties of DHA for lung inflammation.

Soyasaponin A1 inhibits the lipid raft recruitment and dimerization of TLR4, MyD88, and TRIF by maintaining cholesterol homeostasis in palmitic acid-stimulated inflammatory Raw264.7 macrophage cell line

The mechanisms of soyasaponin A1 (SS-A1) on antagonizing inflammation via regulating the lipid raft-mediated toll-like receptor 4 (TLR4) signaling are not completely understood. In this study, SS-A1 inhibited palmitic acid (PA)-induced recruitments of TLR4 and its adaptor molecules into lipid rafts in Raw264.7 macrophage cell line by using ultracentrifugation and confocal microscopy. SS-A1 also suppressed the PA-caused dimerization of TLR4 with and its adaptor molecules by applying immunoprecipitation and fluorescence resonance energy transfer. Meanwhile, SS-A1 modified the formation, clustering, and size of lipid rafts and decreased cholesterol in lipid rafts. Cholesterol replenishment abrogated SS-A1’s inhibition on the lipid rafts recruitment and dimerization of TLR4 and its adaptor molecules and its anti-inflammatory activity. Additionally, SS-A1 inhibited the mature sterol regulatory element-binding protein 2 and increases ATP-binding cassette transporter A1. Collectively, SS-A1 inhibits the lipid raft recruitment and dimerization of TLR4 and its adaptor molecules by maintaining cholesterol homeostasis in PA-stimulated inflammatory Raw264.7 macrophage cell line.

Determination of the size of lipid rafts studied through single-molecule FRET simulations

Fluorescence resonance energy transfer (FRET) is a high-resolution technique that allows the characterization of spatial and temporal properties of biological structures and mechanisms. In this work, we developed an in silico single-molecule FRET methodology to study the dynamics of fluorophores inside lipid rafts. We monitored the fluorescence of a single acceptor molecule in the presence of several donor molecules. By looking at the average fluorescence, we selected events with single acceptor and donor molecules, and we used them to determine the raft size in the range of 5–16 nm. We conclude that our method is robust and insensitive to variations in the diffusion coefficient, donor density, or selected fluorescence threshold.

Soyasaponin A1 Inhibits the Lipid Raft Recruitment and Dimerization of TLR4, MyD88 and TRIF in Palmitate-Stimulated Inflammatory Macrophages

Abstract Objectives Soyasaponin A1 (SSA1) was previously shown to inhibit the palmitate (PA)-induced inflammation via regulating the toll-like receptor 4 (TLR4) signaling in macrophages. Since the lipid raft recruitment and dimerization of TLR4 and its downstream adaptor molecules are vital for PA-initiated TLR4 signaling, we explored whether this process would be modulated by SSA1. Methods Murine macrophage RAW264.7 were stimulated with PA (200 μmol/L) in the presence or absence of SSA1 (40 μmol/L). The lipid raft fractions were separated by sucrose density gradient ultracentrifugation and immunoblotted with anti-flotillin-1, anti-TLR4, anti-myeloid differentiation primary response protein 88 (MyD88), or anti-Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) antibody. Lipid rafts, TLR4, MyD88 and TRIF were fluorescently labeled and analyzed by confocal microscopy to visualize the recruitment of molecules into lipid rafts and investigate the clustering and size of lipid rafts. The complexes of TLR4/MyD88 and TLR4/TRIF were immunoprecipitated by anti-TLR4 antibody first and then immunoblotted by anti-MyD88 or anti-TRIF antibody. Results PA-induced recruitment of TLR4, MyD88 and TRIF into fractions enriched with lipid rafts marker flotillin-1 was inhibited (P < 0.05) by SSA1. Meanwhile, the PA-induced co-localization of TLR4, MyD88, and TRIF with lipid rafts was also decreased (P < 0.05) by SSA1 as visualized by confocal immunofluorescence microscopy. Furthermore, the PA-induced clustering of lipid rafts was diminished (P < 0.05) by SSA1. However, the PA-decreased size of lipid rafts was increased (P < 0.05) by SSA1. The formation of TLR4/MyD88 and TLR4/TRIF complexes was suppressed (P < 0.05) by SSA1, whereas the protein expressions of TLR4, MyD88 and TRIF were not changed (P > 0.05) by SSA1 in PA-stimulated macrophages. Conclusions SSA1 inhibits the recruitment of TLR4 and its adaptor molecules (MyD88 and TRIF) into lipid raft as well as their dimerization (TLR4/MyD88 and TLR4/TRIF) in PA-stimulated inflammatory macrophages. Funding Sources This work was supported by grants from National Natural Science Foundation of China (NSFC).

Regulation of Phagocytosis in Macrophages by Membrane Ethanolamine Plasmalogens.

Macrophages, as professional phagocytes of the immune system, possess the ability to detect and clear invading pathogens and apoptotic cells through phagocytosis. Phagocytosis involves membrane reorganization and remodeling events on the cell surface, which play an essential role in innate immunity and tissue homeostasis and the control of inflammation. In this work, we report that cells deficient in membrane ethanolamine plasmalogen demonstrate a reduced capacity to phagocytize opsonized zymosan particles. Amelioration of plasmalogen deficiency in these cells by incubation with lysoplasmalogen results in a significant augmentation of the phagocytic capacity of the cells. In parallel with these increases, restoration of plasmalogen levels in the cells also increases the number and size of lipid rafts in the membrane, reduces membrane fluidity down to levels found in cells containing normal plasmalogen levels, and improves receptor-mediated signaling. Collectively, these results suggest that membrane plasmalogen level determines characteristics of the plasma membrane such as fluidity and the formation of microdomains that are necessary for efficient signal transduction leading to optimal phagocytosis by macrophages.

Docosahexaenoic acid regulates the formation of lipid rafts: A unified view from experiment and simulation

Docosahexaenoic acid (DHA, 22:6) is an n−3 polyunsaturated fatty acid (n−3 PUFA) that influences immunological, metabolic, and neurological responses through complex mechanisms. One structural mechanism by which DHA exerts its biological effects is through its ability to modify the physical organization of plasma membrane signaling assemblies known as sphingomyelin/cholesterol (SM/chol)-enriched lipid rafts. Here we studied how DHA acyl chains esterified in the sn-2 position of phosphatidylcholine (PC) regulate the formation of raft and non-raft domains in mixtures with SM and chol on differing size scales. Coarse grained molecular dynamics simulations showed that 1-palmitoyl-2-docosahexaenoylphosphatylcholine (PDPC) enhances segregation into domains more than the monounsaturated control, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC). Solid state 2H NMR and neutron scattering experiments provided direct experimental evidence that substituting PDPC for POPC increases the size of raft-like domains on the nanoscale. Confocal imaging of giant unilamellar vesicles with a non-raft fluorescent probe revealed that POPC had no influence on phase separation in the presence of SM/chol whereas PDPC drove strong domain segregation. Finally, monolayer compression studies suggest that PDPC increases lipid-lipid immiscibility in the presence of SM/chol compared to POPC. Collectively, the data across model systems provide compelling support for the emerging model that DHA acyl chains of PC lipids tune the size of lipid rafts, which has potential implications for signaling networks that rely on the compartmentalization of proteins within and outside of rafts.

Interaction of Soybean 7S Globulin Peptide with Cell Membrane Model via Isothermal Titration Calorimetry, Quartz Crystal Microbalance with Dissipation, and Langmuir Monolayer Study.

To understand the underlying molecular mechanism of the cholesterol-lowering effect of soybean 7S globulins, the interactions of their pepsin-released peptides (7S-peptides) with cell membrane models consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and cholesterol (CHOL) were systematically studied. The results showed that 7S-peptides were bound to DPPC/DOPC/CHOL liposomes mainly through van der Waals forces and hydrogen bonds, and the presence of higher CHOL concentrations enhanced the binding affinity (e.g., DPPC/DOPC/CHOL = 1:1:0, binding ratio = 0.114; DPPC/DOPC/CHOL = 1:1:1, binding ratio = 2.02). Compression isotherms indicated that the incorporation of 7S-peptides increased the DPPC/DOPC/CHOL monolayer fluidity and the lipid raft size. The presence of CHOL accelerated the 7S-peptide accumulation on lipid rafts, which could serve as platforms for peptides to develop into β-sheet rich structures. These results allow us to hypothesize that 7S-peptides may indirectly influence membrane protein functions via altering the membrane organization in the enterocytes.

Raft-Like Domains are Driven Together by Pufa

Fish oils rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as docosahexaenoic acid (DHA, 22:6), have a wide variety of health benefits but a complete molecular mechanism is yet to be elucidated. One model postulates that n-3 PUFAs are incorporated into the plasma membrane and reorganize lipid rafts. To investigate this model, we compared lipid bilayers composed of PDPC (1-palmitoyl-2-docosahexaenoylphosphatidylcholine) or POPC (1-palmitoyl-2-oleoylphosphatidylcholine) in mixtures with raft-forming, SM (sphingomyelin) and cholesterol (1:1:1 mol). A combination of solid state 2H NMR, confocal imaging and coarse grain MD simulations were employed. In the PDPC mixture, individual NMR signals assigned to deuterated analogs of each lipid in ordered raft-like domains and disordered non-raft domains were resolved in the spectra recorded for aqueous multilamellar dispersions. In the POPC mixture, by contrast, separate signals were not resolved. Our interpretation is that PDPC promotes the formation of larger domains between which the diffusion-mediated exchange of lipids is slow, whereas the domains are smaller with POPC and the exchange of lipids between them is fast. This interpretation is supported by imaging studies of GUV (giant unilamellar vesicles) and MD simulations. Controlling the size of lipid rafts may be, we propose, an underlying role for PUFA in the plasma membrane.

Lipid Raft Size and Lipid Mobility in Non-raft Domains Increase during Aging and Are Exacerbated in APP/PS1 Mice Model of Alzheimer's Disease. Predictions from an Agent-Based Mathematical Model.

A connection between lipid rafts and Alzheimer's disease has been studied during the last decades. Mathematical modeling approaches have recently been used to correlate the effects of lipid composition changes in the physicochemical properties of raft-like membranes. Here we propose an agent based model to assess the effect of lipid changes in lipid rafts on the evolution and progression of Alzheimer's disease using lipid profile data obtained in an established model of familial Alzheimer's disease. We have observed that lipid raft size and lipid mobility in non-raft domains are two main factors that increase during age and are accelerated in the transgenic Alzheimer's disease mouse model. The consequences of these changes are discussed in the context of neurotoxic amyloid β production. Our agent based model predicts that increasing sterols (mainly cholesterol) and long-chain polyunsaturated fatty acids (LCPUFA) (mainly DHA, docosahexaenoic acid) proportions in the membrane composition might delay the onset and progression of the disease.

Line Tension and Phase Separation of a Four-Component Phospholipid Bilayer

When phospholipid bilayers have coexisting liquid disordered (Ld) and liquid ordered (Lo) phases, Ld/Lo line tension has strong control over the size of the coexisting liquid domains. In order to minimize light-induced artifacts that involve lipid reactivity, we chose a lipid mixture with minimal double bonds, DSPC, diphytanoyl-PC, DLPC, and cholesterol. Giant unilamellar vesicles (GUVs) were generated using the electroswelling technique and visualized with an inverted fluorescent microscope. Line tension was determined from the boundary fluctuations between Ld and Lo domains using Flicker Spectroscopy. The Ld/Lo line tension is measured for different GUV compositions within the Ld + Lo coexistence region. The ratio of diphytanoyl-PC and DLPC is then adjusted to control the size of coexisting domains. Different fluorescent dyes are compared to find the minimal light-induced artifacts during the line tension measurements. The results presented in this study provide a more detailed picture of how a cell might control lipid raft size.

Keratin impact on PKCδ- and ASMase-mediated regulation of hepatocyte lipid raft size - implication for FasR-associated apoptosis.

Keratins are epithelial cell intermediate filament (IF) proteins that are expressed as pairs in a cell-differentiation-regulated manner. Hepatocytes express the keratin 8 and 18 pair (denoted K8/K18) of IFs, and a loss of K8 or K18, as in K8-null mice, leads to degradation of the keratin partner. We have previously reported that a K8/K18 loss in hepatocytes leads to altered cell surface lipid raft distribution and more efficient Fas receptor (FasR, also known as TNFRSF6)-mediated apoptosis. We demonstrate here that the absence of K8 or transgenic expression of the K8 G62C mutant in mouse hepatocytes reduces lipid raft size. Mechanistically, we find that the lipid raft size is dependent on acid sphingomyelinase (ASMase, also known as SMPD1) enzyme activity, which is reduced in absence of K8/K18. Notably, the reduction of ASMase activity appears to be caused by a less efficient redistribution of surface membrane PKCδ toward lysosomes. Moreover, we delineate the lipid raft volume range that is required for an optimal FasR-mediated apoptosis. Hence, K8/K18-dependent PKCδ- and ASMase-mediated modulation of lipid raft size can explain the more prominent FasR-mediated signaling resulting from K8/K18 loss. The fine-tuning of ASMase-mediated regulation of lipid rafts might provide a therapeutic target for death-receptor-related liver diseases.

Mechanical Properties of Nanoscopic Lipid Domains.

The lipid raft hypothesis presents insights into how the cell membrane organizes proteins and lipids to accomplish its many vital functions. Yet basic questions remain about the physical mechanisms that lead to the formation, stability, and size of lipid rafts. As a result, much interest has been generated in the study of systems that contain similar lateral heterogeneities, or domains. In the current work we present an experimental approach that is capable of isolating the bending moduli of lipid domains. This is accomplished using neutron scattering and its unique sensitivity to the isotopes of hydrogen. Combining contrast matching approaches with inelastic neutron scattering, we isolate the bending modulus of ∼13 nm diameter domains residing in 60 nm unilamellar vesicles, whose lipid composition mimics the mammalian plasma membrane outer leaflet. Importantly, the bending modulus of the nanoscopic domains differs from the modulus of the continuous phase surrounding them. From additional structural measurements and all-atom simulations, we also determine that nanoscopic domains are in-register across the bilayer leaflets. Taken together, these results inform a number of theoretical models of domain/raft formation and highlight the fact that mismatches in bending modulus must be accounted for when explaining the emergence of lateral heterogeneities in lipid systems and biological membranes.

Lenalidomide Induces Lipid Raft Assembly to Enhance Erythropoietin Receptor Signaling in Myelodysplastic Syndrome Progenitors

Erythropoietin receptor (EpoR) signaling is impaired in patients with Myelodysplastic Syndromes (MDS) despite appropriate growth factor production and cellular receptor display. We previously reported that EpoR signaling is dependent upon receptor localization within membrane lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity (McGraw KL, et al. PLoS One 2012). Here, we show that MDS erythroid progenitors display markedly diminished raft assembly (p=0.005) and smaller raft aggregates (p=0.023) compared to normal controls. Because lenalidomide triggers raft coalescence in T-lymphocytes to promote immune synapse formation, we assessed the effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lipid rafts were isolated from UT7 cells using ultracentrifugation and identified by GM-1 dot blot and Lyn kinase western blot. Lenalidomide rapidly induced lipid raft formation in UT7 cells which was confirmed by confocal microscopy visualization of GM-1 fluorescence. Lenalidomide also significantly induced lipid raft formation in pooled MDS erythroid progenitors (CD71+, cKit+) from 11 patients [mean raft size, control (n=569) vs. lenalidomide treatment (n=659), p<0.001], with no significant change observed in pooled erythroids from 3 normal donors (n= 327 for control and n=365 for lenalidomide treated, p=0.37). Interestingly, lipid rafts were significantly larger in erythroid progenitors from patients who responded (n=5) to lenalidomide treatment compared to non-responders (n=3) (75.52 ±13.68 vs. 35.85 ±8.56, p=0.02). Although lenalidomide increased raft size in erythroid progenitors from both responders (p=0.0007) and non-responders (p=0.013), mean raft size was greater in erythroid precursors from responding patients after treatment (p=0.11). Increased raft aggregation after lenalidomide treatment was accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase, whereas the JAK2 phosphatase, CD45, a negative regulator of EpoR signaling, was displaced from raft fractions. Incubation with lenalidomide prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 cells and primary MDS erythroid precursors. Bone marrow specimens from 12 non-del(5q) IPSS lower risk, lenalidomide naive MDS patients were analyzed by flow cytometry to compare changes in STAT5 phosphorylation in response to Epo stimulation in the presence or absence of lenalidomide. We found a 79.1% mean increase in p-STAT5 mean fluorescence intensity (MFI 95th percentile) in CD45dim, CD71high, Glylow erythroid precursors in 7 of the 12 patient specimens following lenalidomide exposure. Furthermore, increased STAT5 phosphorylation was accompanied by increased DNA binding of the transcription factor in UT7 cells, and improved erythroid colony forming capacity in both UT7 and primary MDS bone marrow cells. Raft induction was associated with F-actin polymerization that was blocked by Rho kinase inhibition and confirmed by lipid raft isolation followed by dot blot with western blot and confocal microscopy. These data provide new insight into abnormalities in the EpoR signaling platform that underlie impaired Epo responsiveness in MDS erythroid precursors. Our findings that deficient raft integrity impairs EpoR signaling provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS. These data also warrant investigation in a larger data set to determine whether lipid raft size may be a predictive biomarker for lenalidomide response. DisclosuresList:Celgene: Consultancy.

Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes.

We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells.

Effect of Lipid Recycling on the Finite Size of Lipid Rafts in Symmetric and Asymmetric Bilayers

The plasma membrane of eukaryotic cells is composed of various types of lipids and is thought to form lipid rafts, which are nanoscale domains, as a result of phase separation. It is well documented that giant unilamellar vesicles, composed of a saturated lipid, an unsaturated lipid and cholesterol, exhibit phase separation with large micron-scale domains, limited in size only by the finite size of the vesicle itself. In contrast, lipid domains in the plasma membrane are orders of magnitude smaller. The cause of the finite nanoscale size of these lipid rafts in biological systems remains poorly understood. The present research uses molecular dynamics of an implicit-solvent molecular model of self-assembled multi-component lipid membranes to investigate lipid recycling due to trafficking. In particular, the goal of this research is to investigate whether trafficking affects the size of lipid rafts. Our systematic simulations for varying recycling rate as well as the transbilayer lipid distribution and lipid composition in each leaflet show that the system achieves a non-equilibrium steady state with a microphase separation. The length scale of the micro-phase separated domains is found to decrease with increasing the rate of recycling. An extrapolation of our data to recycling rates comparable to biological values lead to an average domain size approaching the size of lipid rafts in plasma membranes.

Notch3 is activated by chronic hypoxia and contributes to the progression of human prostate cancer

Prostate cancer (PC) is still the second cause of cancer-related death among men. Although patients with metastatic presentation have an ominous outcome, the vast majority of PCs are diagnosed at an early stage. Nonetheless, even among patients with clinically localized disease the outcome may vary considerably. Other than androgen sensitivity, little is known about which other signaling pathways are deranged in aggressive, localized cancers. The elucidation of such pathways may help to develop innovative therapies aimed at specific molecular targets. We report that in a hormone-sensitive PC cell line, LNCaP, Notch3 was activated by hypoxia and sustained cell proliferation and colony formation in soft agar. Hypoxia also modulated cellular cholesterol content and the number and size of lipid rafts, causing a coalescence of small rafts into bigger clusters; under this experimental condition, Notch3 migrated from the non-raft into the raft compartment where it colocalized with the γ-secretase complex. We also looked at human PC biopsies and found that expression of Notch3 positively correlated with Gleason score and with expression of carbonic anhydrase IX, a marker of hypoxia. In conclusion, hypoxia triggers the activation of Notch3, which, in turn, sustains proliferation of PC cells. Notch3 pathway represents a promising target for adjuvant therapy in patients with PC.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking1,2 but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases3. Yet their existence is still a matter of controversy4,5. Indeed, lipid raft size has been estimated to be around 20 nm6, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized7,8. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts9,10. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models11. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently12. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change12.

Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts

BackgroundLipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents.ResultsDetergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen.ConclusionsFindings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

Regulating the Size and Stabilization of Lipid Raft-like Domains and Using Calcium Ions as Their Probe

We apply a means to probe, stabilize, and control the size of lipid raft-like domains in vitro. In biomembranes the size of lipid rafts is ca. 10-30 nm. In vitro, mixing saturated and unsaturated lipids results in microdomains, which are unstable and coalesce. This inconsistency is puzzling. It has been hypothesized that biological line-active surfactants reduce the line tension between saturated and unsaturated lipids and stabilize small domains in vivo. Using solution X-ray scattering, we studied the structure of binary and ternary lipid mixtures in the presence of calcium ions. Three lipids were used: saturated, unsaturated, and a hybrid (1-saturated-2-unsaturated) lipid that is predominant in the phospholipids of cellular membranes. Only membranes composed of the saturated lipid can adsorb calcium ions, become charged, and therefore considerably swell. The selective calcium affinity was used to show that binary mixtures, containing the saturated lipid, phase separated into large-scale domains. Our data suggests that by introducing the hybrid lipid to a mixture of the saturated and unsaturated lipids, the size of the domains decreased with the concentration of the hybrid lipid, until the three lipids could completely mix. We attribute this behavior to the tendency of the hybrid lipid to act as a line-active cosurfactant that can easily reside at the interface between the saturated and the unsaturated lipids and reduce the line tension between them. These findings are consistent with a recent theory and provide insight into the self-organization of lipid rafts, their stabilization, and size regulation in biomembranes.