Fluorescent Membrane Probes Research Articles

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

Location, dynamics and solvent relaxation of a nile red-based phase-sensitive fluorescent membrane probe

Fluorescent membrane probes offer the advantage of high sensitivity, suitable time resolution, and multiplicity of measurable parameters, and provide useful information on model and cell membranes. In this paper, we have explored the location, dynamics, and solvent relaxation characteristics of a novel Nile Red-based phase-sensitive probe (NR12S). Unlike Nile Red, NR12S enjoys unique orientation and location in the membrane, and is localized exclusively in the outer leaflet of the membrane bilayer. By analysis of membrane depth using the parallax approach, we show that the fluorescent group in NR12S is localized at the membrane interface, a region characterized by slow solvent relaxation. Our results show that NR12S exhibits REES (red edge excitation shift), consistent with its interfacial localization. More interestingly, REES of NR12S displays sensitivity to the membrane phase. In addition, fluorescence emission maximum, anisotropy, and lifetime of NR12S are dependent on the membrane phase. We envision that NR12S may prove to be a useful probe in future studies of complex natural membranes.

Amphiphilic ESIPT benzoxazole derivatives as prospective fluorescent membrane probes

Fluorescent amphiphilic benzoxazole derivatives were synthesized and used to produce photoactive phosphatidylcholine (PC) liposomes by reserve-phase evaporation. The dyes absorbed in the UV region and were fluorescent in the blue-green region (determined by solvent polarity). The alkyl chain length seemed to play a fundamental role in the photophysics of the benzoxazole fluorophore in reverse liposomes, and despite the same ESIPT core and phospholipid building block, each amphiphilic dye had a particular emission profile related to the dye location in the liposome. The fluorescence emission spectra from dye 5 showed that its fluorophore experienced a polar environment, due to the single normal emission, while dyes 6–7 had (in part) a normal emission, and the main fluorescent band ascribed to the ESIPT emission indicated a more hydrophobic environment. Despite the complex fluorescent profiles, the benzoxazole derivatives could be successfully introduced into the reverse liposome structure due to the interaction between the alkyl chain and PC bilayer.

Endocytosis of Liposomes Containing Sulfo-Cerebrosides by an Astrocytomal Cell Line: Is it a Charge Mediated Process or Does it Involve a Specific Receptor?

Sulfo-cerebrosides (SCB) are glycosphingolipids found in high concentrations in the central nervous system (CNS), particularly in myelin where they play an important structural role. In addition, these lipids play an important role in various biological processes such as establishing a connection between the neuronal axon and myelin, and regulating cell growth and neuronal plasticity. Moreover, recent in-vitro studies from our group have shown that astrocytes avidly endocyte liposomes containing SCB without inducing a pro-inflammatory response. This indicates that astrocytes are involved in the homeostasis of the SCB in the CNS. In this work, we explore the specificity of the process of endocytosis of liposomes containing SCB, using an astrocytoma cell line as a model. Our hypothesis is that this process is receptor-mediated and not due to an electrostatic interaction with the cell surface. We show that (PG enriched) negatively charged liposomes do enhance endocytosis, but at a significantly lower level compared to SCB. We also show that specific uptake can be inhibited by screening the surface with polyethylene glycol. Additionally, we show through the use of endocytotic inhibitors that the SCB endocytotic route is a clatherin dependent route, which differs from non-specific liposome uptake routes. In these experiments, cells were cultured with liposomes labeled with the membrane fluorescent probe (Texas Red) to measure internalization, and a liposome content marker (calcein) to measure release. Measurements were carried out by flow cytometry and the results confirmed by fluorescence microscopy. Since low density lipoproteins (LDL) have been suggested to participate in SCB transport, we propose that the endocytotic pathway involves the LDL receptor (LDLr). Preliminary results show that SCB endocytosis is inhibited by LDLr specific antibodies.

Planarizable push–pull oligothiophenes: in search of the perfect twist

The “twistome” of push–pull oligothiophenes is covered comprehensively. Particular emphasis is on the development of conceptually innovative fluorescent membrane probes.

Newly synthesized benzanthrone derivatives as prospective fluorescent membrane probes

Fluorescence spectral properties of a series of novel benzanthrone derivatives have been explored in lipid bilayers composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with cholesterol (Chol) and anionic phospholipid cardiolipin (CL). Analysis of partition coefficients showed that all the examined compounds possess rather high lipid-associating ability, with the amidino derivatives exhibiting stronger membrane partitioning compared with the aminobenzanthrones. To understand how benzanthrone partition properties correlate with their structure, quantitative structure property relationship (QSPR) analysis was performed involving a range of quantum chemical molecular descriptors.

Rational design of fluorescent membrane probes for apoptosis based on 3-hydroxyflavone

Environment-sensitive probes constitute powerful tools for monitoring changes in the physico-chemical properties of cell plasma membranes. Among these probes, 3-hydroxyflavone probes are of great interest due to their dual emission and ratiometric response. Here, three probes derived from the parent F2N12S were designed, characterized and applied to monitor the membrane changes occurring during apoptosis. These three probes were designed to orient the dye vertically in the membrane. They differ by the length of their alkyl chains (from 4 to 8 carbons), which were included to optimize their affinity to the lipid membranes. Among these three probes, the one with medium chain length (hexyl) showed the best affinity to model and cell membranes, while the one with the longest alkyl chains (octyl) did not efficiently stain the membranes, probably due to aggregation. The new probes were found to be more sensitive than F2N12S to both the lipid phase and surface charge in lipid vesicles and to loss of lipid order in cell plasma membranes after cholesterol extraction. The one with the shortest (butyl) chains was found to be the most sensitive to apoptosis, while the one with medium-length (hexyl) chains was the brightest. Interestingly, apoptosis induced by different agents led to similar spectroscopic effects to those produced by the loss of lipid order and change in the surface charge, confirming that apoptosis decreases the lipid order and increases the negative surface charge in the outer leaflet of cell membranes. In conclusion, these studies report the relationship between the probe structures and their sensitivity to lipid order, surface charge and apoptosis and propose new probes for membrane research.

Quantum chemical simulation of the interaction of membrane fluorescent probe 4-dimethylaminochalcone with hydroxy groups of the environment

The quantum chemical simulation of the ground and electron-excited states of diverse complexes of fluorescent probe 4-dimethylaminochalcone (DMAC) and water in vacuo was performed by the HF/MP2 and RI-CC2 methods. Molecules of the DMAC probe and water can form five types of stable complexes. The geometries corresponding to the potential energy minimum and dipole moments for two lowest singlet and one lowest triplet states were calculated for each type of the complexes. The partial charges on the DMAC atoms and their changes due to the intramolecular charge transfer upon photoexcitation were determined. The coordination of the water molecule at the carbonyl group of DMAC is preferable in vacuo. The formation of hydrogen bonds between the carbonyl group of DMAC and water molecules decreases the energy of the excited state of the complex 1(π, π*), due to which the fluorescence yield increases upon photoexcitation. The calculation results are confirmed by the experimental data on studying the fluorescence of the probe in binary mixtures of benzene and alcohols.

Differential oligomeric nature of mEos2 and mEos3.2 fluorescent proteins is consequential to diffusion and confinement of membrane probes

Lipid‐mediated membrane heterogeneity, often referred to as lipid rafts, is proposed to be an important organizing principle in mammalian cells. Using super‐resolution fluorescence localization imaging we track the diffusion of a large panel of fluorescent membrane probes ranging in size, mode of membrane anchoring, and putative ‘raft’ or ‘non‐raft’ phase‐association. Our expressed probes include fluorescently‐tagged palmitoylated or non‐palmitoylated versions of transmembrane domains including linker of activated T‐cell and hemagglutinin, and fluorescent protein anchored by GPI, palmitate and myristoyl moieties, or a geranyl‐geranyl moiety. The recent advent of photoconvertible fluorescent protein mEos3.2, a “true monomeric” mutant of its mEos2 predecessor1, has enabled us to compare directly monomeric and oligomeric versions of the same probes. Our results indicate that in some cases rates of diffusion are more than two fold lower in the mEos2 probes when compared to their mEos3.2 counterparts. Many mEos2‐labeled probes also differ from mEos3.2‐labeled probes in their exponents of anomalous diffusion. We attribute changes in mobility to the proteins’ differential propensity to oligomerize, and find that larger probes differentiate themselves more than smaller ones with respect to phase‐mediated mobility differences. We are using the clustering effect of mEos2, therefore, as a tool to elucidate phase heterogeneity. Funding for this project was provided by NIH: SV R00GM087810 and a start up grant from the University of Michigan.

Improved membrane fluidity of ionic polysaccharide bead-supported phospholipid bilayer membrane systems

Supported phospholipid bilayer membranes on polysaccharide-based cationic polymer beads (cationic group: –[OCH2CH(OH)CH2]2N+(CH3)3·X−, 45–165μm in diameter) were prepared using small unilamellar vesicles from mixtures of phosphatidylserine (PS) and phosphatidylcholine (PC). Confocal fluorescence microscopic observations with a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the phospholipid-bead complexes were along the outer surface of the beads. The fluidity of the phospholipid bilayer membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after photobleaching (FRAP) technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were lower than that for the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supports. Such less fluid membranes in the complexes appeared to be due to the immobilization of the phospholipid bilayer membranes by electrostatic attractive forces between PS and the bead. The D values for the PS/PC-bead complexes were dependent on the phospholipid composition; the PS(100mol%)/PC(0mol%)-bead complex had the least fluid membranes among the PS/PC-bead complexes tested in this study. The phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer beads were much more fluid than those on a polystyrene-based one. Furthermore, such fluid phospholipid bilayer membranes formed on the polysaccharide-based cationic polymer bead were maintained for 10 days, even though the complex sample was stood in plain buffer (pH 8.5) at ambient temperature.

The New Fluorescent Membrane Probe Ahba: A Comparative Study with the Largely Used Laurdan

Lipid bilayers have been largely used as model systems for biological membranes. Hence, their structures, and alterations caused on them by biological active molecules, have been the subject of many studies. Accordingly, fluorescent probes incorporated into lipid bilayers have been extensively used for characterizing lipid bilayer fluidity and/or polarity. However, for the proper analysis of the alterations undergone by a membrane, a comprehensive knowledge of the fluorescent properties of the probe is fundamental. Therefore, the present work compares fluorescent properties of a relative new fluorescent membrane probe, 2-amino-N-hexadecyl-benzamide (Ahba), with the largely used probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan), using both static and time resolved fluorescence. Both Ahba and Laurdan have the fluorescent moiety close to the bilayer surface; Ahba has a rather small fluorescent moiety, which was shown to be very sensitive to the bilayer surface pH. The main goal was to point out the fluorescent properties of each probe that are most sensitive to structural alterations on a lipid bilayer. The two probes were incorporated into bilayers of the well-studied zwitterionic lipid dimyristoyl phosphatidylcholine (DMPC), which exhibits a gel-fluid transition around 23 °C. The system was monitored between 5 and 50 °C, hence allowing the study of the two different lipid structures, the gel and fluid bilayer phases, and the transition between them. As it is known, the fluorescent emission spectrum of Laurdan is highly sensitive to the bilayer gel-fluid transition, whereas the Ahba fluorescence spectrum was found to be insensitive to changes in bilayer structure and polarity, which are known to happen at the gel-fluid transition. However, both probes monitor the bilayer gel-fluid transition through fluorescence anisotropy measurements. With time-resolved fluorescence, it was possible to show that bilayer structural variations can be monitored by Laurdan excited state lifetimes changes, whereas Ahba lifetimes were found to be insensitive to bilayer structural modifications. Through anisotropy time decay measurements, both probes could monitor structural bilayer changes, but the limiting anisotropy was found to be a better parameter than the rotational correlation time. It is interesting to have in mind that the relatively small fluorophore of Ahba (o-Abz) could possibly be bound to a phospholipid hydrocarbon chain, not disturbing much the bilayer packing and being a sensitive probe for the bilayer core.

Characterization of a New Series of Fluorescent Probes for Imaging Membrane Order

Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.

Combined Use of Several Fluorescent Membrane Probes to Study the Subgel Phase and the Effects of Cholesterol Thereon

Several lines of evidence have indicated that a highly ordered and condensed phase occurs for phosphatidylcholines at low temperatures. Generation of this lamellar subgel phase (Lc) with dipalmitoylphosphatidylcholine (DPPC) depends on the thermal history of the sample; vesicles must be cooled at ∼4°C for at least 48 h. Similarly, samples exit the Lc phase slowly at the sub-transition temperature (18°C). Consequently, vestiges of Lc behavior can still be detected at 25°C within 15 min of passing through the transition temperature. We used this hysteresis as an assay for detecting the Lc phase with fluorescent membrane probes by recording the emission spectrum or anisotropy on refrigerated vesicles at 15, 25, 35, and 45°C followed by a return to 35, 25, and finally 15°C. In each case, samples were incubated for 10 min at the new temperature before data acquisition. The presence of a Lc or Lc-like phase was recognized by lack of reversal of fluorescent properties at 25 or 15°C. The Lc phase was observed for pure DPPC vesicles by merocyanine 540, prodan, and patman but not laurdan or diphenylhexatriene; fluorescence properties of the latter two probes were completely reversible at all temperatures. Inclusion of cholesterol in the vesicles up to 20 mol% caused a linear reduction in the magnitude of the difference between membrane properties detected by these probes before and after vesicle heating. In contrast to previous reports using other techniques, the lack of reversal was not completely eradicated by cholesterol in the range of 20 to 45 mol% suggesting that an Lc-like phase exists for DPPC/cholesterol mixtures. The distinction between probes that can and cannot detect these phases has implications for interpreting the nature of them.

Octyl-β-D-Glucopyranoside Shows Composition Dependent Disordering Effects in Ternary Lipid Bilayers

Solubilization of lipid bilayers by the nonionic detergent OG has been well characterized in one and two component lipid systems, but has not been extensively studied in ternary membranes containing mixed fluid phases. In order to understand the effects of membrane order on solubilization by OG, LUVs of pure POPC were compared to those composed of POPC plus varying fractions of sphingomyelin (SM) and cholesterol. Detergent partitioning from the aqueous phase into the bilayer and membrane solubilization were monitored by ITC. The fluorescent membrane probe, DPH, was observed by time-resolved fluorescence intensity and polarization decay in the presence of increasing detergent concentrations to determine the effects on membrane order and dynamics. The results of partitioning experiments showed approximately a 50% decrease in the partition constant between pure POPC and the most ordered samples. ITC solubilization experiments showed clear boundaries for the micelle-bilayer mixed aggregate coexistence region in samples of low initial order, but the distinct thermodynamic signature associated with this coexistence region was not present in the cholesterol and SM rich samples. The average excited state lifetime of DPH, an indicator of water penetration, in vesicles rich in POPC showed a rapid increase at the onset of solubilization, whereas vesicles rich in SM and cholesterol showed a corresponding rapid decrease. Dynamic fluorescence depolarization data analyzed in terms of a Brownian rotational diffusion model revealed an increase in the occupancy of the bilayer midplane by DPH prior to solubilization in less ordered membranes coupled with a constant rotational correlation time. Membranes with higher initial order showed little increase in midplane occupancy and increasing rotational correlation time in the same detergent concentration range, suggesting that two modes of membrane disruption are possible and depend on the lipid makeup of the bilayer.

The Apoptotic Bile Acid DCA has Preference for Association to Liquid Disordered Lipid Domains and Inhibits the Rigidifying Effect of Cholesterol in Membranes

Hydrophobic bile acids (BAs) such as deoxycholic acid (DCA) induce cell death via multiple pathways, including both the intrinsic and extrinsic pathways of apoptosis. On the other hand, it has been shown that hydrophilic BAs, particularly ursodeoxycholic (UDCA) and tauroursodeoxycholic (TUDCA) acids prevent apoptosis. Still, the mechanisms by which these very similar BAs trigger opposite signaling effects remain unclear. We hypothesize that cellular membranes may constitute a primary target for the modulation of BAs during apoptosis.Here, fluorescent derivatives of DCA and UDCA were studied. Both BAs partitioned with higher affinity to liquid disordered than to the cholesterol-enriched liquid ordered domains. From studies with membrane fluorescent probes, unlabeled BAs are shown to have a superficial location in the lipid membrane upon binding. Additionally, the interaction of DCA with lipid membranes resulted in a significant expansion in membrane surface area, as observed by a FRET experiment. This effect might be related to changes in membrane permeability observed after incubation of both mitochondria and model membranes with hydrophobic BAs. Importantly, it is shown that only DCA is able to significantly disrupt the ordering of the membrane by cholesterol, as seen by the fluorescence anisotropy of membrane probes. In this way, it is possible that the apoptotic effects of BAs are achieved through modulation of lipid raft formation. Alternatively, since it has been reported that the oligomerization of the apoptotic protein Bax is dependent on membrane fluidity, DCA-induced apoptosis could be the result of an increase in mitochondrial membrane fluidity, which promotes Bax oligomerization and triggers apoptosis.Acknowledgements: FCT Portugal is acknowledged for financial support (PTDC/QUI-BIQ/119494/2010; PTDC/QUI-BIQ/112067/2009; SFRH/BPD/64320/2009).

Differential Oligomeric Nature of mEos2 and mEos3.2 Fluorescent Proteins is Consequential to Diffusion and Confinement of Membrane Probes

Lipid-mediated membrane heterogeneity is proposed to be an important organizing principle in mammalian cells. using super-resolution fluorescence localization imaging we track the diffusion of a large panel of fluorescent membrane probes ranging in size, mode of membrane anchoring, and putative phase-association. Our expressed probes include fluorescently-tagged palmitoylated or non-palmitoylated versions of transmembrane domains including linker of activated T-cell and haemagglutinin, and fluorescent protein anchored by GPI, palmitate-myristoyl moieties, or a geranyl-geranyl moiety. The recent advent of photoconvertible fluorescent protein mEos3.2, a “truly monomeric” mutant of its mEos2 predecessor1, has enabled us to compare directly monomeric and oligomeric versions of the same probes. Our results indicate that in some cases rates of diffusion are more than two fold lower in the mEos2 probes when compared to their mEos3.2 counterparts. Many mEos2-labeled probes also differ from mEos3.2-labeled probes in their exponents of anomalous diffusion. We attribute changes in mobility to the proteins' differential propensity to oligomerize, and are using the clustering effect of mEos2 to investigate whether phase heterogeneity influences cluster mobility.1 Zhang, M. et al. Rational design of true monomeric and bright photoactivatable fluorescent proteins. Nat Methods 9, 727-729. (2012).

Selectivity for and destruction of Salmonella typhimurium via a membrane damage mechanism of a cell-penetrating peptide ppTG20 analogue

P7, an analogue of the cell-penetrating peptide (CPP) ppTG20, was derived by replacing Phe and Trp with Arg based on the structure–activity relationships of CPPs and antimicrobial peptides (AMPs). P7 showed antimicrobial activity against Salmonella typhimurium at 4μM and possessed broad antimicrobial activity greater than its parent peptide. P7 displayed good selectivity, with low haemolysis below its minimum inhibitory concentration range, but displayed cytotoxic activity against the HT29 and MDA-MB231 mammalian cell lines. Studies of calcein leakage from egg yolk l-α-phosphatidylcholine/egg yolk l-α-phosphatidyl-dl-glycerol (EYPC/EYPG) (bacterial membrane mimic) and EYPC/cholesterol (eukaryotic membrane mimic) vesicles also demonstrated that P7 exhibited high selectivity and caused pore formation in the bacterial membrane. Circular dichroism experiments suggested that the conformation of P7 underwent transitions from a random coil in sodium phosphate buffer to an α-helical conformation in bacterial model membranes. P7 induced influx of the membrane fluorescent probe 1-N-phenylnaphthylamine (NPN) and the nucleic acid fluorescent probe SYTOX® Green by increasing live S. typhimurium cell outer membrane and plasma membrane permeability, respectively. P7 also induced ion channel formation in the cell plasma membrane causing leakage of potassium ions. Flow cytometric analysis demonstrated that S. typhimurium cell membrane integrity was destroyed following incubation with P7. These results indicated that P7 exhibited good bacterial selectivity and exerted its antibacterial activity by a membrane damage mechanism. Furthermore, these results suggested that CPPs may represent a source of templates for AMP design.

Biophysical properties of ergosterol-enriched lipid rafts in yeast and tools for their study: characterization of ergosterol/phosphatidylcholine membranes with three fluorescent membrane probes

In this work, binary mixtures of phospholipid/ergosterol (erg) were studied using three fluorescent membrane probes. The phospholipid was either saturated (1,2-dipalmitoyl-sn-glycero-3-phosphocholine, DPPC) or monounsaturated (1-palmitoyl-2-dioleoyl-sn-glycero-3-phosphocholine, POPC) phosphatidylcholine, to evaluate the fluorescence properties of the probes in gel, liquid ordered (lo) and liquid disordered (ld) phases. The probes have been used previously to study cholesterol-enriched domains, but their photophysical properties in erg-enriched membranes have not been characterized.N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-DPPE) presents modest blue-shifts upon erg addition, and the changes in the fluorescence lifetime are mainly due to differences in the efficiency of its fluorescence dynamic self-quenching. However, the steady-state fluorescence anisotropy of NBD-DPPE presents well-defined values in each lipid phase.N-(lissamine rhodamine B sulfonyl)-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (Rhod-DOPE) presents a close to random distribution in erg-rich membranes. There are no appreciable spectral shifts and the steady-state fluorescence anisotropy presents complex behavior, as a result of different photophysical processes. The probe is mostly useful to label ld domains in yeast membranes.4-(2-(6-(Dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium (di-4-ANEPPS) is an electrochromic dye with excitation spectra largely insensitive to the presence of erg, but presenting a strong blue-shift of its emission with increasing concentrations of this sterol. Its partition coefficient is favorable to lo domains in POPC/erg mixtures. Although the fluorescence properties of di-4-ANEPPS are less sensitive to erg than to chol, in both cases the fluorescence lifetime responds monotonically to sterol mole fraction, becoming significantly longer in the presence of sterol as compared to pure POPC or DPPC bilayers. The probe displays a unique sensitivity to sterol–lipid interaction due to the influence of hydration and H-bonding patterns at the membrane/water interface on its fluorescence properties. This makes di-4-ANEPPS (and possibly similar probes) potentially useful in the study of erg-enriched domains in more complex lipid mixtures and in the membranes of living yeast cells.

Reorganization of lipid domain distribution in giant unilamellar vesicles upon immobilization with different membrane tethers

Characterization of phase coexistence in biologically relevant lipid mixtures is often carried out through confocal microscopy of giant unilamellar lipid vesicles (GUVs), loaded with fluorescent membrane probes. This last analysis is generally limited to the vesicle hemisphere further away from the coverslip, in order to avoid artifacts induced by the interaction with the solid surface, and immobilization of vesicles is in many cases required in order to carry out intensity, lifetime or single‐molecule based microscopy. This is generally achieved through the use of membrane tethers adhering to a coverslip surface. Here, we aimed to determine whether GUV immobilization through membrane tethers induces changes in lipid domain distribution within liposomes displaying coexistence of lipid lamellar phases. Confocal imaging and a Förster resonance energy transfer (FRET) methodology showed that biotinylated phospholipids present significantly different membrane phase partition behavior upon protein binding, depending on the presence or absence of a linker between the lipid headgroup and the biotinyl moiety. Membrane phases enriched in a membrane tether displayed in some cases a dramatically increased affinity for the immobilization surface, effectively driving sorting of lipid domains to the adherent membrane area, and in some cases complete sequestering of a lipid phase to the interaction surface was observed. On the light of these results, we conclude that tethering of lipid membranes to protein surfaces has the potential to drastically reorganize the distribution of lipid domains, and this reorganization is solely dictated by the partition properties of the protein–tether complex.

Fluorescence microscopic characterization of ionic polymer bead-supported phospholipid bilayer membrane systems

Supported phospholipid membrane structures on cationic organic polymer beads were prepared using mixtures of dioleoylphosphatidylserine (PS) and egg yolk phosphatidylcholine (PC). Confocal fluorescence microscopic observations using a fluorescent membrane probe (N-4-nitrobenzo-2-oxa-1,3-diazole-phosphatidylethanolamine) revealed that the phospholipid molecules in the PS/PC-bead complexes were along the outer surface of the beads, but not inside the beads. The anionic PS on the most outer surface of the PS/PC-bead complexes was responsible for the binding of a positively charged macromolecule, rhodamine isothiocyanate dextran (Mw 70,000) by electrostatic attractive forces. The fluidity of the membranes in the PS/PC-bead complexes was investigated by the fluorescence recovery after a photobleaching technique. The lateral diffusion coefficients (D) for the PS/PC-bead complexes were one-half or less than that for 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine giant unilamellar vesicles without solid supporting materials. Such a constrain of the phospholipid bilayer membrane in the complexes appeared to be due to its immobilization on the cationic polymer bead by electrostatic attractive forces between the PS and ammonium group on the surface of the bead. The D values for the complexes were dependent on the phospholipid composition; the PS(25mol%)/PC(75mol%)-bead complex produced a more fluid membrane than the PS(50mol%)/PC(50mol%)-bead one. Thus, the fluidity of the phospholipid bilayer membranes formed on the cationic polymer beads was significantly affected by the anionic phospholipid fraction used for the preparation of the complexes.