Domains In Giant Unilamellar Vesicles Research Articles

Imaging Giant Vesicle Membrane Domains with a Luminescent Europium Tetracycline Complex.

Microdomains in lipid bilayer membranes are routinely imaged using organic fluorophores that preferentially partition into one of the lipid phases, resulting in fluorescence contrast. Here, we show that membrane microdomains in giant unilamellar vesicles (GUVs) can be visualized with europium luminescence using a complex of europium III (Eu3+) and tetracycline (EuTc). EuTc is unlike typical organic lipid probes in that it is a coordination complex with a unique excitation/emission wavelength combination (396/617 nm), a very large Stokes shift (221 nm), and a very narrow emission bandwidth (8 nm). The probe preferentially interacts with liquid disordered domains in GUVs, which results in intensity contrast across the surface of phase-separated GUVs. Interestingly, EuTc also alters GM1 ganglioside partitioning. GM1 typically partitions into liquid ordered domains, but after labeling phase-separated GUVs with EuTc, cholera toxin B-subunit (CTxB), which binds GM1, labels liquid disordered domains. We also demonstrate that EuTc, but not free Eu3+ or Tc, significantly reduces lipid diffusion coefficients. Finally, we show that EuTc can be used to label cellular membranes similar to a traditional membrane probe. EuTc may find utility as a membrane imaging probe where its large Stokes shift and sharp emission band would enable multicolor imaging.

Defining raft domains in the plasma membrane

Many plasma membrane (PM) functions depend on the cholesterol concentration in the PM in strikingly nonlinear, cooperative ways: fully functional in the presence of physiological cholesterol levels (35~45 mol%), and nonfunctional below 25 mol% cholesterol; namely, still in the presence of high concentrations of cholesterol. This suggests the involvement of cholesterol-based complexes/domains formed cooperatively. In this review, by examining the results obtained by using fluorescent lipid analogs and avoiding the trap of circular logic, often found in the raft literature, we point out the fundamental similarities of liquid-ordered (Lo)-phase domains in giant unilamellar vesicles, Lo-phase-like domains formed at lower temperatures in giant PM vesicles, and detergent-resistant membranes: these domains are formed by cooperative interactions of cholesterol, saturated acyl chains, and unsaturated acyl chains, in the presence of >25 mol% cholesterol. The literature contains evidence, indicating that the domains formed by the same basic cooperative molecular interactions exist and play essential roles in signal transduction in the PM. Therefore, as a working definition, we propose that raft domains in the PM are liquid-like molecular complexes/domains formed by cooperative interactions of cholesterol with saturated acyl chains as well as unsaturated acyl chains, due to saturated acyl chains' weak multiple accommodating interactions with cholesterol and cholesterol's low miscibility with unsaturated acyl chains and TM proteins. Molecules move within raft domains and exchange with those in the bulk PM. We provide a logically established collection of fluorescent lipid probes that preferentially partition into raft and non-raft domains, as defined here, in the PM.

Micron-sized domains in quasi single-component giant vesicles

Giant unilamellar vesicles (GUVs), are a convenient tool to study membrane-bound processes using optical microscopy. An increasing number of studies highlights the potential of these model membranes when addressing questions in membrane biophysics and cell-biology. Among them, phase transitions and domain formation, dynamics and stability in raft-like mixtures are probably some of the most intensively investigated. In doing so, many research teams rely on standard protocols for GUV preparation and handling involving the use of sugar solutions. Here, we demonstrate that following such a standard approach can lead to the abnormal formation of micron-sized domains in GUVs grown from only a single phospholipid. The membrane heterogeneity is visualized by means of a small fraction (0.1 mol%) of a fluorescent lipid dye. For dipalmitoylphosphatidylcholine GUVs, different types of membrane heterogeneities were detected. First, the unexpected formation of micron-sized dye-depleted domains was observed upon cooling. These domains nucleated about 10 K above the lipid main phase transition temperature, TM. In addition, upon further cooling of the GUVs down to the immediate vicinity of TM, stripe-like dye-enriched structures around the domains are detected. The micron-sized domains in quasi single-component GUVs were observed also when using two other lipids. Whereas the stripe structures are related to the phase transition of the lipid, the dye-excluding domains seem to be caused by traces of impurities present in the glucose. Supplementing glucose solutions with nm-sized liposomes at millimolar lipid concentration suppresses the formation of the micron-sized domains, presumably by providing competitive binding of the impurities to the liposome membrane in excess. It is likely that such traces of impurities can significantly alter lipid phase diagrams and cause differences among reported ones.

White-Fluorescent Dual-Emission Mechanosensitive Membrane Probes that Function by Bending Rather than Twisting.

Bent N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine amphiphiles are introduced as mechanosensitive membrane probes that operate by an unprecedented mechanism, namely, unbending in the excited state as opposed to the previously reported untwisting in the ground and twisting in the excited state. Their dual emission from bent or “closed” and planarized or “open” excited states is shown to discriminate between micelles in water and monomers in solid‐ordered (So), liquid‐disordered (Ld) and bulk membranes. The dual‐emission spectra cover enough of the visible range to produce vesicles that emit white light with ratiometrically encoded information. Strategies to improve the bent mechanophores with expanded π systems and auxochromes are reported, and compatibility with imaging of membrane domains in giant unilamellar vesicles by two‐photon excitation fluorescence (TPEF) microscopy is demonstrated.

Fluid domain patterns in free-standing membranes captured on a solid support

We devise a methodology to fixate and image dynamic fluid domain patterns of giant unilamellar vesicles (GUVs) at sub-optical length scales. Individual GUVs are rapidly transferred to a solid support forming planar bilayer patches. These are taken to represent a fixated state of the free standing membrane, where lateral domain structures are kinetically trapped. High-resolution images of domain patterns in the liquid-ordered (lo) and liquid-disordered (ld) co-existence region in the phase-diagram of ternary lipid mixtures are revealed by atomic force microscopy (AFM) scans of the patches. Macroscopic phase separation as known from fluorescence images is found, but with superimposed fluctuations in the form of nanoscale domains of the lo and ld phases. The size of the fluctuating domains increases as the composition approaches the critical point, but with the enhanced spatial resolution, such fluctuations are detected even deep in the coexistence region. Agreement between the area-fraction of domains in GUVs and the patches respectively, supports the assumption that the thermodynamic state of the membrane remains stable. The approach is not limited to specific lipid compositions, but could potentially help uncover lateral structures in highly complex membranes.

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Synthesizing GUV from Wet Lipid Film to Achieve Better Uniformity in Lipid Composition

Giant Unilamellar Vesicles (GUVs) are cell-sized model membrane systems that allow direct investigation of membrane-related phenomena using fluorescence microscopy. Understanding the organization and dynamics of lipid bilayers is important for understanding the processes taking place in cell membranes. Previous work showed that micron-scale domains in GUV can be observed in some ternary lipid mixtures composed of a high melting temperature lipid, a low melting temperature lipid and cholesterol. However, it is a well-known problem that GUVs synthesized by the electroformation from dry lipid films have a wide distribution of transition temperature and lipid composition. In this study, we synthesized DOPC/DSPC/cholesterol GUVs and diPhyPC/DPPC/cholesterol GUVs by the standard electroformation method from dry lipid films as well as by a modified method using wet liposomes made from the Rapid Solvent Exchange (RSE) method. We quantified the lipid composition distributions of GUVs synthesized by the two methods by measuring the transition temperature distributions of GUVs using fluorescence microscopy; since a narrower distribution of transition temperature should correspond to a more uniform distribution in GUV lipid composition. Our results show that GUVs synthesized from wet lipid film have a better uniformity in lipid composition, and the standard deviations of transition temperature are about 3 times smaller than that of GUVs prepared from dry lipid films. This improved method not only gives a better control of GUV lipid composition, but also has a potential of synthesizing GUVs from cell membranes containing native proteins without going through a dry film state.

Translational and rotational diffusion of micrometer-sized solid domains in lipid membranes

We use simultaneous observation of translational and rotational Brownian motion of domains in lipid membranes to test the hydrodynamics-based theory for the viscous drag on the membrane inclusion. We find that translational and rotational diffusion coefficients of micrometer-sized solid (gel-phase) domains in giant unilamellar vesicles showing fluid–gel phase coexistence are in excellent agreement with the theoretical predictions.

The Phase Diagram for the Four-Component DSPC/DOPC/POPC/CHOL Mixture

When liquid-ordered (Lo) and liquid-disordered (Ld) phases coexist in mixtures of DSPC/DOPC/Chol, phase domains appear as large round domains in giant unilamellar vesicles (GUVs) examined by fluorescence microscopy. However, when liquid-ordered and liquid-disordered phases coexist in DSPC/POPC/Chol mixtures, domain size is too small to be detected by fluorescence microscopy. The phase diagram of the four-component DSPC/DOPC/POPC/Chol mixture allows for the exploration of the transition from macroscopic-to-nanoscopic domains. We have found complex shapes (modulated phase morphology) in a particular region of composition within the liquid-ordered and liquid-disordered coexistence region in DSPC/DOPC/POPC/Chol mixtures. By controlling lipid composition, we see distinct types of modulated liquid-liquid phase morphologies, including linear, irregular, and angular features in GUVs. These studies show that both the size and morphology of membrane rafts can be controlled by adjusting the composition and the type of low-melting lipid in mixtures with high-melting lipid and cholesterol.

Making a Tool of an Artifact: The Application of Photoinduced Lo Domains in Giant Unilamellar Vesicles to the Study of Lo/Ld Phase Spinodal Decomposition and Its Modulation by the Ganglioside GM1

Electroformed giant unilamellar vesicles containing liquid-ordered Lo domains are important tools for the modeling of the physicochemical properties and biological functions of lipid rafts. Lo domains are usually imaged using fluorescence microscopy of differentially phase-partionioning membrane-embedded probes. Recently, it has been shown that these probes also have a photosensitizing effect that leads to lipid chemical modification during the fluorescence microscopy experiments. Moreover, the lipid reaction products are able as such to promote Lo microdomain formation, leading to potential artifacts. We show here that this photoinduced effect can also purposely be used as a new approach to study Lo microdomain formation in giant unilamellar vesicles. Photosensitized lipid modification can promote Lo microdomain appearance and growth uniformly and on a faster time scale, thereby yielding new information on such processes. For instance, in egg phosphatidylcholine/egg sphingomyelin/cholesterol 50:30:20 (mol/mol) giant unilamellar vesicles, photoinduced Lo microdomain formation appears to occur by the rarely observed spinodal decomposition process rather than by the common nucleation process usually observed for Lo domain formation in bilayers. Moreover, temperature and the presence of the ganglioside GM1 have a profound effect on the morphological outcome of the photoinduced phase separation, eventually leading to features such as bicontinuous phases, phase percolation inversions, and patterns evoking double phase separations. GM1 also has the effect of destabilizing Lo microdomains. These properties may have consequences for Lo nanodomains stability and therefore for raft dynamics in biomembranes. Our data show that photoinduced Lo microdomains can be used to obtain new data on fast raft-mimicking processes in giant unilamellar vesicles.

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: Implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (ld) domains on the surface of GUVs composed of 40:60mol% DOPC/DPPC, over a wide range of temperatures (14–25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of LissamineTM rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.

The use of Light-Induced lo Domains in Giant Unilamellar Vesicles to Mimick raft Dynamics: Application to the Effect of the Ganglioside GM1

Cholesterol and sphingolipid-enriched raft lipid domains play important roles in numerous cellular processes. Recently, giant unilamellar vesicles containing lo domains have become valuable tools for the modeling of raft properties. One topic which has been adressed is the dynamics of micrometer-sized lo domains formation in GUVs. This is usually done by varying the lipid composition and temperature. However, these slow procedures cannot reproduce the very dynamics of raft formation and size modulation in biomembranes, that can occur in subsecond timescales. Here, we propose a simple method which allows one to monitor the dynamics of lo domain formation in GUVs on a faster timescale. The method is derived from the initial observation by several authors of a photosensitizing effect of the fluorescent probes used for lo domain detection in GUVs, which promotes lipid oxidation. Such oxidized lipids are able as such to induce raft formation. It has been relevantly emphasized that such photooxidation may lead to artifacts. Here, we show that such oxidation-induced lo domain formation can also be purposely used, since it provides a way to trigger raft-type microdomain growth in GUVs on a faster timescale, relevant to cellular processes, and to study the effect of any parameter on such dynamics. We illustrate the usefulness of this approach by studying the effect of the ganglioside GM1, an essential component of cellular rafts. We show that GM1 has a profound influence on lo domain formation, dynamics and stability in GUVs, which may bear relevance to physiological situations.

Lipid domains in giant unilamellar vesicles and their correspondence with equilibrium thermodynamic phases: A quantitative fluorescence microscopy imaging approach

We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram's tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.

Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative

Cholesterol-rich, liquid-ordered (L o) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (L d) domains, the number of such analogs with a high affinity for biologically relevant L o domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The L d phase domains were also labeled with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI-C 12) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 ± 0.08 ns, compared with 4.1 ± 0.4 ns in the presence of DiI-C 12. The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C 12 (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 ± 0.2 nm. These results also indicate preferential partitioning ( K p = 1.88) of Bdp-Chol into the L o phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence correlation spectroscopy, is (7.4 ± 0.3) × 10 −8 cm 2/s and (5.0 ± 0.2) × 10 −8 cm 2/s in the L d and L o phases, respectively. Experimental translational/rotational diffusion coefficient ratios are compared with theoretical predictions using the hydrodynamic model (Saffman-Delbrück). The results suggest that Bdp-Chol is likely to form a complex with other lipid molecules during its macroscopic diffusion in GUV lipid bilayers at room temperature. Our integrated, multiscale results demonstrate the potential of this cholesterol analog for studying lipid-lipid interactions, lipid order, and membrane fluidity of biologically relevant L o domains.

The fluorescent cholesterol analog dehydroergosterol induces liquid-ordered domains in model membranes

The fluorescent sterol dehydroergosterol (DHE) is often used as a marker for cholesterol in cellular studies. We show by vesicle fluctuation analysis that DHE has a lower ability than cholesterol to stiffen lipid bilayers suggesting less efficient packing with phospholipid acyl chains. Despite this difference, we found by fluorescence and atomic force microscopy, that DHE induces liquid-ordered/-disordered coexistent domains in giant unilamellar vesicles (GUVs) and supported bilayers made of dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylcholine (DOPC) and DHE or cholesterol. DHE-induced phases have a height difference of 0.9–1 nm similar as known for cholesterol-containing domains. DHE not only promotes formation of liquid–liquid immiscibility but also shows strong partition preference for the liquid-ordered phase further supporting its suitability as cholesterol probe.

Detection of Lipid Domains in Model and Plasma Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate that lipid domains in plasma membranes of mammalian cells are short lived and in the submicron range. To unravel the lateral heterogeneity of cellular membranes, in particular of mammalian plasma membranes, at this scale various techniques of fluorescence spectroscopy and microscopy have been applied. Measuring fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in Giant Unilamellar Vesicles (GUVs) and the plasma membrane of mammalian cells by Fluorescence Lifetime Imaging (FLIM). For GUVs forming microscopically visible lipid domains the fluorescence lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct being about 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM. Application of this approach to mammalian cells revealed that while the fluorescence lifetime is sensitive to the composition of the plasma membrane, distinct lipid domains as found for GUVs were not detected. A broad distribution of the long lifetime was found for C6-NBD-PC inserted in the plasma membrane of these cells centred around 11 ns. However, FLIM studies on lipid domain forming giant vesicles derived from the plasma membrane of HeLa-cells rather support a recent hypothesis that an ensemble of lipid domains being of submicroscopic size exist in the plasma membrane.

Lipid Domains In Giant Vesicles Composed Of Ternary Lipid Mixtures Containing Cholesterol And Their Relationship With Thermodynamic Phases

Fluorescence microscopy related techniques provide a powerful tool for direct observation of lipid domains in giant unilamellar vesicles (GUVs) [1]. Using these techniques it was reported that liquid-ordered (lo) - liquid-disordered (ld) phase coexistence can be observed in GUVs composed of cholesterol containing ternary lipid mixtures [1,2]. However, still it is not rigorously established if the lipid domains observed in these GUVs correspond to real thermodynamic phases. Recently we introduced a new method to measure the area fractions of the coexisting lipid domains in GUVs [3]. This novel procedure that involves deconvolution and segmentation of the individual GUV's fluorescence image stacks (including fitting with 3D surface models), allows reconstruction of GUVs 3D structure, permitting to retrieve, at the level of single vesicles, the area fractions of the coexisting lipid domains. The last procedure allowed us to demonstrate quantitatively the accomplishment of the lever rule in GUVs composed of binary phospholipid mixtures displaying solid ordered/liquid disordered domains [3].In this work we measured the relative areas of the two observed distinct regions (lipid domains) at the reported lo/ld coexisting region in GUVs composed of POPC/DPPC/cholesterol mixtures (approximately 20 different compositions). We explore subsequently if the relationship between the measured areas are consistent with that expected for coexistence of real thermodynamic phases. In particular our method provides a mean of characterizing the tie lines in the lo/ld coexistence region, providing evidences of a connection between what is observed in GUVs and what is predicted from the 3-component phase diagram of the lipid mixture.1) Bagatolli, L.A, 2006, Biochim Biophys Acta 1758:1541-1556.2) Veatch, S.L and Keller S.L, 2005, Biochim Biophys Acta. 1746:172-85.3) M. Fidorra et al., 1198-Pos Biophys. J. 2008 94:1198.

Combining Fluorescence Lifetime and Polarization Microscopy to Discriminate Phase Separated Domains in Giant Unilamellar Vesicles

Using fluorescence lifetime microscopy we study the structure of lipid domains in giant unilamellar vesicles made from sphingomyelin, 1,2-dioleoyl-sn-glycero-3-phosphocholine, and cholesterol. Lifetimes and orientation of a derivative of the fluorescent probe DPH embedded in the membrane were measured for binary and ternary lipid mixtures incorporating up to 42mol % of cholesterol. The results show that adding cholesterol always increases the lifetime of the probe studied. In addition, the analysis of the probe orientation indicates that cholesterol has little influence on the ordering of the sphingomyelin alkyl chains whereas it has a noticeable effect on the structure of the 1,2-dioleoyl-sn-glycero-3-phosphocholine chains. The measurements made on the orientation and lifetime of the probe show the structure of the membrane in its liquid ordered and liquid disordered domains.

Visualization of lipid domains in giant unilamellar vesicles using an environment-sensitive membrane probe based on 3-hydroxyflavone

We characterized the recently introduced environment-sensitive fluorescent membrane probe based on 3-hydroxyflavone, F2N12S, in model lipid membranes displaying liquid disordered (Ld) phase, liquid ordered (Lo) phase, or their coexistence. Steady-state fluorescence studies in large unilamellar vesicles show that the probe dual emission drastically changes with the lipid bilayer phase, which can be correlated with the difference in their hydration. Using two-photon excitation microscopy on giant unilamellar vesicles, the F2N12S probe was found to bind both Ld and Lo phases, allowing visualization of the individual phases from the fluorescence intensity ratio of its two emission bands. By using a linearly polarized excitation light, a strong photoselection was observed for F2N12S in the Lo phase, indicating that its fluorophore is nearly parallel to the lipid chains of the bilayer. In contrast, the absence of the photoselection with the Ld phase indicated no predominant orientation of the probe in the Ld phase. Comparison of the present results with those reported previously for F2N12S in living cells suggests a high content of the Lo phase in the outer leaflet of the cell plasma membranes. Taking into account the high selectivity of F2N12S for the cell plasma membranes and its suitability for both single- and two-photon excitation, applications of this probe to study membrane lateral heterogeneity in biological membranes are foreseen.

Detection of Lipid Domains in Model and Cell Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.