Determination of lipid raft partitioning of fluorescently-tagged probes in living cells by Fluorescence Correlation Spectroscopy (FCS)

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. They play a role in cellular physiological processes such as signalling, and trafficking but are also thought to be key players in several diseases including viral or bacterial infections and neurodegenerative diseases. Yet their existence is still a matter of controversy. Indeed, lipid raft size has been estimated to be around 20 nm, far under the resolution limit of conventional microscopy (around 200 nm), thus precluding their direct imaging. Up to now, the main techniques used to assess the partition of proteins of interest inside lipid rafts were Detergent Resistant Membranes (DRMs) isolation and co-patching with antibodies. Though widely used because of their rather easy implementation, these techniques were prone to artefacts and thus criticized. Technical improvements were therefore necessary to overcome these artefacts and to be able to probe lipid rafts partition in living cells. Here we present a method for the sensitive analysis of lipid rafts partition of fluorescently-tagged proteins or lipids in the plasma membrane of living cells. This method, termed Fluorescence Correlation Spectroscopy (FCS), relies on the disparity in diffusion times of fluorescent probes located inside or outside of lipid rafts. In fact, as evidenced in both artificial membranes and cell cultures, probes would diffuse much faster outside than inside dense lipid rafts. To determine diffusion times, minute fluorescence fluctuations are measured as a function of time in a focal volume (approximately 1 femtoliter), located at the plasma membrane of cells with a confocal microscope (Fig. 1). The auto-correlation curves can then be drawn from these fluctuations and fitted with appropriate mathematical diffusion models. FCS can be used to determine the lipid raft partitioning of various probes, as long as they are fluorescently tagged. Fluorescent tagging can be achieved by expression of fluorescent fusion proteins or by binding of fluorescent ligands. Moreover, FCS can be used not only in artificial membranes and cell lines but also in primary cultures, as described recently. It can also be used to follow the dynamics of lipid raft partitioning after drug addition or membrane lipid composition change.     

Fluorescence resonance energy transfer between lipid probes detects nanoscopic heterogeneity in the plasma membrane of live cells

Fluorescence resonance energy transfer (FRET) between matched carbocyanine lipid analogs in the plasma membrane outer leaflet of RBL mast cells was used to investigate lateral distributions of lipids and to develop a general method for quantitative measurements of lipid heterogeneity in live cell membranes. FRET measured as fluorescence quenching of long-chain donor probes such as DiO-C18 is greater with long-chain, saturated acceptor probes such as DiI-C16 than with unsaturated or shorter-chain acceptors with the same chromophoric headgroup compared at identical concentrations. FRET measurements between these lipid probes in model membranes support the conclusion that differential donor quenching is not caused by nonideal mixing or spectroscopic differences. Sucrose gradient analysis of plasma membrane-labeled, Triton X-100-lysed cells shows that proximity measured by FRET correlates with the extent of lipid probe partitioning into detergent-resistant membranes. FRET between DiO-C16 and DiI-C16 is sensitive to cholesterol depletion and disruption of liquid order (Lo) by short-chain ceramides, and it is enhanced by cross linking of Lo-associated proteins. Consistent results are obtained when homo-FRET is measured by decreased fluorescence anisotropy of DiI-C16. These results support the existence of nanometer-scale Lo/liquid disorder heterogeneity of lipids in the outer leaflet of the plasma membrane in live cells.     

Pyrene-labeled lipids: versatile probes of membrane dynamics in vitro and in living cells

Pyrene-labeled analogs of fatty acids have been studied as probes of lipid metabolism in vitro and in cultured cells. Procedures for the synthesis of complex pyrenyl lipids and the analytical methods for their separation and quantification are described. Pyrenyl-lipids have been used to quantify the relationship between lipid structure and the rates of spontaneous lipid transfer. Modifications of these methods have also been used to monitor protein-mediated lipid transfer, lipolysis and lipid translocation across bilayer membranes. According to several criteria, pyrene dodecanoic acid has been identified as a good analog of some naturally occurring fatty acids. Digital imaging microscopy has been used to monitor the rate of accumulation of pyrenyl lipids in living cells.     

Probes of membrane electrostatics: synthesis and voltage-dependent partitioning of negative hydrophobic ion spin labels in lipid vesicles.

Two spin-labeled derivatives of the hydrophobic anion trinitrophenol have been synthesized and characterized in lipid vesicles. In the presence of lipid vesicles, the electron paramagnetic resonance (EPR) spectra of these probes are a composite of both membrane-bound and aqueous populations; as a result, the membrane-aqueous partitioning can be determined from their electron paramagnetic resonance spectra. The effect of transmembrane potentials on the membrane-aqueous partitioning of these spin-labeled hydrophobic ions was examined in phosphatidylcholine vesicles formed by extrusion. Inside positive membrane potentials promote an increase in the binding of these probes that is quantitatively accounted for by a simple thermodynamic model used previously to describe the partitioning of paramagnetic phosphonium ions. The transmembrane migration rates of these ions are dependent on the dipole potential, indicating that these ions transit the membrane in a charged form. The partitioning of the probe is also sensitive to the membrane surface potential, and this dependence is accurately accounted for using the Gouy-Chapman Stern formalism. As a result of the membrane dipole potential, these probes exhibit a stronger binding and a more rapid transmembrane migration rate compared with positive hydrophobic ion spin labels and provide a new set of negatively charged hydrophobic ion probes to investigate membrane electrostatics.     

Localization of the lipid probe 1,6-diphenyl-1,3,5 hexatriene (DPH) in intact cells by fluorescence microscopy

The lipophilic fluorescent probe DPH, generally used to determine the microviscosity of membrane lipids, has been visualized in intact cells by fluorescence microscopy. All lipid material of the cells, including cytoplasmic lipid droplets, was found to be labelled with DPH. The fluorescent signal from inside the cells contributes to a large extent to the total cell fluorescence. The results indicate that fluorescence polarization data obtained from intact cells, using DPH as probe, give information on the total lipid material of the cells rather than exclusive information on microviscosity and fluidity of plasma membranes of these cells, as has been repeatedly suggested.     

Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ². From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.     

Inositol lipids: receptor-stimulated hydrolysis and cellular lipid pools

Our current knowledge of the process by which receptors stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) has its origin in the discovery by Hokin & Hokin (J. biol. Chem. 263, 967 (1953] that some pancreatic secretagogues not only elicit exocrine secretion but also stimulate the metabolism of membrane phospholipids. Despite the recent elucidation of many aspects of this widespread signalling system, there is still little information on the control of the supply of its substrate, PtdIns(4,5)P2. In particular, some studies have suggested that inositol-lipid-mediated signalling involves much or all of the inositol lipid complement of the stimulated cells, whereas other observations have equally clearly implicated the receptor-activated hydrolysis of an inositol phospholipid pool that comprises only a small fraction of the total cellular complement of these lipids. These studies, which have largely employed radiochemical analyses using single isotopes, are briefly reviewed. In addition, we report the first information obtained by a new procedure for analysing the metabolic characteristics of the inositol lipids that are broken down during stimulation. This technique employs cells that are doubly labelled in the inositol moiety of their lipids (to isotopic equilibrium with 14C and only briefly with 3H) to search for functional metabolic heterogeneity among the inositol lipids of stimulated cells. Using this method, we have found that the inositol phosphates liberated in stimulated cells during brief stimulation of V1a-vasopressin receptors or prostaglandin F2 alpha receptors come from phospholipid that has a turnover rate typical of the bulk of the cellular inositol lipids.     

Stoichiometry of lipid interactions with transmembrane proteins--Deduced from the 3D structures

The stoichiometry of the first shell of lipids interacting with a transmembrane protein is defined operationally by the population of spin-labeled lipid chains whose motion is restricted directly by the protein. Interaction stoichiometries have been determined experimentally for a wide range of alpha-helical integral membrane proteins by using spin-label ESR spectroscopy. Here, we determine the spatially defined number of first-shell lipids at the hydrophobic perimeter of integral membrane proteins whose 3D structure has been determined by X-ray crystallography and lipid-protein interactions characterized by spin-labeling. Molecular modeling is used to build a single shell of lipids surrounding transmembrane structures derived from the PDB. Constrained energy optimization of the protein-lipid assemblies is performed by molecular mechanics. For relatively small proteins (up to 7-12 transmembrane helices), the geometrical first shell corresponds to that defined experimentally by perturbation of the lipid-chain dynamics. For larger, multi-subunit alpha-helical proteins, the lipids perturbed directly by the protein may either exceed or be less in number than those that can be accommodated at the intramembranous perimeter. In these latter cases, the motionally restricted spin-labeled lipids can be augmented by intercalation, or can correspond to a specific subpopulation at the protein interface, respectively. For monomeric beta-barrel proteins, the geometrical lipid stoichiometry corresponds to that determined from lipid mobility for a 22-stranded barrel, but fewer lipids are motionally restricted than can be accommodated around an eight-stranded barrel. Deviations from the geometrical first shell, in the beta-barrel case, are for the smaller protein with a highly curved barrel.     

Photo-crosslinking analysis of preferential interactions between a transmembrane peptide and matching lipids

In this study, a novel method is presented by which the molecular environment of a transmembrane peptide can be investigated directly. This was achieved by incorporating a photoactivatable crosslinking probe in the hydrophobic segment of a model transmembrane peptide. When this peptide was incorporated into lipid bilayers and irradiated with UV light, a covalent bond was formed between the crosslinking probe and a lipid. This crosslinking reaction could be visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resulting product could be characterized by mass spectrometry. By use of phospholipases, it was demonstrated that the peptide crosslinks to both acyl chains of the lipids. The peptide showed a clear preference to partition into fluid lipids and was excluded from lipids in the gel phase. However, when the peptide was incorporated into bilayers containing two lipid species with different acyl chain lengths, molecular sorting of the lipids around the peptide based on hydrophobic matching was not observed. It is proposed that the size of the transmembrane part plays an important role in the dynamic interactions of membrane proteins with the surrounding lipids and hence in determining whether molecular sorting can occur.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles

The structural basis for organizational heterogeneity of lipids and proteins underlies fundamental questions about the plasma membrane of eukaryotic cells. A current hypothesis is the participation of liquid ordered (Lo) membrane domains (lipid rafts) in dynamic compartmentalization of membrane function, but it has been difficult to demonstrate the existence of these domains in live cells. Recently, giant plasma membrane vesicles (GPMVs) obtained by chemically induced blebbing of cultured cells were found to phase separate into optically resolvable, coexisting fluid domains containing Lo-like and liquid disordered (Ld)-like phases as identified by fluorescent probes. In the present study, we used these GPMVs to investigate the structural bases for partitioning of selected lipids and proteins between coexisting Lo-like/Ld-like fluid phases in compositionally complex membranes. Our results with lipid probes show that the structure of the polar headgroups, in addition to acyl chain saturation, can significantly affect partitioning. We find that the membrane anchor of proteins and the aggregation state of proteins both significantly influence their distributions between coexisting fluid phases in these biological membranes. Our results demonstrate the value of GPMVs for characterizing the phase preference of proteins and lipid probes in the absence of detergents and other perturbations of membrane structure.     

Solvent relaxation in lipid bilayers with dansyl probes

The solvent relaxation properties of the dansyl group attached to two lipids (dansylphosphatidylethanolamine and dansylphosphatidylserine), a fatty acid (dansylundecanoic acid), and two drugs (dansylbenzocaine and dansylpropranolol) were compared in a variety of different lipid systems. Several methods for characterising solvent relaxation were compared in detail for dansylpropranolol in bilayer vesicles of egg phosphatidylcholine. It was shown that the relaxation process is non-monoexponential; nevertheless, for comparative purposes, a model was adopted in which the lifetime associated with the negative exponent in a two exponential decay analysis, obtained at a particular energy on the red edge of emission, was taken as an approximation to a 'solvent relaxation' rate. A negative exponent, indicative of solvent relaxation processes, occurring in the nanosecond time-scale, was found only for dansylpropranolol, dansylPE and dansylundecanoic acid. On addition of the spin probe, 5-doxylstearate, the negative exponent was unaffected in liquid-crystalline phase lipids but was no longer found in gel-phase lipid in the case of dansylpropranolol, while for dansylPE the relaxation time was reduced. On the basis of these types of measurement it was possible to distinguish between different lipid environments using the same probe or between different dansyl environments of the different probes in the same lipid in cases where this would have been difficult or impossible solely on the basis of steady-state or fluorescence lifetime measurements.     

Aggregation, lipid exchange, and metastable phases of dimyristoylphosphatidylethanolamine vesicles

A new fluorescent lipid analogue, bimanephosphatidylcholine, has been synthesized for use in lipid bilayers. This probe is well suited as an energy-transfer donor with N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine as the acceptor. Dimyristoylphosphatidylethanolamine vesicles are prepared by sonication at pH 9 and characterized by electron microscopy and other methods. Resonance energy transfer between separately labeled donor and acceptor vesicles is monitored during HCl-induced aggregation to determine the kinetics of lipid randomization. Light scattering is also monitored to measure the kinetics of aggregation. The light scattering shows a marked reversal with NaOH while the energy transfer does not, indicating lipid exchange during a reversibly aggregated state; the extent of energy transfer suggests that only lipids in the outer monolayers exchange. The gel to liquid-crystalline phase transition temperature in HCl-treated vesicles is found to be 47 degrees C with diphenylhexatriene. The initial sonicated dispersion does not show a sharp phase transition. In vesicles labeled with both donor and acceptor probes, a small, irreversible increase in energy transfer is obtained upon lowering and then restoring the pH. These results suggest a metastable phase in the sonicated vesicles containing a randomized distribution of lipid and probes within the bilayers; the thermodynamically favored phase, whose formation is triggered by the pH shock, contains domains within which the probe lipids are more highly concentrated.     

The rate of lipid transfer during fusion depends on the structure of fluorescent lipid probes: a new chain-labeled lipid transfer probe pair.

A number of fluorescent probes have been used to follow membrane fusion events, particularly intermixing of lipids. None of them is ideal. The most popular pair of probes is NBD-PE and Rh-PE, in which the fluorescent groups are attached to the lipid headgroups, making them sensitive to changes in the surrounding medium. Here we present a new assay for monitoring lipid transfer during membrane fusion using the acyl chain tagged fluorescent probes BODIPY500-PC and BODIPY530-PE. Like the NBD-PE/Rh-PE assay, this assay is based on fluorescence resonance energy transfer (FRET) between the donor, BODIPY500, and the acceptor, BODIPY530. The magnitude of FRET is sensitive to the probe surface concentration, allowing one to detect movement of probes from labeled to unlabeled vesicles during fusion. The high quantum yield of fluorescence, high efficiency of FRET (R(o) is estimated to be approximately 60 A), photostability, and localization in the central hydrophobic region of a bilayer all make this pair of probes quite promising for detecting fusion. We have compared this and two other lipid mixing assays for their abilities to detect the initial events of poly(ethylene glycol) (PEG)-mediated fusion of small unilamellar vesicles (SUVs). We found that the BODIPY500/530 assay showed lipid transfer rates consistent with those obtained using the DPHpPC self-quenching assay, while lipid mixing rates measured with the NBD-PE/Rh-PE RET assay were significantly slower. We speculate that the bulky labeled headgroups of NBD-PE and especially Rh-PE molecules hamper movement of probes through the stalk between fusing vesicles, and thus reduce the apparent rate of lipid mixing.     

Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments

Exchangeable phospho- and sphingolipid probes (phosphatidylcholine, -ethanolamine, -serine, and -glycerol, phosphatidic acid, sphingomyelin, cerebroside, and sulfatide) have been synthesized in which one acyl chain is substituted with a fluorescent bimanyl, 7-(dimethylamino)coumarin-3-yl, or diphenyl-hexatrienyl group. The distribution of these probes between two different populations of lipid vesicles can be readily monitored by fluorescence intensity measurements, as described by Nichols and Pagano [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726], when one of the vesicle populations contains a low mole fraction of a nonexchangeable quencher, (12-DABS)-18-PC. The probes examined in this study exchange between phospholipid vesicles on a time scale of minutes, with kinetics indicating that the transfer process takes place by diffusion of probe monomers through the aqueous phase. As expected, lipid probes with different charges differ markedly in their equilibrium distributions between neutral and charged lipid vesicles. However, probes with different polar headgroups differ only modestly in their relative affinities for vesicles composed of "hydrogen-bonding" lipids (PE and PS) vs "non-hydrogen-bonding" lipids (PC and PG or O-methyl-PA). Probes with different headgroups also show modest, albeit reproducible, differences in their relative affinities for cholesterol-containing vs cholesterol-free PC/PG vesicles. Our results suggest that lipids with different headgroup structures may mix more nearly ideally in liquid-crystalline lipid bilayers than would be predicted from previous analyses of the phase diagrams for binary lipid mixtures.     

Fluidity of bacterial membrane lipids monitored by intramolecular excimerization of 1.3-di(2-pyrenyl)propane

Intramolecular excimer formation of 1,3-di(2-pyrenyl)propane was used to study the fluidity of liposomes prepared from membrane polar lipids of Bacillus stearothermophilus. On the basis of spectral data, local polarity and polarizability parameters were established suggesting that the probe molecules are located well inside the membranes, but displaced towards the polar head groups of the phospholipid molecules. The excimerization rate is very sensitive to lipid phase transitions and pretransitions of synthetic pure lipid bilayers. In bacterial lipids from cultures grown at 55 and 68 degrees C, thermal profiles of excimer to monomer intensity ratios (I'/I) show a broad transition which is displaced to higher temperatures in response to the increase of the growth temperature; these results correlate well with differential scanning calorimetry data and fluorescence polarization of diphenylhexatriene. Additionally, lipid bilayers of bacteria grown at 68 degrees C exhibit a decreased membrane fluidity, as monitored by both fluorescent probes.     

ATR-FTIR spectroscopy: a chemometric approach for studying the lipid organisation of the stratum corneum

The barrier function of skin resides in the lipid components of the stratum corneum, particularly their spatial organisation. FTIR spectroscopy has already been used as a relevant tool to study this lipid organisation: IR vibration band shifts have been attributed to the variations in lipid organisation induced by temperature. Our study included a stratum corneum model, composed of the three main lipids: palmitic acid as an example of fatty acids, cholesterol and ceramide III as an example of ceramide. Different films with various ratios of these lipids were studied. In our analytical strategy, the interest of using a chemometric analysis of global data obtained from ATR-FTIR spectra to highlight the main interactions involved in the molecular organisation of lipids has been demonstrated. Two kinds of interaction between the three main lipids have been shown: a non polar interaction between the long hydrocarbon chains and a polar interaction as the hydrogen bonding between polar functional groups. By varying the lipid ratio, we have shown first that the relative importance of each interaction was modified, second, that the induced modification of organisation can be detected by chemometric analysis of the ATR-FTIR spectra. The role of each kind of lipid in the organisation has been discussed. In conclusion, associating the ATR-FTIR with chemometric treatment is a promising tool: firstly, to understand the consequence of lipid relative compositions on the structural organisation of the stratum corneum, secondly, to show the relationship between lipid organisation and percutaneous penetration data. Indeed, this methodology will be transposed to in vivo studies with IR measurements through a probe.     

Azide- and diazirine-modified membrane lipids: Physicochemistry and applicability to study peptide/lipid interactions via cross-linking/mass spectrometry

Although the incorporation of photo-activatable lipids into membranes potentially opens new avenues for studying interactions with peptides and proteins, the question of whether azide- or diazirine-modified lipids are suitable for such studies remains controversial. We have recently shown that diazirine-modified lipids can indeed form cross-links to membrane peptides after UV activation and that these cross-links can be precisely determined in their position by mass spectrometry (MS). However, we also observed an unexpected backfolding of the lipid's diazirine-containing stearoyl chain to the membrane interface challenging the potential application of this modified lipid for future cross-linking (XL)-MS studies of protein/lipid interactions. In this work, we compared an azide- (AzidoPC) and a diazirine-modified (DiazPC) membrane lipid regarding their self-assembly properties, their mixing behavior with saturated bilayer-forming phospholipids, and their reactivity upon UV activation using differential scanning calorimetry (DSC), dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), transmission electron microscopy (TEM), and MS. Mixtures of both modified lipids with DMPC were further used for photo-chemically induced XL experiments with a transmembrane model peptide (KLAW23) to elucidate similarities and differences between the azide and the diazirine moiety. We showed that both photo-reactive lipids can be used to study lipid/peptide and lipid/protein interactions. The AzidoPC proved easier to handle, whereas the DiazPC had fewer degradation products and a higher cross-linking yield. However, the problem of backfolding occurs in both lipids; thus, it seems to be a general phenomenon.     

Rotational relaxation of 1,6-diphenylhexatriene in membrane lipids of cells acclimated to high and low growth temperatures.

Measurement of the time-resolved fluorescence depolarization of 1,6-diphenylhexatriene (DPH) in artificial bilayers of microsomal membrane lipids from Tetrahymena gives detailed information concerning the molecular motion of this probe and fluid properties of the membrane lipids which are obscured with steady-state methods. The rotational motion of DPH in these lipids from cells acclimated to 15 and 39.5 degrees C growth temperatures was anisotropic, which agrees with recent time-resolved studies of this probe in synthetic phospholipid systems. Evaluation of DPH polarization data obtained from these lipid fractions at their respective growth temperatures showed differences in physical properties which suggest that "viscosity", per se, of the microsomal lipids is not a strictly regulated as it is in prokaryotic systems. Rotational relaxation of DPH in 39.5 degrees C microsomal lipids measured at 15 degrees C is more complex than that of either lipid fraction measured at its actual growth temperature, suggesting that the probe has partitioned into two dissimilar environments within the bilayer. Similar effects are observed in the microsomes of 39.5 degrees C cells by freeze-fracture electron microscopy following rapid cooling to 15 degrees C. Under these conditions, two distinct regions are observed on the fracture faces, suggesting a correlation between lipid phase changes and alterations in membrane structure.     

Fluorescent lipid probes in the study of viral membrane fusion

Fluorescent lipid probes are widely used in the observation of viral membrane fusion, providing a sensitive method to study fusion mechanism(s). Due to the wealth of data concerning liposome fusion, a variety of fusion assays has been designed including fluorescent probe redistribution, fluorescence dequenching, fluorescence resonance energy transfer and photosensitized labeling. These methods can be tailored for different virus fusion assays. For instance, virions can be loaded with membrane dye which dequenches at the moment of membrane merger. This allows for continuous observation of fusion and therefore kinetic information can be acquired. In the case of cells expressing viral envelope proteins, dye redistribution studies of lipidic and water-soluble fluorophores yield information about fusion intermediates. Lipid probes can be metabolically incorporated into cell membranes, allowing observation of membrane fusion in vitro with minimal chance of flip flop, non-specific transfer and formation of microcrystals. Fluorescent lipid probes have been incorporated into liposomes and/or reconstituted viral envelopes, which provide a well-defined membrane environment for fusion to occur. Interactions of the viral fusion machinery with the membrane can be observed through the photosensitized labeling of the interacting segments of envelope proteins with a hydrophobic probe. Thus, fluorescent lipid probes provide a broad repertoire of fusion assays and powerful tools to produce precise, quantitative data in real time required for the elucidation of the complex process of viral fusion.