Confocal microscopy of giant vesicles supports the absence of HIV-1 neutralizing 2F5 antibody reactivity to plasma membrane phospholipids

The broadly neutralizing anti-HIV-1 2F5 monoclonal antibody recognizes a gp41 epitope proximal to the viral membrane. Potential phospholipid autoreactivity at cell surfaces has raised concerns about the use of this antibody for development of vaccines or immunotherapy. In this study, confocal microscopy of giant unilamellar vesicles (GUVs) was used to assess 2F5 reactivity with phospholipids assembled into bilayers with surface charge and curvature stress approximating those of the eukaryotic plasma membranes. Antibody partitioning into lipid bilayers required the specific recognition of membrane-inserted epitope, indicating that 2F5 was unable to directly react with GUV phospholipids, even under fluid phase segregation conditions. Our results thus support the feasibility of raising 2F5-like neutralizing responses through vaccination, and the medical safety of mAb infusions.     

The reactivation of DnaA(L366K) requires less acidic phospholipids supporting their role in the initiation of chromosome replication in Escherichia coli

DnaA(L366K), in concert with a wild-type DnaA (wtDnaA) protein, restores the growth of Escherichia coli cells arrested in the absence of adequate levels of cellular acidic phospholipids. In vitro and in vivo studies showed that DnaA(L366K) alone does not induce the initiation of replication, and wtDnaA must also be present. Hitherto the different behavior of wt and mutant DnaA were not understood. We now demonstrate that this mutant may be activated at significantly lower concentrations of acidic phospholipids than the wild-type protein, and this may explain the observed growth restoration in vivo.     

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.     

Oxidised LDL modulate adipogenesis in 3T3-L1 preadipocytes by affecting the balance between cell proliferation and differentiation

The effects of oxidised LDL (oxLDL) on cell proliferation, apoptosis and hormone-induced differentiation have been evaluated for the first time in 3T3-L1 preadipocytes. Unlike control cells, oxLDL-treated preadipocytes showed a high proliferation rate, a low apoptosis level, and an impaired differentiation process with an increased preadipocyte factor-1 (Pref-1) mRNA expression at late times. By silencing Pref-1 mRNA or inhibiting its expression with an increased dexamethasone concentration, differentiation occurred as usual, which demonstrates the key role of Pref-1 overexpression. The results suggest a specific action of oxLDL on the adipogenesis inhibitor Pref-1, as indicated also by its reappearance in mature adipocytes treated with oxLDL. The inhibitory effects of oxLDL on differentiation required oxLDL uptake by CD36, and were associated with lipoprotein lipids. These results point to oxLDL as a modulator of adipose tissue mass and as possible link between obesity and its clinical complications.     

Line tension at lipid phase boundaries regulates formation of membrane vesicles in living cells

Ternary lipid compositions in model membranes segregate into large-scale liquid-ordered (L_o) and liquid-disordered (L_d) phases. Here, we show μm-sized lipid domain separation leading to vesicle formation in unperturbed human HaCaT keratinocytes. Budding vesicles in the apical portion of the plasma membrane were predominantly labelled with L_d markers 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-dilinoleyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate, 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate and weakly stained by L_o marker fluorescein-labeled cholera toxin B subunit which labels ganglioside GM₁ enriched plasma membrane rafts. Cholesterol depletion with methyl-β-cyclodextrin enhanced DiI vesiculation, GM₁/DiI domain separation and was accompanied by a detachment of the subcortical cytoskeleton from the plasma membrane. Based on these observations we describe the energetic requirements for plasma membrane vesiculation. We propose that the decrease in total ‘L_o/L_d’ boundary line tension arising from the coalescence of smaller L_d-like domains makes it energetically favourable for L_d-like domains to bend from flat μm-sized surfaces to cap-like budding vesicles. Thus living cells may utilize membrane line tension energies as a control mechanism of exocytic events.     

Tunneling nanotube (TNT)-like structures facilitate a constitutive, actomyosin-dependent exchange of endocytic organelles between normal rat kidney cells

Tunneling nanotube (TNT)-like structures are intercellular membranous bridges that mediate the transfer of various cellular components including endocytic organelles. To gain further insight into the magnitude and mechanism of organelle transfer, we performed quantitative studies on the exchange of fluorescently labeled endocytic structures between normal rat kidney (NRK) cells. This revealed a linear increase in both the number of cells receiving organelles and the amount of transferred organelles per cell over time. The intercellular transfer of organelles was unidirectional, independent of extracellular diffusion, and sensitive to shearing force. In addition, during a block of endocytosis, a significant amount of transfer sustained. Fluorescence microscopy revealed TNT-like bridges between NRK cells containing F-actin but no microtubules. Depolymerization of F-actin led to the disappearance of TNT and a strong inhibition of organelle exchange. Partial ATP depletion did not affect the number of TNT but strongly reduced organelle transfer. Interestingly, the myosin II specific inhibitor S-(−)-blebbistatin strongly induced both organelle transfer and the number of TNT, while the general myosin inhibitor 2,3-butanedione monoxime induced the number of TNT but significantly inhibited transfer. Taken together, our data indicate a frequent and continuous exchange of endocytic organelles between cells via TNT by an actomyosin-dependent mechanism.     

Enhancement of angiogenic and vasculogenic potential of endothelial progenitor cells by haptoglobin

Endothelial progenitor cells (EPCs) were transfected with the haptoglobin (Hp) gene to investigate the effect of Hp on cell function. Hp potentiated the gene expression of various pro-angiogenic factors in the EPCs. The Hp-modified EPCs also increased in vitro tube formation on Matrigel compared with control cells. In hindlimb ischaemia models, Hp-EPCs showed a greater ability for improving blood perfusion and recovery from ischaemic injury. These results indicate that Hp improves EPC function in neovasculogenesis, which suggests that ex vivo modification of EPCs with the Hp gene can be applied to the treatment of vascular damage.     

Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

Tanshinone IIA increases recruitment of bone marrow mesenchymal stem cells to infarct region via up-regulating stromal cell-derived factor-1/CXC chemokine receptor 4 axis in a myocardial ischemia model

Systemic administration with bone marrow mesenchymal stem cells (BMSCs) is a promising approach to cure myocardial ischemia (MI), while the efficacy of cell transplantation is limited by the low engraftment of BMSCs. Tanshinone IIA (Tan IIA) has been reported many times for the treatment of MI. Therefore, the present study was performed to investigate whether Tan IIA could increase the migration of BMSCs to ischemic region and its potential mechanisms. In our study, we found that combination treatment with Tan IIA and BMSCs significantly alleviated the infarct size when compared with control group (31.46 ± 3.00% vs. 46.95 ± 6.51%, p < 0.05). Results of real-time PCR showed that Tanshinone IIA (Tan IIA) did increase the migration of BMSCs to ischemic region in vivo, which was correlated with cardiac function recovery after MI. Furthermore, 2 μM Tan IIA could enhance the migration capability of BMSCs in vitro (3.69-fold of control), and this enhancement could be blocked by AMD3100 (a CXC chemokine receptor 4 blocker). CXCR4, together with its specific receptor, stromal cell-derived factor-1 (SDF-1) plays a critical role in the stem cell recruitment. Our experiment indicated that Tan IIA could promote SDF-1α expression in the infarct area and enhance the CXCR4 expression of BMSCs in vitro. Therefore, we postulated that Tan IIA could increase the BMSCs migration via up-regulating SDF1/CXCR4 axis.     

Inhibition of mTOR down-regulates scavenger receptor,class B,type I (SR-BI) expression,reduces endothelial cell migration and impairs nitric oxide production

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.     

Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol

We have undertaken a series of experiments to examine the behavior of individual components of cell membranes. Here we report an initial stage of these experiments, in which the properties of a chemically simple lipid mixture are carefully mapped onto a phase diagram. Four different experimental methods were used to establish the phase behavior of the 3-component mixture DSPC/DOPC/chol: (1) confocal fluorescence microscopy observation of giant unilamellar vesicles, GUVs; (2) FRET from perylene to C20:0-DiI; (3) fluorescence of dilute dyes C18:2-DiO and C20:0-DiI; and (4) wide angle X-ray diffraction. This particular 3-component mixture was chosen, in part, for a high level of immiscibility of the components in order to facilitate solving the phase behavior at all compositions. At 23 °C, a large fraction of the possible compositions for this mixture give rise to a solid phase. A region of 3-phase coexistence of {Lα + Lβ + Lo} was detected and defined based on a combination of fluorescence microscopy of GUVs, FRET, and dilute C20:0-DiI fluorescence. At very low cholesterol concentrations, the solid phase is the tilted-chain phase Lβ′. Most of the phase boundaries have been determined to be within a few percent of the composition. Measurements of the perturbations of the boundaries of this accurate phase diagram could serve as a means to understand the behaviors of a range of added lipids and proteins.     

Specific lipids supply critical negative spontaneous curvature--an essential component of native Ca2+-triggered membrane fusion

The Ca²⁺-triggered merger of two apposed membranes is the defining step of regulated exocytosis. CHOL is required at critical levels in secretory vesicle membranes to enable efficient, native membrane fusion: CHOL-sphingomyelin enriched microdomains organize the site and regulate fusion efficiency, and CHOL directly supports the capacity for membrane merger by virtue of its negative spontaneous curvature. Specific, structurally dissimilar lipids substitute for CHOL in supporting the ability of vesicles to fuse: diacylglycerol, αT, and phosphatidylethanolamine support triggered fusion in CHOL-depleted vesicles, and this correlates quantitatively with the amount of curvature each imparts to the membrane. Lipids of lesser negative curvature than cholesterol do not support fusion. The fundamental mechanism of regulated bilayer merger requires not only a defined amount of membrane-negative curvature, but this curvature must be provided by molecules having a specific, critical spontaneous curvature. Such a local lipid composition is energetically favorable, ensuring the necessary “spontaneous” lipid rearrangements that must occur during native membrane fusion—Ca²⁺-triggered fusion pore formation and expansion. Thus, different fusion sites or vesicle types can use specific alternate lipidic components, or combinations thereof, to facilitate and modulate the fusion pore.