Vesicle fusion with bilayer lipid membrane controlled by electrostatic interaction

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO₂ substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.     

Interaction of tetrasubstituted cationic aluminum phthalocyanine with artificial and natural membranes

A study of the properties of water-soluble tetrasubstituted cationic aluminum phthalocyanine (AlPcN₄) revealed efficient binding of this photosensitizer to phospholipid membranes as compared with tetrasulfonated aluminum and zinc phthalocyanine complexes. This also manifested itself in enhanced photodynamic activity of AlPcN₄ as measured by the photosensitized damage of gramicidin channels in a planar bilayer lipid membrane. The largest difference in the photodynamic activity of cationic and anionic phthalocyanines was observed in a membrane containing negatively charged lipids, thereby pointing to significant contribution of electrostatic interactions to the binding of photosensitizers to a membrane. Fluoride anions suppressed the photodynamic activity and binding to membrane of both tetraanionic and tetracationic aluminum phthalocyanines, which supports our hypothesis that interaction of charged metallophthalocyanines with phospholipid membranes is mostly determined by coordination of the central metal atom with the phosphate group of lipid.     

Photosensitizer binding to lipid bilayers as a precondition for the photoinactivation of membrane channels

The photodynamic activity of sulfonated aluminum phthalocyanines (AlPcS(n), 1 相似文献   

Study of the interaction of an α-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K₂-L₂₄-K₂-amide (L₂₄), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L₂₄ decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L₂₄ in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L₂₄:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L₂₄. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and ²H NMR spectroscopy study of the interaction of the L₂₄ and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between ²H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L₂₄ suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature

Asymmetry of inner and outer leaflet lipid composition is an important characteristic of eukaryotic plasma membranes. We previously described a technique in which methyl-β-cyclodextrin-induced lipid exchange is used to prepare biological membrane-like asymmetric small unilamellar vesicles (SUVs). Here, to mimic plasma membranes more closely, we used a lipid-exchange-based method to prepare asymmetric large unilamellar vesicles (LUVs), which have less membrane curvature than SUVs. Asymmetric LUVs in which sphingomyelin (SM) or SM + 1-palmitoyl-2-oleoyl-phosphatidylcholine was exchanged into the outer leaflet of vesicles composed of 1,2-dioleoyl-phosphatidylethanolamine (DOPE) and 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS) were prepared with or without cholesterol. Approximately 80–100% replacement of outer leaflet DOPE and POPS was achieved. At room temperature, SM exchange into the outer leaflet increased the inner leaflet lipid order, suggesting significant interleaflet interaction. However, the SM-rich outer leaflet formed an ordered state, melting with a midpoint at ∼37°C. This was about the same value observed in pure SM vesicles, and was significantly higher than that observed in symmetric vesicles with the same SM content, which melted at ∼20°C. In other words, ordered state formation by outer-leaflet SM in asymmetric vesicles was not destabilized by an inner leaflet composed of DOPE and POPS. These properties suggest that the coupling between the physical states of the outer and inner leaflets in these asymmetric LUVs becomes very weak as the temperature approaches 37°C. Overall, the properties of asymmetric LUVs were very similar to those previously observed in asymmetric SUVs, indicating that they do not arise from the high membrane curvature of asymmetric SUVs.     

Interaction of meso-tetrakis (4-N-methylpyridyl) porphyrin in its free base and as a Zn(II) derivative with large unilamellar phospholipid vesicles

Our aim was to investigate the interaction of the cationic meso-tetrakis (4-N-methylpyridyl) porphyrin, a photosensitizer used for photodynamic therapy, in its free base form (TMPyP) and complexed with Zn(II) (ZnTMPyP), with large unilamellar vesicles (LUVs), as a model for the gram-negative bacterial cell wall. Mixtures of the zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (POPG) phospholipids, at different molar percentages, were used as LUVs. A significant increase of porphyrin affinity at higher POPG molar concentrations was observed from the binding constant values, K _b, estimated by optical absorption and steady-state fluorescence. Besides, as demonstrated by time-resolved fluorescence, this affinity increase is also followed by a higher fraction of vesicle-bound porphyrin in the LUVs. Moreover, based on the K _b values, we have observed a higher affinity of the ZnTMPyP to the POPG containing LUVs as compared to the TMPyP. Steady-state fluorescence quenching and zeta potential studies revealed that both porphyrins are possibly located at the LUVs Stern layer region. Therefore, the electrostatic attraction between the positively charged porphyrin peripheral groups and the negatively charged outer surface of the LUVs plays an important role in porphyrin association and localization. Our results have improved the understanding of the successful application of cationic porphyrins on the photo-inactivation of gram-negative bacteria. Since a higher accumulation of the ZnTMPyP in the bacterial cell wall would be expected, this porphyrin could be a more efficient therapeutic drug for this treatment.     

The broad-spectrum antibiofilm activity of amyloid-forming hexapeptides

Evidence suggests that short amyloid-forming peptides derived from bacterial proteomes have functional roles; however, the reported activities are diverse and the underlying mechanisms remain unclear. In this study, we simulated short amyloid-forming peptides from the amyloid-forming truncated protein C123 of Streptococcus mutans (S. mutans), studied their biological functions in microbial proliferation and biofilm formation, and further investigated the underlying mechanism. Fourteen hexapeptides were simulated, 13 of which were successfully synthesized. We found that the amyloid-forming hexapeptides (AFhPs) displayed efficient broad-spectrum antibiofilm activity against the Gram-positive bacteria S. mutans, Streptococcus sanguis and Staphylococcus aureus, Gram-negative bacteria Escherichia coli and fungus Candida albicans, by aggregating into rigid amyloid fibres agglutinating microbes, whereas the non-amyloid-forming hexapeptides (non-AFhPs) did not. The AFhPs did not kill microbes and showed little or no cytotoxicity. Furthermore, a set of AFhPs displayed broad-spectrum antibiofilm activity, regardless of its source. The microbial cell wall carbohydrates, peptidoglycan (PGN), lipoteichoic acid (LTA), glucan and zymosan A, mediated AFhP binding and triggered significant AFhP fibrillation. Although amyloid fibres agglutinated lipid membrane model – large unilamellar vesicles (LUVs) – and LUVs facilitated AFhP fibrillation, the roles of lipid membranes in AFhP antibiofilm activities remain to be elucidated. We highlight the potential use of AFhPs as novel antibiofilm agents.     

Stability of giant unilamellar vesicles and large unilamellar vesicles of liquid-ordered phase membranes in the presence of Triton X-100

We have investigated the stability of giant unilamellar vesicles (GUVs) and large unilamellar vesicles (LUVs) of lipid membranes in the liquid-ordered phase (lo phase) against a detergent, Triton X-100. We found that in the presence of high concentrations of Triton X-100, the structure of GUVs and LUVs of dipalmitoyl-PC (DPPC)/cholesterol (chol) and sphingomyelin (SM)/chol membranes in the lo phase was stable and no leakage of fluorescent probes from the vesicles occurred. We also found that ether-linked dihexadecylphosphatidylcholine (DHPC) membranes containing more than 20 mol% cholesterol were in the lo phase, and that DHPC/chol-GUV and DHPC/chol-LUV in the lo phase were stable and no leakage of internal contents occurred in the presence of Triton X-100. In contrast, octylglucoside solution could easily break these GUVs and LUVs of the lo phase membranes and induced internal contents leakage. These data indicate that GUVs and LUVs of the lo phase membranes are very valuable for practical use.     

Thermodynamics of the interactions of tryptophan-rich cathelicidin antimicrobial peptides with model and natural membranes

Tritrpticin and indolicidin are short 13-residue tryptophan-rich antimicrobial peptides that hold potential as future alternatives for antibiotics. Isothermal titration calorimetry (ITC) has been applied as the main tool in this study to investigate the thermodynamics of the interaction of these two cathelicidin peptides as well as five tritrpticin analogs with large unilamellar vesicles (LUVs), representing model and natural anionic membranes. The anionic LUVs were composed of (a) 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPE/POPG) (7:3) and (b) natural E. coli polar lipid extract. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used to make model zwitterionic membranes. Binding isotherms were obtained to characterize the antimicrobial peptide binding to the LUVs, which then allowed for calculation of the thermodynamic parameters of the interaction. All peptides exhibited substantially stronger binding to anionic POPE/POPG and E. coli membrane systems than to the zwitterionic POPC system due to strong electrostatic attractions between the highly positively charged peptides and the negatively charged membrane surface, and results with tritrpticin derivatives further revealed the effects of various amino acid substitutions on membrane binding. No significant improvement was observed upon increasing the Tritrp peptide charge from + 4 to + 5. Replacement of Arg residues with Lys did not substantially change peptide binding to anionic vesicles but moderately decreased the binding to zwitterionic LUVs. Pro to Ala substitutions in tritrpticin, allowing the peptide to adopt an α-helical structure, resulted in a significant increase of the binding to both anionic and zwitterionic vesicles and therefore reduced the selectivity for bacterial and mammalian membranes. In contrast, substitution of Trp with other aromatic amino acids significantly decreased the peptide's ability to bind to anionic LUVs and essentially eliminated binding to zwitterionic LUVs. The ITC results were consistent with the outcome of fluorescence spectroscopy membrane binding and perturbation studies. Overall, our work showed that a natural E. coli polar lipid extract as a bacterial membrane model was advantageous compared to the simpler and more widely used POPE/POPG lipid system.     

Observation of the main phase transition of dinervonoylphosphocholine giant liposomes by fluorescence microscopy

The phase heterogeneity of giant unilamellar dinervonoylphosphocholine (DNPC) vesicles in the course of the main phase transition was investigated by confocal fluorescence microscopy observing the fluorescence from the membrane incorporated lipid analog, 1-palmitoyl-2-(N-4-nitrobenz-2-oxa-1,3-diazol)aminocaproyl-sn-glycero-3-phosphocholine (NBDPC). These data were supplemented by differential scanning calorimetry (DSC) of DNPC large unilamellar vesicles (LUV, diameter ∼0.1 and 0.2 μm) and multilamellar vesicles (MLV). The present data collected upon cooling reveal a lack of micron-scale gel and fluid phase coexistence in DNPC GUVs above the temperature of 20.5 °C, this temperature corresponding closely to the heat capacity maxima (T_em) of DNPC MLVs and LUVs (T_em ≈21 °C), measured upon DSC cooling scans. This is in keeping with the model for phospholipid main transition inferred from our previous fluorescence spectroscopy data for DMPC, DPPC, and DNPC LUVs. More specifically, the current experiments provide further support for the phospholipid main transition involving a first-order process, with the characteristic two-phase coexistence converting into an intermediate phase in the proximity of T_em. This at least macroscopically homogenous intermediate phase would then transform into the liquid crystalline state by a second-order process, with further increase in acyl chain trans→gauche isomerization.     

Antifungal properties of a peptide derived from the signal peptide of the HIV-1 regulatory protein, Rev

Antifungal effects of nuclear entry inhibitory signal peptide of HIV-1 Rev protein (Rev-NIS) were investigated. Rev-NIS contained potent antifungal activities without hemolytic effects. To understand the antifungal mechanism(s), in vivo and in vitro fluorescent studies were conducted. Flow cytometric analysis with bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] and calcein-leakage measurement from large unilamellar vesicles (LUVs) indicated that Rev-NIS depolarized and disrupted the fungal membranes. These results were further confirmed by using giant unilamellar vesicles (GUVs). The current study suggests that Rev-NIS exerts its antifungal activity with membrane-active mechanism(s).     

An Electron Paramagnetic Resonance Method for Measuring the Affinity of a Spin-Labeled Analog of Cholesterol for Phospholipids

Cholesterol (chol)–lipid interactions are thought to play an intrinsic role in determining lateral organization within cellular membranes. Steric compatibility of the rigid steroid moiety for ordered saturated chains contributes to the high affinity that holds chol and sphingomyelin together in lipid rafts whereas, conversely, poor affinity of the sterol for highly disordered polyunsaturated fatty acids (PUFAs) is hypothesized to drive the formation of PUFA-containing phospholipid domains depleted in chol. Here, we describe a novel method using electron paramagnetic resonance (EPR) to measure the relative affinity of chol for different phospholipids. We monitor the partitioning of 3β-doxyl-5α-cholestane (chlstn), a spin-labeled analog of chol, between large unilamellar vesicles (LUVs) and cyclodextrin (mβCD) through analysis of EPR spectra. Because the shape of the EPR spectrum for chlstn is sensitive to the very different tumbling rates of the two environments, the ratio of the population of chlstn in LUVs and mβCD can be determined directly from spectra. Partition coefficients ( $ K_{\text{B}}^{\text{A}} $ K B A ) between lipids derived from our results for chlstn agree with values obtained for chol and confirm that decreased affinity for the sterol accompanies increasing acyl chain unsaturation. The virtue of this EPR method is that it provides a measure of chol binding that is quick, employs a commercially available probe and avoids the necessity for physical separation of LUVs and mβCD.     

Spontaneous transfer of ganglioside GM1 from its micelles to lipid vesicles of differing size.

The spontaneous incorporation of II3-N-acetylneuraminosylgangliotetraosylceramide (GM1) from its micelles into phospholipid bilayer vesicles has been investigated to determine whether curvature-induced changes in membrane lipid packing influence ganglioside uptake. Use of conventional liquid chromatography in conjunction with technically-improved molecular sieve gels permits ganglioside micelles to be separated from phospholipid vesicles of different average size including vesicles with diameters smaller than 40 nm and, thus, allows detailed study of native ganglioside GM1 incorporation into model membranes under conditions where complicating processes like fusion are readily detected if present. At 45 degrees C, the spontaneous transfer rate of GM1 from its micelles to small unilamellar vesicles (SUVs) comprised of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) is at least 3-fold faster than that to similar composition large unilamellar vesicles (LUVs) prepared by octyl glucoside dialysis. Careful analysis of ganglioside GM1 distribution among vesicle populations of differing average size reveals that GM1 preferentially incorporates into the smaller vesicles of certain populations. This behavior is observed in SUVs as well as in LUV-SUV mixtures and actually serves as a sensitive indicator for the presence of trace quantities of SUVs in various LUV preparations. Analysis of the results shows that both differences in the diffusional collision frequency between GM1 monomers and either SUVs or LUVs and curvature-induced changes in the interfacial lipid packing in either SUVs or LUVs can dramatically influence spontaneous ganglioside uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impact of Ca2+ on membrane catalyzed IAPP amyloid formation and IAPP induced vesicle leakage

Human islet amyloid polypeptide (hIAPP, also known as amylin) is a 37 amino acid pancreatic polypeptide hormone that plays a role in regulating glucose levels, but forms pancreatic amyloid in type-2 diabetes. The process of amyloid formation by hIAPP contributes to β-cell death in the disease. Multiple mechanisms of hIAPP induced toxicity of β-cells have been proposed including disruption of cellular membranes. However, the nature of hIAPP membrane interactions and the effect of ions and other molecules on hIAPP membrane interactions are not fully understood. Many studies have used model membranes with a high content of anionic lipids, often POPS, however the concentration of anionic lipids in the β-cell plasma membrane is low. Here we study the concentration dependent effect of Ca²⁺ (0 to 50 mM) on hIAPP membrane interactions using large unilamellar vesicles (LUVs) with anionic lipid content ranging from 0 to 50 mol%. We find that Ca²⁺ does not effectively inhibit hIAPP amyloid formation and hIAPP induced membrane leakage from binary LUVs with a low percentage of POPS, but has a greater effect on LUVs with a high percentage of POPS. Mg²⁺ had very similar effects, and the effects of Ca²⁺ and Mg²⁺ can be largely rationalized by the neutralization of POPS charge. The implications for hIAPP-membrane interactions are discussed.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP^Sc, an abnormally misfolded, protease-resistant, and β-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP^Sc and presents similar neurotoxicity as PrP^Sc. By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.     

Riboflavin spraying impairs the antioxidant defense system but induces waterlogging tolerance in tobacco

Riboflavin, which causes plants to produce reactive oxygen species (ROS) when exposed to light, is an excellent photosensitizer for biocidal reactions. This study explores the possible protective role of riboflavin against waterlogging stress in tobacco plants. Tobacco seedlings (4 weeks old) were divided into four groups and pretreated with 0, 0.2, 0.5 or 1.0 mM riboflavin for 1 week, after which all groups were exposed to waterlogging stress for 7 days. We observed delayed leaf senescence and extended survival time, suggesting that riboflavin can confer increased waterlogging tolerance to plants as compared with the control (0 mM riboflavin). Enhanced stomatal closure was observed in the riboflavin-pretreated tobacco. We evaluated the levels of oxidative damage (H₂O₂ and lipid peroxidation), antioxidant enzyme (superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase) activity and antioxidant metabolites (including ascorbate and glutathione) in tobacco leaves that were pretreated with riboflavin. However, the results show that riboflavin pretreatment caused a decrease in chlorophyll content, antioxidant enzyme activity and redox values (AsA/DHA and GSH/GSSG), while causing a significant increase in lipid peroxidation, H₂O₂ accumulation and total ascorbate or glutathione content. In addition, the survival time and stomatal aperture of riboflavin-treated plants were significantly modified by exogenous application of GSH, well-known ROS scavenger. To explain the stomatal closure observed in tobacco plants, we propose a “damage avoidance” hypothesis based on riboflavin-mediated ROS toxicity. The protective function of the photosensitizer riboflavin may be highly significant for farming in frequently waterlogged areas.     

Peptide:lipid ratio and membrane surface charge determine the mechanism of action of the antimicrobial peptide BP100. Conformational and functional studies

The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism.     

Characterization and application of a new optical probe for membrane lipid domains

In this article, we characterize the fluorescence of an environmentally sensitive probe for lipid membranes, di-4-ANEPPDHQ. In large unilamellar lipid vesicles (LUVs), its emission spectrum shifts up to 30 nm to the blue with increasing cholesterol concentration. Independently, it displays a comparable blue shift in liquid-ordered relative to liquid-disordered phases. The cumulative effect is a 60-nm difference in emission spectra for cholesterol containing LUVs in the liquid-ordered state versus cholesterol-free LUVs in the liquid-disordered phase. Given these optical properties, we use di-4-ANEPPDHQ to image the phase separation in giant unilamellar vesicles with both linear and nonlinear optical microscopy. The dye shows green and red fluorescence in liquid-ordered and -disordered domains, respectively. We propose that this reflects the relative rigidity of the molecular packing around the dye molecules in the two phases. We also observe a sevenfold stronger second harmonic generation signal in the liquid-disordered domains, consistent with a higher concentration of the dye resulting from preferential partitioning into the disordered phase. The efficacy of the dye for reporting lipid domains in cell membranes is demonstrated in polarized migrating neutrophils.     

Biophysical properties of membrane lipids of anammox bacteria: I. Ladderane phospholipids form highly organized fluid membranes

Anammox bacteria that are capable of anaerobically oxidizing ammonium (anammox) with nitrite to nitrogen gas produce unique membrane phospholipids that comprise hydrocarbon chains with three or five linearly condensed cyclobutane rings. To gain insight into the biophysical properties of these ‘ladderane’ lipids, we have isolated a ladderane phosphatidylcholine and a mixed ladderane phosphatidylethanolamine/phosphatidylglycerol lipid fraction and reconstituted these lipids in different membrane environments. Langmuir monolayer experiments demonstrated that the purified ladderane phospholipids form fluid films with a relatively high lipid packing density. Fluid-like behavior was also observed for ladderane lipids in bilayer systems as monitored by cryo-electron microscopy on large unilamellar vesicles (LUVs) and epi-fluorescence microscopy on giant unilamellar vesicles (GUVs). Analysis of the LUVs by fluorescence depolarization revealed a relatively high acyl chain ordering in the hydrophobic region of the ladderane phospholipids. Micropipette aspiration experiments were applied to study the mechanical properties of ladderane containing lipid bilayers and showed a relatively high apparent area compressibility modulus for ladderane containing GUVs, thereby confirming the fluid and acyl chain ordered characteristics of these lipids. The biophysical findings in this study support the previous postulation that dense membranes in anammox cells protect these microbes against the highly toxic and volatile anammox metabolites.     

Cellular uptake and subcellular distribution of chlorin e6 as functions of pH and interactions with membranes and lipoproteins

The uptake and more importantly the subcellular distribution of photosensitizers are major determinants of their efficacy. In this paper, the cellular internalization of chlorin e6 (Ce6), a photosensitizer bearing three carboxylic chains, is considered with emphasize on pH effects. Small unilamellar vesicles are used as models to investigate the dynamics of interactions of Ce6 with membranes. The entrance and exit steps from the outer lipid hemileaflet are very fast (∼ ms). A slow transfer of Ce6 through the membrane was observed only for thin bilayers made of dimyristoleoyl-phosphatidylcholine. Ce6 did not permeate through bilayers consisting of longer phospholipids more representative of biological membranes. These results along with previous data on the interactions of Ce6 with low-density lipoproteins (LDL) are correlated with cellular studies. After 15 min incubation of HS68 human fibroblasts with Ce6, fluorescence microscopy revealed labeling of the plasma membrane and cytosolic vesicles different from lysosomes. When vectorized by LDL, Ce6 was mainly localized in lysosomes but absent from the plasma membrane. Internalization of LDL bound photosensitizer via ApoB/E receptor mediated pathway was demonstrated by overexpression experiments. A pH decrease from 7.4 to 6.9 did not affect the intracellular distribution of Ce6, but significantly increased its overall cellular uptake.