Plasma membrane microdomains containing vesicular stomatitis virus M protein are separate from microdomains containing G protein and nucleocapsids

Immunogold electron microscopy and analysis were used to determine the organization of the major structural proteins of vesicular stomatitis virus (VSV) during virus assembly. We determined that matrix protein (M protein) partitions into plasma membrane microdomains in VSV-infected cells as well as in transfected cells expressing M protein. The sizes of the M-protein-containing microdomains outside the virus budding sites (50 to 100 nm) were smaller than those at sites of virus budding (approximately 560 nm). Glycoprotein (G protein) and M protein microdomains were not colocalized in the plasma membrane outside the virus budding sites, nor was M protein colocalized with microdomains containing the host protein CD4, which efficiently forms pseudotypes with VSV envelopes. These results suggest that separate membrane microdomains containing either viral or host proteins cluster or merge to form virus budding sites. We also determined whether G protein or M protein was colocalized with VSV nucleocapsid protein (N protein) outside the budding sites. Viral nucleocapsids were observed to cluster in regions of the cytoplasm close to the plasma membrane. Membrane-associated N protein was colocalized with G protein in regions of plasma membrane of approximately 600 nm. In contrast to the case for G protein, M protein was not colocalized with these areas of nucleocapsid accumulation. These results suggest a new model of virus assembly in which an interaction of VSV nucleocapsids with G-protein-containing microdomains is a precursor to the formation of viral budding sites.     

Molecular interactions between the photoreceptor G protein and rhodopsin

1. The visual transduction system of the vertebrate retina is a well-studied model for biochemical and molecular studies of signal transduction. The structure and function of rhodopsin, a prototypical G protein-coupled receptor, and transducin or Gt, the photoreceptor G protein, have been particularly well studied. Mechanisms of rhodopsin-Gt interaction are discussed in this review. 2. The visual pigment rhodopsin contains a chromophore, and thus conformational changes leading to activation can be monitored spectroscopically. A model of the conformational changes in the activated receptor is presented based on biophysical and biochemical data. 3. The current information on sites of interaction on receptors and cognate G proteins is summarized. Studies using synthetic peptides from amino acid sequences corresponding to Gt and rhodopsin have provided information on the sites of rhodopsin-Gt interaction. Synthetic peptides from the carboxyl terminal region of alpha t mimic Gt by stabilizing the active conformation of rhodopsin, Metarhodopsin II. 4. The conformation of one such peptide when it is bound to Metarhodopsin II was determined by 2D NMR. The model based on the NMR data was tested using peptide analogs predicted to stabilize or break the structure. These studies yield molecular insight into why toxin-treated and mutant G proteins are uncoupled from receptors.     

Interaction between Brucella abortus and cellular prion protein in lipid raft microdomains

A wide variety of pathogens employ lipid raft microdomains to infect host cells. Here, we review selected aspects of interaction between Brucella abortus and cellular prion protein, one of the lipid raft-associated molecules on the plasma membrane, when bacteria infect macrophages, and discuss the correlates of proliferation in mice.     

Role of lipid microdomains in TLR-mediated signalling

Over the last twenty years, evidence has been provided that the plasma membrane is partitioned with microdomains, laterally mobile in the bilayer, providing the necessary microenvironment to specific membrane proteins for signalling pathways to be initiated. We discuss here the importance of such microdomains for Toll-like receptors (TLR) localization and function. First, lipid microdomains favour recruitment and clustering of the TLR machinery partners, i.e. receptors and co-receptors previously identified to be required for ligand recognition and signal transmission. Further, the presence of the so-called Cholesterol Recognition Amino-Acid Consensus (CRAC) sequences in the intracellular juxtamembrane domain of several Toll-like receptors suggests a direct role of cholesterol in the activation process. This article is part of a Special Issue entitled: Lipid-protein interactions.     

LDL uptake by Leishmania amazonensis: involvement of membrane lipid microdomains

Leishmania amazonensis lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this lipid from the host environment. In this study we show that the L. amazonensis takes up and metabolizes human LDL(1) particles in both a time and dose-dependent manner. This mechanism implies the presence of a true LDL receptor because the uptake is blocked by both low temperature and by the excess of non-labelled LDL. This receptor is probably associated with specific microdomains in the membrane of the parasite, such as rafts, because this process is blocked by methyl-β-cyclodextrin (MCBD). Cholesteryl ester fluorescently-labeled LDL (BODIPY-cholesteryl-LDL) was used to follow the intracellular distribution of this lipid. After uptake it was localized in large compartments along the parasite body. The accumulation of LDL was analyzed by flow cytometry using FITC-labeled LDL particles. Together these data show for the first time that L. amazonensis is able to compensate for its lack of lipid synthesis through the use of a lipid importing machinery largely based on the uptake of LDL particles from the host. Understanding the details of the molecular events involved in this mechanism may lead to the identification of novel targets to block Leishmania infection in human hosts.     

Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains

Supported cell-membrane sheets are promising in vitro systems to investigate the properties of membranes with native protein/lipid composition, in particular their sub-compartmentalization and the differential localization of proteins associated to them. While such studies are usually performed using static microscopy techniques, we demonstrate here the potential offered by dynamic diffusion measurements. Whereas the overall fluidity of the lipid bilayer was preserved, the preparation of the membrane sheets led to the selective immobilization of extracellular and transmembrane (TM) glycosylated proteins and the anchored proteins/lipids associated with them. Taking advantage of this, we investigated the association of the G protein Gq with TM proteins, in particular G-protein coupled receptors (GPCRs), by monitoring the changes in diffusion occurring after preparation of the supported membranes. Two fluorescently tagged Galphaq proteins were constructed, which remained either mostly monomeric in the plasma membrane or associated with Gbetagamma in heterotrimers. While both constructs diffused similarly in living cells, the preparation of the supported membranes led to the selective immobilization of the heterotrimers with minimal changes of the diffusion of the monomeric Galphaq. The diverse mobility of monomeric and heterotrimeric Galphaq was a result of their different lipid anchors as demonstrated by monitoring the diffusion of the corresponding anchors alone. We propose that the immobilization of the heterotrimer was caused by its partitioning inside membrane microdomains surrounding GPCRs.     

Cholesterol rich lipid raft microdomains are gateway for acute phase protein,SERPINA1

Cholesterol is the most abundant lipid component of the plasma membrane, and thus the equilibrium between free cholesterol and raft cholesterol act as a determinant of raft function and cell signalling. The mechanisms that regulate the lipid raft cholesterol levels are largely unknown. Here we demonstrate that SERPINA1 (α1-antitrypsin), an acute phase protein and the classical neutrophil elastase inhibitor, is localized within lipid rafts in primary human monocytes in vitro. SERPINA1 association with monocytes is inhibited by cholesterol depleting/efflux-stimulating agents (nystatin, filipin, MβCD (methyl-beta-cyclodextrin) and oxidized low-density lipoprotein (oxLDL) and conversely, enhanced by free cholesterol. Furthermore, SERPINA1/monocyte association per se depletes lipid raft cholesterol as characterized by the activation of extracellular signal-regulated kinase 2, formation of cytosolic lipid droplets, and a complete inhibition of oxLDL uptake by monocytes. Our findings for the first time highlight that the entry and cell association of SERPINA1 is dependent on lipid raft cholesterol and that SERPINA1 depletes lipid raft cholesterol.     

Regulation of integrin growth factor interactions in oligodendrocytes by lipid raft microdomains

Individual growth factors can regulate multiple aspects of behavior within a single cell during differentiation, with each signaling pathway controlled independently and also responsive to other receptors such as cell surface integrins. The mechanisms by which this is achieved remain poorly understood. Here we use myelin-forming oligodendrocytes and their precursors to examine the role of lipid rafts, cholesterol and sphingolipid-rich microdomains of the cell membrane implicated in cell signaling. In these cells, the growth factor PDGF has sequential and independent roles in proliferation and survival. We show that the oligodendrocyte PDGFalpha receptor becomes sequestered in a raft compartment at the developmental stage when PDGF ceases to promote proliferation, but is now required for survival. We also show that laminin-2, which is expressed on axons in the CNS and which provides a target-dependent signal for oligodendrocyte survival by amplification of PDGFalphaR signaling, induces clustering of the laminin binding integrin alpha6beta1 with the PDGFalphaR-containing lipid raft domains. This extracellular matrix-induced colocalization of integrin and growth factor receptor generates a signaling environment within the raft for survival-promoting PI3K/Akt activity. These results demonstrate novel signaling roles for lipid rafts that ensure the separation and amplification of growth factor signaling pathways during development.     

Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalisation by macrophages

Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc–macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc–macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc–macrophage interaction. Using optical tweezers to study cell‐to‐cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide‐glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1^?/? mice) showed a deficient ability to interact with Hc. Coincubation of oligo‐GM1 and treatment with Cholera toxin Subunit B, which recognises the ganglioside GM1, also reduced Hc association. Although purified GM1 did not alter Hc binding, treatment with P4 significantly increased the time required for Hc binding to macrophages. The content of CD18 was displaced from lipid microdomains in B4galnt1^?/? macrophages. In addition, macrophages with reduced CD18 expression (CD18^low) were associated with Hc at levels similar to wild‐type cells. Finally, CD11b and CD18 colocalised with GM1 during Hc–macrophage interaction. Our results indicate that lipid rafts and particularly complex gangliosides that reside in lipid rafts stabilise Hc–macrophage adhesion and mediate efficient internalisation during histoplasmosis.     

Caveolae and non-caveolae lipid raft microdomains of human umbilical vein endothelial cells contain utrophin-associated protein complexes

Several studies have shown the importance of dystrophin-associated protein complex in the development of muscular dystrophies and dilated cardiomyopathy associated to vascular dysfunction. In vascular endothelium, dystrophin is substituted for utrophin (autosomal homolog of dystrophin); however, its role in this tissue is unknown. Therefore, it is important to obtain a more extensive knowledge of utrophin and its associated proteins in endothelial cells. In a previous study, we demonstrated the presence of utrophin-associated protein complex (UAPC) in human umbilical vein endothelial cells HUVEC, which interacts with caveolin-1 (Cav-1) and endothelial nitric oxide synthase (eNOS). Also, some of our observations suggested the presence of this complex in distinct membrane domains. Therefore, the aim of this study was to analyze the presence of the UAPC in caveolae and non-caveolae lipid rafts domains of HUVEC at baseline and with a mechanical stimulus. It was demonstrated, by subcellular fractionation and co-immunoprecipitation assays, the association of UAPC with Cav-1 and eNOS in caveolae domains, as well as its interaction with eNOS in non-caveolae lipid raft domains. Additionally, it was also observed that mechanical stress on endothelial cells induced activation and release of eNOS from both caveolae and non-caveolae lipid raft associated to UAPC. Together these results suggest that UAPC located in caveolae and non-caveolae lipid raft domains of HUVECs may have a mechanosensory function that could participate in the control of eNOS activity.     

Transition from nanodomains to microdomains induced by exposure of lipid monolayers to air

The morphology of monolayers prepared from ternary lipid mixtures that have coexisting fluid phases has been examined by atomic force microscopy for samples transferred to mica before and after exposure to air. Mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol with either egg sphingomyelin or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied at several surface pressures. Both lipid mixtures have a combination of small islands and large microdomains at low surface pressure (5-10 mN/m) for monolayers deposited in either air or nitrogen. By contrast, monolayers have small interconnected nanodomains when deposited under nitrogen at 30 mN/m but mixtures of large microdomains and small nanodomains when transferred after exposure to air. These results are consistent with an earlier report that concluded that the formation of large domains at high surface pressures (>30 mN/m) for monolayers exposed to air is caused by lipid oxidation. However, the higher spatial resolution available with atomic force microscopy indicates that exposure of the monolayers to air leads to an increase in the size of preexisting nanodomains, rather than a change in the miscibility pressure. Examination of changes in surface morphology as a function of surface pressure demonstrate a gradual evolution in size and surface coverage for both nano- and microdomains, before formation of a network of interconnected nanodomains. Similar studies for binary mixtures in the absence of cholesterol indicate that lipid oxidation results in analogous changes in domain size for monolayers with coexisting gel and fluid phases. These results illustrate the importance of using techniques capable of probing the nanoscale organization of membranes.     

On the evaluation of photoreceptor properties by micro-fluorimetric measurements of fluorochrome diffusion

By use of the microfluorimetric technique it is possible to study the diffusion of the fluorochrome di-dansylcystine (DDC) within isolated frog rod outer segments (ros) which are immobilysed in agarose gel. For this purpose, by a short hypotonic shock a leak is applied to one end of the ros. By this open end the DDC enters the rod and migrates through the whole outer segment. Following the propagation of the fluorescence boundary with time the cytoplasmatic diffusion constant can be determined if a Chromatographic model is used to allow for the considerable binding of DDC to the inner membrane surface. With a binding constant K=5·10^–4 cm the cytoplasmatic diffusion constant was found to be D= 1.3·10^–6cm²/s whereas D _g =2·10^–6cm²/s and D _r =3.5·10^–6cm²/s were found in agarose gel or ringer solution, respectively. Using the mobility reduction factor given by D/D _r 0.4 to calculate the cytoplasmatic conductivity an inner resistance per length of 1.7 M / could be calculated for a frog rod which is in good agreement with corresponding data obtained from electrophysiological measurements.     

Restricted lateral diffusion of luteinizing hormone receptors in membrane microdomains

Single particle tracking was used to evaluate lateral motions of individual FLAG-tagged human luteinizing hormone (LH) receptors expressed on CHO cells and native LH receptors on both KGN human granulosa-derived tumor cells and M17 human neuroblastoma cells before and after exposure to human chorionic gonadotropin (hCG). Compared with LH receptors on untreated cells, LH receptors on cells treated with 100 nm hCG exhibit restricted lateral diffusion and are confined in small, nanometer-scale, membrane compartments. Similar to LH receptors labeled with Au-hCG, LH receptors labeled with gold-deglycosylated hCG, an hCG antagonist, also exhibit restricted lateral diffusion and are confined in nanoscale membrane compartments on KGN cells treated with 100 nm hCG. LH receptor point mutants lacking potential palmitoylation sites remain in large compartments despite treatment with 100 nm hCG as do LH receptors on cells treated with cytochalasin D. Finally, both polarization homotransfer fluorescence resonance energy transfer imaging and photon counting histogram analysis indicate that treatment with hCG induces aggregation of YFP-coupled LH receptors stably expressed on CHO cells. Taken together, our results demonstrate that binding of hCG induces aggregation of LH receptors within nanoscale, cell surface membrane compartments, that hCG binding also affects the lateral motions of antagonist binding LH receptors, and that receptor surface densities must be considered in evaluating the extent of hormone-dependent receptor aggregation.     

Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy

The lateral motion of single fluorescence labeled lipid molecules was imaged in native cell membranes on a millisecond time scale and with positional accuracy of approximately 50 nm, using 'single dye tracing'. This first application of single molecule microscopy to living cells rendered possible the direct observation of lipid-specific membrane domains. These domains were sensed by a lipid probe with saturated acyl chains as small areas in a liquid-ordered phase: the probe showed confined but fast diffusion, with high partitioning (approximately 100-fold) and long residence time (approximately 13 s). The analogous probe with mono-unsaturated chains diffused predominantly unconfined within the membrane. With approximately 15 saturated probes per domain, the locations, sizes, shapes and motions of individual domains became clearly visible. Domains had a size of 0.7 micrometer (0.2-2 micrometer), covering approximately 13% of total membrane area. Both the liquid-ordered phase characteristics and the sizes of domains match properties of membrane fractions described as detergent-resistant membranes (DRMs), strongly suggesting that the domains seen are the in vivo correlate of DRMs and thus may be identified as lipid rafts.     

Control of the photosynthetic electron transport by PQ diffusion microdomains in thylakoids of higher plants

We investigate the role of plastoquinone (PQ) diffusion in the control of the photosynthetic electron transport. A control analysis reveals an unexpected flux control of the whole chain electron transport by photosystem (PS) II. The contribution of PSII to the flux control of whole chain electron transport was high in stacked thylakoids (control coefficient, CJ(PSII) =0.85), but decreased after destacking (CJ(PSII)=0.25). From an 'electron storage' experiment, we conclude that in stacked thylakoids only about 50 to 60% of photoreducable PQ is involved in the light-saturated linear electron transport. No redox equilibration throughout the membrane between fixed redox groups at PSII and cytochrome (cyt) bf complexes, and the diffusable carrier PQ is achieved. The data support the PQ diffusion microdomain concept by Lavergne et al. [J. Lavergne, J.-P. Bouchaud, P. Joliot, Biochim. Biophys. Acta 1101 (1992) 13-22], but we come to different conclusions about size, structure and size distribution of domains. From an analysis of cyt b6 reduction, as a function of PSII inhibition, we conclude that in stacked thylakoids about 70% of PSII is located in small domains, where only 1 to 2 PSII share a local pool of a few PQ molecules. Thirty percent of PSII is located in larger domains. No small domains were found in destacked thylakoids. We present a structural model assuming a hierarchy of specific, strong and weak interactions between PSII core, light harvesting complexes (LHC) II and cyt bf. Peripheral LHCII's may serve to connect PSII-LHCII supercomplexes to a flexible protein network, by which small closed lipid diffusion compartments are formed. Within each domain, PQ moves rapidly and shuttles electrons between PSII and cyt bf complexes in the close vicinity. At the same time, long range diffusion is slow. We conclude, that in high light, cyt bfcomplexes located in distant stromal lamellae (20 to 30%) are not involved in the linear electron transport.     

All-trans retinol in rod photoreceptor outer segments moves unrestrictedly by passive diffusion

The visual pigment protein of vertebrate rod photoreceptors, rhodopsin, contains an 11-cis retinyl moiety that is isomerized to all-trans upon light absorption. Subsequently, all-trans retinal is released from the protein and reduced to all-trans retinol, the first step in the recycling of rhodopsin's chromophore group through the series of reactions that constitute the visual cycle. The concentration of all-trans retinol in photoreceptor outer segments can be monitored from its fluorescence. We have used two-photon excitation (720 nm) of retinol fluorescence and fluorescence recovery after photobleaching to characterize the mobility of all-trans retinol in frog photoreceptor outer segments. Retinol produced after rhodopsin bleaching moved laterally in the disk membrane bilayer with an apparent diffusion coefficient of 2.5 +/- 0.3 micro m(2) s(-1). The diffusion coefficient of exogenously added retinol was 3.2 +/- 0.5 micro m(2) s(-1). These diffusion coefficients are in close agreement with those reported for lipids, suggesting that retinol is not tightly bound to protein sites that would be diffusing much more slowly in the plane of the membrane. In agreement with this interpretation, a fluorescent-labeled C-16 fatty acid diffused laterally with a similar diffusion coefficient, 2.2 +/- 0.2 micro m(2) s(-1). Retinol also moved along the length of the rod outer segment, with an apparent diffusion coefficient of 0.07 +/- 0.01 micro m(2) s(-1), again suggesting that it is not tightly bound to proteins that would confine it to the disks. The axial diffusion coefficient of exogenously added retinol was 0.05 +/- 0.01 micro m(2) s(-1). In agreement with passive diffusion, the rate of axial movement was inversely proportional to the square of the length of the rod outer segment. Diffusion of retinol on the plasma membrane of the outer segment can readily account for the measured value of the axial diffusion coefficient, as the plasma membrane comprises approximately 1% of the total outer-segment membrane. The values of both the lateral and axial diffusion coefficients are consistent with most of the all-trans retinol in the outer segments moving unrestricted and not being bound to carrier proteins. Therefore, and in contrast to other steps of the visual cycle, there does not appear to be any specialized processing for all-trans retinol within the rod outer segment.     

Lysosome membrane lipid microdomains: novel regulators of chaperone-mediated autophagy

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins in lysosomes. The limiting step of this type of autophagy is the binding of substrates to the lysosome-associated membrane protein type 2A (LAMP-2A). In this work, we identify a dynamic subcompartmentalization of LAMP-2A in the lysosomal membrane, which underlies the molecular basis for the regulation of LAMP-2A function in CMA. A percentage of LAMP-2A localizes in discrete lysosomal membrane regions during resting conditions, but it exits these regions during CMA activation. Disruption of these regions by cholesterol-depleting agents or expression of a mutant LAMP-2A excluded from these regions enhances CMA activity, whereas loading of lysosomes with cholesterol significantly reduces CMA. Organization of LAMP-2A into multimeric complexes, required for translocation of substrates into lysosomes via CMA, only occurs outside the lipid-enriched membrane microdomains, whereas the LAMP-2A located within these regions is susceptible to proteolytic cleavage and degradation. Our results support that changes in the dynamic distribution of LAMP-2A into and out of discrete microdomains of the lysosomal membrane contribute to regulate CMA.     

Segregation of heterotrimeric G proteins in cell surface microdomains. G(q) binds caveolin to concentrate in caveolae, whereas G(i) and G(s) target lipid rafts by default

Select lipid-anchored proteins such as glycosylphosphatidylinositol (GPI)-anchored proteins and nonreceptor tyrosine kinases may preferentially partition into sphingomyelin-rich and cholesterol-rich plasmalemmal microdomains, thereby acquiring resistance to detergent extraction. Two such domains, caveolae and lipid rafts, are morphologically and biochemically distinct, contain many signaling molecules, and may function in compartmentalizing cell surface signaling. Subfractionation and confocal immunofluorescence microscopy reveal that, in lung tissue and in cultured endothelial and epithelial cells, heterotrimeric G proteins (G(i), G(q), G(s), and G(betagamma)) target discrete cell surface microdomains. G(q) specifically concentrates in caveolae, whereas G(i) and G(s) concentrate much more in lipid rafts marked by GPI-anchored proteins (5' nucleotidase and folate receptor). G(q), apparently without G(betagamma) subunits, stably associates with plasmalemmal and cytosolic caveolin. G(i) and G(s) interact with G(betagamma) subunits but not caveolin. G(i) and G(s), unlike G(q), readily move out of caveolae. Thus, caveolin may function as a scaffold to trap, concentrate, and stabilize G(q) preferentially within caveolae over lipid rafts. In N2a cells lacking caveolae and caveolin, G(q), G(i), and G(s) all concentrate in lipid rafts as a complex with G(betagamma). Without effective physiological interaction with caveolin, G proteins tend by default to segregate in lipid rafts. The ramifications of the segregated microdomain distribution and the G(q)-caveolin complex without G(betagamma) for trafficking, signaling, and mechanotransduction are discussed.     

Proteomic analysis of lipid microdomains from lipopolysaccharide-activated human endothelial cells

The endothelium plays a critical role in orchestrating the inflammatory response seen during sepsis. Many of the inflammatory effects of Gram-negative sepsis are elicited by lipopolysaccharide (LPS), a glycolipid component of bacterial cell walls. Lipid-rich microdomains have been shown to concentrate components of the LPS signaling system. However, much remains to be learned about which proteins are constituents of lipid microdomains, and how these are regulated following cell activation. Progress in this area would be accelerated by employing global proteomic analyses, but the hydrophobicity of membrane proteins presents an analytical barrier to the effective application of such approaches. Herein, we describe a method to isolate detergent-resistant membranes from endothelial cells, and prepare these samples for proteomic analysis in a way that is compatible with subsequent separations and mass spectrometric (MS) analysis. In the application of these sample preparation and MS analyses, 358 proteins from the lipid-rich microdomains of LPS-activated endothelial cell membranes have been identified of which half are classified as membrane proteins by Gene Ontology. We also demonstrate that the sample preparation method used for solubilization and trypsin digestion of lipid-rich microdomains renders the membrane spanning sequences of transmembrane proteins accessible for endoproteolytic hydrolysis. This analysis sets the analytical foundation for an in-depth probing of LPS signaling in endothelial cells.     

Resolving the kinetics of lipid,protein and peptide diffusion in membranes

Abstract

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.