Spectroscopic signatures of bilayer ordering in native biological membranes

Membrane proteins and polycyclic lipids like cholesterol and hopanoids coordinate phospholipid bilayer ordering. This phenomenon manifests as partitioning of the liquid crystalline phase into liquid-ordered (L_o) and liquid-disordered (L_d) regions. In Eukaryotes, microdomains are rich in cholesterol and sphingolipids and serve as signal transduction scaffolds. In Prokaryotes, L_o microdomains increase pathogenicity and antimicrobial resistance. Previously, we identified spectroscopically distinct chemical shift signatures for all-trans (AT) and trans-gauche (TG) acyl chain conformations, cyclopropyl ring lipids (CPR), and hopanoids in prokaryotic lipid extracts and used Polarization Transfer (PT) SSNMR to investigate bilayer ordering. To investigate how these findings relate to native bilayer organization, we interrogate whole cell and whole membrane extract samples of Burkholderia thailendensis to investigate bilayer ordering in situ. In ¹³C-¹³C 2D SSNMR spectra, we assigned chemical shifts for lipid species in both samples, showing conservation of lipids of interest in our native membrane sample. A one-dimensional temperature series of PT SSNMR and transverse relaxation measurements of AT versus TG acyl conformations in the membrane sample confirm bilayer ordering and a broadened phase transition centered at a lower-than-expected temperature. Bulk protein backbone Cα dynamics and correlations consistent with lipid-protein contacts within are further indicative of microdomain formation and lipid ordering. In aggregate, these findings provide evidence for microdomain formation in vivo and provide insight into phase separation and transition mechanics in biological membranes.     

Cholesterol Partition and Condensing Effect in Phase-Separated Ternary Mixture Lipid Multilayers

The cholesterol partitioning and condensing effect in the liquid-ordered (L_o) and liquid-disordered (L_d) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the L_o phase was observed for total cholesterol concentration of 20∼30%.     

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (L_o) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38°C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20°–50°C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.     

Response of isolated thylakoid membranes with altered fluidity to short term heat stress

The effect of alterations of lipid phase order of thylakoid membranes on the thermosensitivity of photosystem I (PS I) and photosystem II (PS II) was studied. Plant sterols stigmasterol and cholesterol were applied to decrease the fluidity in isolated membranes. After sterol treatment, a decrease of the temperature of 50 % inhibition of PSII activity was observed. Heat stress-induced stimulation of PSI-mediated electron transport rate was registered for control, but not for sterol-treated membranes. Effect of altered lipid order on oxygen evolving complex was evaluated by means of flash oxygen yields revealing changes in the stoichiometry of PSII_α and PSII_β centers. The effect of sterol incorporation on the changes in the thermotropic behavior of the main pigment-protein complexes was studied by differential scanning calorimetry (DSC). DSC traces of control thylakoids in the temperature range 20–98 °C exhibited several irreversible endothermic transitions. Incorporation of cholesterol and stigmasterol results in superimposition of the transitions and only two main bands could be resolved. While high temperature band peaks at the same temperature after treatment with both sterols, the band that combines low temperature transitions shows different melting temperature (T_m): 70 °C for stigmasterol- and 65 °C for cholesterol-treated membranes. The data presented here emphasise the crucial role of lipid order for the response of thylakoids to high temperatures, mediated not only by changes in the fluidity of bulk lipid phase as result of sterol incorporation but also by changes in the thermotropic properties of pigment-protein complexes.Key words: Cholesterol, Fluidity, Heat stress, Oxygen flash yields, Thylakoid membrane, Stigmasterol     

In Situ Determination of Structure and Fluctuations of Coexisting Fluid Membrane Domains

Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (L_o) and liquid-disordered (L_d) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. L_o domains were found to be ∼10 Å thicker, and laterally up to 20 Å²/lipid more condensed than L_d domains. Their bending fluctuations were also reduced by ∼65%. Increase of cholesterol concentration caused significant changes in structural properties of L_d, while its influence on L_o properties was marginal. We further observed that temperature-induced melting of L_o domains is associated with a diffusion of cholesterol to L_d domains and controlled by L_o/L_d thickness differences.     

Lo/Ld phase coexistence modulation induced by GM1

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” L_o domain size and the search for L_o nanodomains as precursors in L_o microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the L_o/L_d lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1 mol % abolishes the formation of the micrometer-scale L_o domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a L_o/L_d lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C₁₂NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale L_o phase domains over an L_d phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to L_o/L_d micrometric phase separation, L_o/L_d nanometric phase separation, and a homogeneous L_d phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing L_o nanodomains.     

The Effects of Ca2+ and MgADP on Force Development during and after Muscle Length Changes

The goal of this study was to compare the effects of Ca²⁺ and MgADP activation on force development in skeletal muscles during and after imposed length changes. Single fibres dissected from the rabbit psoas were (i) activated in pCa²⁺4.5 and pCa²⁺6.0, or (ii) activated in pCa²⁺4.5 before and after administration of 10 mM MgADP. Fibres were activated in sarcomere lengths (SL) of 2.65 µm and 2.95 µm, and subsequently stretched or shortened (5%SL at 1.0 SL.s⁻¹) to reach a final SL of 2.80 µm. The kinetics of force during stretch were not altered by pCa²⁺ or MgADP, but the fast change in the slope of force development (P₁) observed during shortening and the corresponding SL extension required to reach the change (L₁) were higher in pCa²⁺6.0 (P₁ = 0.22±0.02 P_o; L₁ = 5.26±0.24 nm.HS^.1) than in pCa²⁺4.5 (P₁ = 0.15±0.01 P_o; L₁ = 4.48±0.25 nm.HS^.1). L₁ was also increased by MgADP activation during shortening. Force enhancement after stretch was lower in pCa²⁺4.5 (14.9±5.4%) than in pCa²⁺6.0 (38.8±7.5%), while force depression after shortening was similar in both Ca²⁺ concentrations. The stiffness accompanied the force behavior after length changes in all situations. MgADP did not affect the force behavior after length changes, and stiffness did not accompany the changes in force development after stretch. Altogether, these results suggest that the mechanisms of force generation during and after stretch are different from those obtained during and after shortening.     

Acute Simvastatin Inhibits KATP Channels of Porcine Coronary Artery Myocytes

Background

Statins (3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors) consumption provides beneficial effects on cardiovascular systems. However, effects of statins on vascular K_ATP channel gatings are unknown.

Methods

Pig left anterior descending coronary artery and human left internal mammary artery were isolated and endothelium-denuded for tension measurements and Western immunoblots. Enzymatically-dissociated/cultured arterial myocytes were used for patch-clamp electrophysiological studies and for [Ca²⁺]_i, [ATP]_i and [glucose]_o uptake measurements.

Results

The cromakalim (10 nM to 10 µM)- and pinacidil (10 nM to 10 µM)-induced concentration-dependent relaxation of porcine coronary artery was inhibited by simvastatin (3 and 10 µM). Simvastatin (1, 3 and 10 µM) suppressed (in okadaic acid (10 nM)-sensitive manner) cromakalim (10 µM)- and pinacidil (10 µM)-mediated opening of whole-cell K_ATP channels of arterial myocytes. Simvastatin (10 µM) and AICAR (1 mM) elicited a time-dependent, compound C (1 µM)-sensitive [³H]-2-deoxy-glucose uptake and an increase in [ATP]_i levels. A time (2–30 min)- and concentration (0.1–10 µM)-dependent increase by simvastatin of p-AMPKα-Thr¹⁷² and p-PP2A-Tyr³⁰⁷ expression was observed. The enhanced p-AMPKα-Thr¹⁷² expression was inhibited by compound C, ryanodine (100 µM) and KN93 (10 µM). Simvastatin-induced p-PP2A-Tyr³⁰⁷ expression was suppressed by okadaic acid, compound C, ryanodine, KN93, phloridzin (1 mM), ouabain (10 µM), and in [glucose]_o-free or [Na⁺]_o-free conditions.

Conclusions

Simvastatin causes ryanodine-sensitive Ca²⁺ release which is important for AMPKα-Thr¹⁷² phosphorylation via Ca²⁺/CaMK II. AMPKα-Thr¹⁷² phosphorylation causes [glucose]_o uptake (and an [ATP]_i increase), closure of K_ATP channels, and phosphorylation of AMPKα-Thr¹⁷² and PP2A-Tyr³⁰⁷ resulted. Phosphorylation of PP2A-Tyr³⁰⁷ occurs at a site downstream of AMPKα-Thr¹⁷² phosphorylation.     

Adhesion Stabilizes Robust Lipid Heterogeneity in Supercritical Membranes at Physiological Temperature

Regions of contact between cells are frequently enriched in or depleted of certain protein or lipid species. Here, we explore a possible physical basis that could contribute to this membrane heterogeneity using a model system of a giant vesicle tethered to a planar supported bilayer. Vesicles contain coexisting liquid-ordered (L_o) and liquid-disordered (L_d) phases at low temperatures and are tethered using trace quantities of adhesion molecules that preferentially partition into one liquid phase. We find that the L_d marker DiI-C₁₂ is enriched or depleted in the adhered region when adhesion molecules partition into L_d or L_o phases, respectively. Remarkably, adhesion stabilizes an extended zone enriched or depleted of DiI-C₁₂ even at temperatures >15°C above the miscibility phase transition when membranes have compositions that are in close proximity to a critical point. A stable adhesion zone is also observed in plasma membrane vesicles isolated from living RBL-2H3 cells, and probe partitioning at 37°C is diminished in vesicles isolated from cells with altered cholesterol levels. Probe partitioning is in good quantitative agreement with predictions of the two-dimensional Ising model with a weak applied field for both types of model membranes. These studies experimentally demonstrate that large and stable domain structure can be mediated by lipids in single-phase membranes with supercritical fluctuations.     

Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts

Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10⁻⁵ molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F₇₄₀/F₆₈₅ was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F_o) was virtually eliminated in ABA-treated (10⁻⁵ molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F_o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F_o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F_o was maximal at 45°C. In contrast, chloroplasts treated with 10⁻⁵ molar ABA did not exhibit a heat-induced increase in F_o until 50°C.     

Stationary phase lipophagy as a cellular mechanism to recycle sterols during quiescence

Delivery of cellular contents to yeast vacuoles/mammalian lysosomes via autophagy ensures long-term cell survival and extends life span. When cultured yeast cells are grown for a prolonged period of time to enter stationary phase, a nondividing state mimicking quiescence, vacuolar membrane proteins partition into either one of the vacuolar microdomains, liquid-ordered (Lo) or liquid-disordered (Ld). We show that during the transition to stationary phase, lipid droplets (LDs), organelles originated from the endoplasmic reticulum (ER), undergo lateral movement to reach the vacuolar surface and are confined within the specific Lo microdomain underlying the network of vacuolar quasi-symmetrical micodomains. Stationary phase lipophagy uses the autophagy machineries to modify the sterol-enriched Lo microdomain to engulf LDs and subsequently deposits the LD-containing vesicles inside the vacuole lumen, which is a pathway morphologically resembling microautophagy. Moreover, stationary phase lipophagy supplies quiescent yeast cells with sterols to sustain phase partitioning of lipids for vacuolar microdomain maintenance. A feed forward loop model was proposed to depict that the sterols boosted by LDs via stationary phase lipophagy promote the Lo microdomain maintenance that in turn stimulates lipophagy.     

Stationary phase lipophagy as a cellular mechanism to recycle sterols during quiescence

Delivery of cellular contents to yeast vacuoles/mammalian lysosomes via autophagy ensures long-term cell survival and extends life span. When cultured yeast cells are grown for a prolonged period of time to enter stationary phase, a nondividing state mimicking quiescence, vacuolar membrane proteins partition into either one of the vacuolar microdomains, liquid-ordered (Lo) or liquid-disordered (Ld). We show that during the transition to stationary phase, lipid droplets (LDs), organelles originated from the endoplasmic reticulum (ER), undergo lateral movement to reach the vacuolar surface and are confined within the specific Lo microdomain underlying the network of vacuolar quasi-symmetrical micodomains. Stationary phase lipophagy uses the autophagy machineries to modify the sterol-enriched Lo microdomain to engulf LDs and subsequently deposits the LD-containing vesicles inside the vacuole lumen, which is a pathway morphologically resembling microautophagy. Moreover, stationary phase lipophagy supplies quiescent yeast cells with sterols to sustain phase partitioning of lipids for vacuolar microdomain maintenance. A feed forward loop model was proposed to depict that the sterols boosted by LDs via stationary phase lipophagy promote the Lo microdomain maintenance that in turn stimulates lipophagy.     

Membrane microdomain switching: a regulatory mechanism of amyloid precursor protein processing

Neuronal activity has an impact on β cleavage of amyloid precursor protein (APP) by BACE1 to generate amyloid-β peptide (Aβ). However, the molecular mechanisms underlying this effect remain to be elucidated. Cholesterol dependency of β cleavage prompted us to analyze immunoisolated APP-containing detergent-resistant membranes from rodent brains. We found syntaxin 1 as a key molecule for activity-dependent regulation of APP processing in cholesterol-dependent microdomains. In living cells, APP associates with syntaxin 1–containing microdomains through X11–Munc18, which inhibits the APP–BACE1 interaction and β cleavage via microdomain segregation. Phosphorylation of Munc18 by cdk5 causes a shift of APP to BACE1-containing microdomains. Neuronal hyperactivity, implicated in Aβ overproduction, promotes the switching of APP microdomain association as well as β cleavage in a partially cdk5-dependent manner. We propose that microdomain switching is a mechanism of cholesterol- and activity-dependent regulation of APP processing in neurons.     

Unraveling Sterol-dependent Membrane Phenotypes by Analysis of Protein Abundance-ratio Distributions in Different Membrane Fractions Under Biochemical and Endogenous Sterol Depletion

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-β-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.The plasma membrane incorporates a broad spectrum of proteins covering mainly different structural, signaling or transport functionalities. Being the first semipermeable cell barrier to its surrounding environment the plasma membrane is important for metabolite transport as well as initiation point of several signaling processes (1–4). To maintain cell homeostasis, protein activity as well as complex formation through protein protein interactions (PPI) need to be tightly regulated. The major regulating mechanisms are postranslational modification of proteins and modulated abundances of proteins present in the plasma membrane. Another potential regulating mechanism became apparent with the discovery of sterol and sphingolipid enriched domains (microdomains) in the plasma membrane (5–8, 3). Microdomain like structures have been shown to form spontaneously in artificial plasma membranes (9). After a decade of research on these structures, microdomains turned out to be particularly involved in signaling and transport processes incorporating a specific set of proteins. Microdomains provide subcompartments in the plasma membrane with specific physicochemical properties that on specific sterol protein interactions might alter protein activity or PPIs. With the discovery of microdomains the fluid lipid mosaic model was extended by distinguishing two plasma membrane phases, an ordered phase of lower density (L_o phase) enriched in sterols, sphingolipids and long chain fatty acids and a disordered phase of higher density (L_d phase). From isolated plasma membranes a lower density and a higher density membrane fraction can be separated in a sucrose gradient after treatment with non-ionic detergents. The resulting detergent resistant membrane fraction (DRM)¹ is related to L_o phase and high density detergent soluble membrane fraction (DSF) relates to L_o phases. Although it is still under debate how well DRMs represent native plasma membrane microdomains (10–12), research on protein-sterol interactions is possible by usage of sterol depleting agents like methyl-β-cyclodextrin mβcd (13). Therefore mβcd is suitable for detecting false positive cholesterol protein interactions in DRM studies (14–19). Proteins depleted on mβcd treatment are finally considered to be sterol dependent (15–17). To compare the mβcd treatment for disturbing the sterol distribution in the L_o fraction, we studied the sterol biosynthesis deficient mutant smt1. (20) smt1 carries a point mutation in the smt1 locus, encoding the sterol methyltransferase 1 and it exhibits a dwarf-like phenotype on whole plant level (20). In total, three sterol methyl transferases are encoded in Arabidopsis where SMT1 catalyzes the first step in the sterol biosynthesis by adding a methyl group at C24 of the sterol precursor cycloartenol. SMT2 and SMT3 act at later steps and were shown to be functionally redundant as C-24 sterol methyltransferases at the branching in sterol synthesis that either leads to sitosterol or campesterol (21). The total sterol composition in smt1 mutants was shown to be different from wild type, with the major phytosterols like sitosterol, stigmasterol, and brassicasterol being strongly depleted. In contrast, other sterol species remained unaltered and some even increased (20, 21). So far, it remains unclear how the altered sterol-composition of the smt1 mutant affects sterol-protein interactions. In this study, using the newly developed algorithm Unicorn, we compared changes in protein distributions between DRM and DSF after biochemical mβcd treatment and on endogenous alterations in sterol composition in smt1 to improve understanding of sterol–protein interactions.     

Polyunsaturated fatty acids promote the rapid fusion of lipid droplets in Caenorhabditis elegans

Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal β-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal β-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element–binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal β-oxidation–defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.     

Cytoplasmic Body Component TRIM5α Requires Lipid-enriched Microdomains for Efficient HIV-1 Restriction

TRIM5α is a member of the tripartite motif (TRIM) family of proteins and affects both early and late phases of the retroviral life cycle. Although TRIM5α multimerizes to form cytoplasmic bodies, which are thought to play an important role in viral restriction, the identity of TRIM5α-containing cytoplasmic bodies remains elusive. To better understand TRIM5α cytoplasmic body constituents and the cellular proteins that could be involved in the TRIM5α-mediated antiviral activities, we sought TRIM5α-binding factors. We identified a lipid microdomain protein flotillin-1/Reggie-2 as an interacting partner of TRIM5α via co-immunoprecipitation. Immunohistochemistry studies confirmed the co-localization of rhesus monkey TRIM5α (TRIM5αrh) cytoplasmic bodies with flotillin-1/Reggie-2. Caveolin-1, another lipid microdomain-associated protein, also co-localized with TRIM5α cytoplasmic bodies. Intriguingly, disruption of cellular cholesterol by cyclodextrin perturbed TRIM5α cytoplasmic body formation. Furthermore, lipid starvation partially relieved the endogenous post-entry restriction of HIV-1 infection, which could be subsequently restored by lipid repletion. These observations indicate the involvement of cellular lipids in TRIM5α-mediated antiviral activities. Given that many viruses utilize cellular lipid microdomains for viral entry and assembly, it is plausible that lipid-enriched domains provide microenvironments where TRIM5α recognizes retroviral components.     

Cooperative Binding of Annexin A2 to Cholesterol- and Phosphatidylinositol-4,5-Bisphosphate-Containing Bilayers

Biological membranes are organized into dynamic microdomains that serve as sites for signal transduction and membrane trafficking. The formation and expansion of these microdomains are driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Annexin A2 (AnxA2) is a peripherally associated membrane protein that can support microdomain formation in a Ca²⁺-dependent manner and has been implicated in membrane transport processes. Here, we performed a quantitative analysis of the binding of AnxA2 to solid supported membranes containing the annexin binding lipids phosphatidylinositol-4,5-bisphosphate and phosphatidylserine in different compositions. We show that the binding is of high specificity and affinity with dissociation constants ranging between 22.1 and 32.2 nM. We also analyzed binding parameters of a heterotetrameric complex of AnxA2 with its S100A10 protein ligand and show that this complex has a higher affinity for the same membranes with K_d values of 12 to 16.4 nM. Interestingly, binding of the monomeric AnxA2 and the AnxA2-S100A10 complex are characterized by positive cooperativity. This cooperative binding is mediated by the conserved C-terminal annexin core domain of the protein and requires the presence of cholesterol. Together our results reveal for the first time, to our knowledge, that AnxA2 and its derivatives bind cooperatively to membranes containing cholesterol, phosphatidylserine, and/or phosphatidylinositol-4,5-bisphosphate, thus providing a mechanistic model for the lipid clustering activity of AnxA2.     

Focal junctions retard lateral movement and disrupt fluid phase connectivity in the plasma membrane

In cultured keratinocytes, focal junctions are enriched in major constituents of lipid rafts, such as GM₁ ganglioside, phosphoinositides, caveolins and flotillins. We have therefore speculated that focal junctions represent superrafts formed by coalescence of microdomains into large areas containing liquid-ordered (L_o) lipids. Indeed, values of maximal fluorescence recovery after photobleaching revealed that the long-range mobility of cholera toxin B subunit (CTB, marker of L_o) was ∼1.5-fold retarded within the focal junctions compared to the surrounding membrane. However, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI-C_18:0), which specifically partitions to the liquid-disordered (L_d), non-raft phase, was also enriched in focal junctions and its mobility was slightly retarded. Cross-linking of GM₁ by CTB or raft aggregation by methyl-β-cyclodextrin further decreased the recovery of DiI-C_18:0. We propose a model in which focal junctions impose lateral heterogeneity in the plasma membrane by entrapment of lipid microdomains between dense arrays of immobilized transmembrane molecules which can enmesh otherwise freely percolating L_d phase lipids.     

Crystal Structure,Cytotoxicity and Interaction with DNA of Zinc (II) Complexes with o-Vanillin Schiff Base Ligands

Two new zinc complexes, Zn(HL¹)₂ (1) and [Zn₂(H₂L²)(OAc)₂]₂ (2) [H₂L¹ = Schiff base derived from o-vanillin and (R)-(+)-2-amino-3-phenyl-1-propanol, H₃L² = Schiff base derived from o-vanillin and 2-amino-2-ethyl-1,3-propanediol], have been synthesized and characterized by single crystal X-ray diffraction, elemental analyses, TG analyses, solid fluorescence, IR, UV-Vis and circular dichroism spectra. The structural analysis shows that complex 1 has a right-handed double helical chain along the crystallographic b axis. A homochiral 3D supramolecular architecture has been further constructed by intermolecular C-H··· π, O-H···O and C-H···O interactions. Complex 2 includes two crystallographically independent binuclear zinc molecules. The two binuclear zinc molecules are isostructural. The 2-D sheet supramolecular structure was formed by intermolecular hydrogen bonding interaction. The fluorescence of ligands and complexes in DMF at room temperature are studied. The interactions of two complexes with calf thymus DNA (CT-DNA) are investigated using UV-Vis, CD and fluorescence spectroscopy. The results show that complex 1 exhibits higher interaction with CT-DNA than complex 2. In addition, in vitro cytotoxicity of the complexes towards four kinds of cancerous cell lines (A549, HeLa, HL-60 and K562) were assayed by the MTT method. Investigations on the structures indicated that the chirality and nuclearity of zinc complexes play an important role on cytotoxic activity.