Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy

The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein-protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.     

Photophysical properties of hydroxyphenyl benzazoles and their applications as fluorescent probes to study local environment in DNA,protein and lipid

Fluorescence techniques have drawn increasing attention because they provide crucial information about molecular interactions in protein–ligand systems beyond that obtained by other methods. The advantage of fluorescence spectroscopy stems from the fact that the majority of molecules in biological systems do not exhibit fluorescence, making fluorescent probes useful with high sensitivity. Also, the fluorescence emission is highly sensitive to the local environment, providing a valuable tool to investigate the nature of binding sites in macromolecules. In this review, we discuss some of the important applications of a class of molecules that have been used as fluorescent probes in a variety of studies. Hydroxyphenyl benzazoles (HBXs) show distinct spectroscopic features that make them suitable probes for the study of certain biological mechanisms in DNA, protein and lipid. In particular, the complex photophysics of 2‐(2′‐hydroxyphenyl)benzoxazole (HBO) and the distinguished fluorescence signatures of its different tautomeric forms make this molecule a useful probe in several applications. Among these are probing the DNA local environment, study of the flexibility and specificity of protein‐binding sites, and detecting the heterogeneity and ionization ability of the head groups of different lipidic phases. The spectroscopy of HBX molecules and some of their chemically modified structures is also reviewed. Copyright © 2016 John Wiley & Sons, Ltd.     

Fluorescent lipid probes: some properties and applications (a review)

Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the size of an average lipid molecule, special care is needed for the selection of probes, since if the size and intramolecular localization of the probe is not specifically taken into account, it may dramatically affect the properties of the lipids. The latter is particularly important in cell biological studies of lipid trafficking and sorting, where the probed lipid should resemble its natural counterpart as closely as possible. On the other hand, for biophysical applications, these considerations may be less critical. Here we provide a brief overview of the application of several lipid probes in cell biological and biophysical research, and critically analyze their validity in the various fields.     

Fluorescent probe study of the spatial structure of membranes and lipoproteins

A review is devoted to principles of studies in spatial structure of the model and biological membranes and lipoproteins on the basis of measuring radiationless energy transfer between fluorescent probes and from proteins to the probes. Recently the theory has been developed for energy transfer in membranes of various geometry and in lipoproteins of different size and structure. Special fluorescent probes are designed and made. The measurement procedure was tested in simulated systems and used to study a series of membranes as well as blood plasma lipoproteins of main classes. Everything above-mentioned resulted in obtaining data on the size of protein molecules in membranes and lipoproteins, proteins location relative to the lipid phase, on the surface area of the membranes (isolated and directly in a cell), association of protein molecules, state of near-protein lipid layer, membrane asymmetry, spreading of proteins on the lipoprotein surface, on the cholesterol effect on the lipid bilayer size etc.     

Use of liposomes as membrane models to evaluate the contribution of drug–membrane interactions to antioxidant properties of etodolac

Abstract

This work stresses the need to combine antioxidant assays and drug–membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO?) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO?) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties – fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

Visualization of Sterol-Rich Membrane Domains with Fluorescently-Labeled Theonellamides

Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.     

Reaction Fields in the Environment of Fluorescent Probes: Polarity Profiles in Membranes

Fluorescent probes in biological systems are sensitive to environmental polarity by virtue of their response to the reaction field created by polarization of the dielectric medium. Classically, fluorophore solvatochromism is analyzed in terms of the Lippert equation and later variants, all of which rely upon the original reaction field of Onsager. A recent survey of the solvent dependence of EPR spin-label probes, which are responsive solely to the reaction field in the ground state without the complication of excited states, shows that the reaction field of Block and Walker performs best in describing the polarity dependence. In this model, the step-function transition to the bulk dielectric medium used by Onsager is replaced by a graded transition. Analysis of the Stokes shifts for representative fluorescent membrane probes, such as PRODAN, DANSYL, and anthroyl fatty acid, reveals that, of several different reaction fields (including that of Onsager), the Block-Walker model best describes the dependence on solvent dielectric constant and refractive index for the different probes simultaneously. This is after full allowance is made for all contributions involving polarizability of the fluorophore, a point that is frequently neglected or treated incorrectly in studies using biological fluorescent probes. By using the full range of polar and apolar solvents, it is then possible to establish a common reference for the polarity dependence of different fluorophores and to relate this also to the polarity dependence of biologically relevant spin-label EPR probes. An important application is calibration of the transmembrane polarity profile recorded by fluorescent probes in terms of the high-resolution profile obtained from site-specifically spin-labeled lipid chains.     

Use of liposomes as membrane models to evaluate the contribution of drug-membrane interactions to antioxidant properties of etodolac

This work stresses the need to combine antioxidant assays and drug-membrane interaction studies to describe more accurately the antioxidant profile of non-steroidal anti-inflammatory drugs (NSAIDs). Different experiments performed in liposomes and aqueous solution were compared and used to evaluate the protective effect of etodolac in lipid peroxidation. Lipid peroxidation was induced by the peroxyl radical (ROO*) derived from 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydroxyl radical (HO*) generated by the Fenton reaction and was assessed by the fluorescence intensity decay of three fluorescence probes with distinct lipophilic properties--fluorescein; hexadecanoyl aminofluorescein (HDAF) and diphenylhexatriene propionic acid (DPHPA). Membrane fluidity changes due to lipid peroxidation were also evaluated by steady-state anisotropy measurements. Interactions of etodolac with lipid bilayers were evaluated by membrane zeta-potential measurements. Results indicate a drug location near the membrane surface and show that etodolac can scavenge the radicals studied but to a variable extent, depending on the assayed media and reactive species. The use of different probes and liposomes as membrane mimetic systems allowed us to conclude that membrane lipoperoxidation is not only related to the scavenging characteristics of the antioxidants, but also to their ability to interact with lipid bilayers.     

Detection of lipid domains in docasahexaenoic acid-rich bilayers by acyl chain-specific FRET probes

A major problem in defining biological membrane structure is deducing the nature and even existence of lipid microdomains. Lipid microdomains have been defined operationally as heterogeneities in the behavior of fluorescent membrane probes, particularly the fluorescence resonance energy transfer (FRET) probes 7-nitrobenz-2-oxa-1,3-diazol-4-yl-diacyl-sn-glycero-3-phosphoethan olamine (N-NBD-PE) and (N-lissamine rhodamine B sulfonyl)-diacyl-snglycero-3-phosphoethanolamine (N-Rh-PE). Here we test a variety of N-NBD-PEs and N-Rh-PEs containing: (a) undefined acyl chains, (b) liquid crystalline- and gel-state acyl chains, and (c) defined acyl chains matching those of phase separated membrane lipids. The phospholipid bilayer systems employed represent a liquid crystalline/gel phase separation and a cholesterol-driven fluid/fluid phase separation; phase separation is confirmed by differential scanning calorimetry. We tested the hypothesis that acyl chain affinities may dictate the phase into which N-NBD-PE and N-Rh-PE FRET probes partition. While these FRET probes were largely successful at tracking liquid crystalline/gel phase separations, they were less useful in following fluid/fluid separations and appeared to preferentially partition into the liquid-disordered phase. Additionally, partition measurements indicate that the rhodamine-containing probes are substantially less hydrophobic than the analogous NBD probes. These experiments indicate that acyl chain affinities may not be sufficient to employ acyl chain-specific N-NBD-PE/N-Rh-PE FRET probes to investigate phase separations into biologically relevant fluid/fluid lipid microdomains.     

Lipid lateral diffusion and membrane organization

It is shown that investigating the lateral motion of lipids in biological membranes can provide useful information on membrane lateral organization. After labeling membranes with extrinsic or intrinsic lipophilic fluorescent probes, fluorescence recovery after photobleaching experiments strongly suggests that specialized cells like spermatozoa, eggs and epithelia exhibit surface membrane regionalization or macrocompartmentation and that lateral microheterogeneities or lipid microdomains exist in the plasma membrane of many cellular systems.     

The partition of cis-parinaric acid and trans-parinaric acid among aqueous,fluid lipid,and solid lipid phases

Summary In this article, I review the current information concerning the partition of the fluorescent probes, cis-parinaric acid (9, 11, 13, 15-cis, trans, trans, cis-octadecatetraenoic acid) and trans-parinaric acid (9, 11, 13, 15-all trans-octadecatetraenoic acid) among aqueous, solid lipid, and fluid lipid phases. The association of these probes with lipid is described by a mole fraction partition coefficient whose value is typically in the range of 1–5 × 10⁶, a reasonable value in light of partition coefficients for other fatty acids between hydrophobic phases and water. The partition coefficient, in the absence of lipid phase changes, is relatively independent of temperature and only slightly dependent on the total aqueous probe concentration.In lipid samples which contain coexisting fluid and solid phases, trans-parinaric acid preferentially partitions into the solid phase, while cis-parinaric acid distributes nearly equally between fluid and solid phases. This partition behavior probably arises from the molecular shape of the cis and trans parinaric acid isomers. From measurements of the polarization of fluorescence of cis and trans parinaric acid in mixed lipid systems or membranes it is possible to evaluate the proportion of lipid components involved in phase changes or phase separation. From fluorescence energy transfer between protein typtophan residues and the parinaric acid isomers it is possible to gain information about the organization of lipids and proteins in membranes and model systems. I close the review by considering some of the membrane research areas where these probes and their various lipid derivatives may be particularly useful.     

Proteomic profiling of metalloprotease activities with cocktails of active-site probes

Metalloproteases are a large, diverse class of enzymes involved in many physiological and disease processes. Metalloproteases are regulated by post-translational mechanisms that diminish the effectiveness of conventional genomic and proteomic methods for their functional characterization. Chemical probes directed at active sites offer a potential way to measure metalloprotease activities in biological systems; however, large variations in structure limit the scope of any single small-molecule probe aimed at profiling this enzyme class. Here, we address this problem by creating a library of metalloprotease-directed probes that show complementary target selectivity. These probes were applied as a 'cocktail' to proteomes and their labeling profiles were analyzed collectively using an advanced liquid chromatography-mass spectrometry platform. More than 20 metalloproteases were identified, including members from nearly all of the major branches of this enzyme class. These findings suggest that chemical proteomic methods can serve as a universal strategy to profile the activity of the metalloprotease superfamily in complex biological systems.     

Lateral diffusion of phospholipids in the lipid surface of human low-density lipoprotein measured with a pyrenyl phospholipid probe

Human low-density lipoprotein (LDL) was labelled with the excimeric fluorescent phospholipid analogue 1-palmitoyl-2-(1'-pyreneoctanoyl)-sn-glycero-3-phosphocholine by using phosphatidylcholine-specific transfer protein for the probe insertion. The lateral diffusivity of the probe in the phospholipid/cholesterol surface monolayer of LDL was determined from the measured dependence of the pyrene monomer fluorescence yield on probe concentration. The data were analyzed by the milling-crowd model (J. Eisinger et al. (1986) Biophys. J. 49, 987-1001] to obtain the short-range lateral diffusivity of the probe. The lateral mobility of the probe in LDL was compared to that in model lipid systems, i.e. in protein-free LDL-like lipid particles and in small unilamellar vesicles, with a phospholipid/cholesterol composition characteristic of LDL. This analysis with the probability PE = 1 for excimer production between nearest-neighbour probes gives the lower limits for f, the frequency of translational lipid--lipid exchanges of the probe of 0.62 x 10(8), 0.19 x 10(8) and 0.19 x 10(8)s-1 in LDL, LDL-like lipid particles, and small unilamellar vesicles, respectively. The lower limits for the corresponding lateral diffusion constants are 16, 5 and 5 microns 2 s-1. The results suggest that the translational mobility of phospholipid molecules in the lipid--protein surface of LDL is not constrained by the apolipoprotein B-100 moiety or the neutral lipid core of the lipoprotein. Instead, the protein moiety may perturb the lipid order with the lipid--associating peptide domains and thus fluidize the amphiphilic surface monolayer of LDL relative to the protein-free model systems. In general, lateral diffusivity of the pyrenyl phospholipid probe in LDL and the model lipid systems is comparable to the lateral mobility of lipid analogue probes in a variety of model and biological membranes.     

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Δν, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.     

Fluorescent probes for asymmetric lipid bilayers: Synthesis and properties in phosphatidyl choline liposomes and erythrocyte membranes

Summary We have synthesized three sets of fluorescent probes which we believe will be useful in studies of asymmetric membranes and have studied their interactions with model lipid bilayers and erythrocyte membranes. The probes were designed to partition preferentially into one face of a lipid bilayer with asymmetrically disposed phospholipids and to report lipid transitions in that monolayer.We synthesized more than twenty probes containing anthroyl-, dansyl-, or pyrene rings with acidic, basic, and neutral functional groups and alkyl spacers of various lengths. The interactions of these probes with liposomes of phosphatidyl choline and with erythrocyte membranes were characterized to determine whether probe insertion was asymmetric, how deeply the probe penetrated the bilayer, and whether the probe reflected thermotropic phase transitions in model membranes. The set of variously charged anthroyl esters, analogs of local anaesthetics, appears to be promising for studies of asymmetric membranes.Fluorescent probes have been used extensively to provide information on the lipid regions of biological membranes. Membrane fluidity, a composite of molecular packing and motion of acyl chains in lipid bilayers, has been assessed with a variety of fluorescent probes, the fluorescence of which undergoes some measurable change at the temperature of the membrane's thermotropic phase transition. A large number of fluorescent probes have been used for this purpose. Bashford, Morgan and Radda (Bashford, C.L., Morgan, C.G., Radda, G.K. 1976;Biochim. Biophys. Acta 426: 157) and Thulborn and Sawyer (Thulborn, K.R., Sawyer, W.H. 1978;Biochim. Biophys. Acta 511: 125) synthesized several fatty acid derivatives in which an anthracene group is attached (in ester linkage) along the acyl chain at various positions, and have shown that this set of probes may be useful in probing membrane fluidity at differentdepths within the bilayer.This report describes the synthesis and properties of several sets of amphipathic fluorescent probes, which may partition unequally into the two faces of an asymmetric lipid bilayer, and may therefore provide information about membranes complementary to that obtainable with existing probes.     

The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Deltanu, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.     

Effect of cholesterol on lateral diffusion of fluorescent lipid probes in native hippocampal membranes

Cholesterol is an abundant lipid of mammalian membranes and plays a crucial role in membrane organization, dynamics, function and sorting. The role of cholesterol in membrane organization has been a subject of intense investigation that has largely been carried out in model membrane systems. An extension of these studies in natural membranes, more importantly in neuronal membranes, is important to establish a relationship between disease states and changes in membrane physical properties resulting from an alteration in lipid composition. We have monitored the lateral diffusion of lipid probes, DiIC(18)(3) and FAST DiI which are similar in their intrinsic fluorescence properties but differ in their structure, in native and cholesterol-depleted hippocampal membranes using the fluorescence recovery after photobleaching (FRAP) approach. Our results show that the mobility of these probes is in general higher in hippocampal membranes depleted of cholesterol. Interestingly, the increase in mobility of these probes does not linearly correlate with the extent of cholesterol depletion. These results assume significance in the light of recent reports on the requirement of cholesterol to support the function of the G-protein coupled serotonin(1A) receptor present endogenously in hippocampal membranes.     

Laurdan and di-4-ANEPPDHQ do not respond to membrane-inserted peptides and are good probes for lipid packing

Laurdan and di-4-ANEPPDHQ are used as probes for membrane order, with a blue shift in emission for membranes in liquid-ordered (lo) phase relative to membranes in liquid-disordered (ld) phase. Their use as membrane order probes requires that their spectral shifts are unaffected by membrane proteins, which we have examined by using membrane inserting peptides and large unilamellar vesicles (LUVs). The transmembrane polypeptides, mastoparan and bovine prion protein-derived peptide (bPrPp), were added to LUVs of either lo or ld phase, up to 1:10 peptide/total lipid ratio. The excitation and emission spectra of laurdan and di-4-ANEPPDHQ in both lipid phases were unaltered by peptide addition. The integrity and size distribution of the LUVs upon addition of the polypeptides were determined by dynamic light scattering. The insertion efficiency of the polypeptides into LUVs was determined by measuring their secondary structure by circular dichroism. Mastoparan had an α-helical and bPrPp a β-strand conformation compatible with insertion into the lipid bilayer. Our results suggest that the presence of proteins in biological membranes does not influence the spectra of laurdan and di-4-ANEPPDHQ, supporting that the dyes are appropriate probes for assessing lipid order in cells.     

‘Design,synthesis, and strategic use of small chemical probes toward identification of novel targets for drug development’

Chemical probes are essential tools used to study and modulate biological systems. Here, we describe some of the recent scientific advancement in the field of chemical biology, as well as how the advent of new technologies is redefining the criteria of ‘good’ chemical probes and influencing the discovery of valuable drug leads. In this review, we report selected examples of the usage of linkered and linker-free chemical probes for target identification, biological discovery, and general mechanistic understanding. We also discuss the promises of chemogenomics libraries in phenotypic screens, as well as the limitation of their usage to identify the modulation of new targets and biology.     

Protein kinase C penetration into lipid bilayers

Physical characteristics of the association and subsequent penetration of protein kinase C into defined lipid bilayers were analyzed using four different fluorescence probes. The enzyme demonstrated strong hydrophobic and electrostatic interactions with the bilayer as suggested by its ability to increase permeability of carboxyfluorescein-filled unilamellar vesicles. The intensity of interaction was dependent on the concentration of phosphatidylserine. The hydrophilic quencher, N-methylpicolinium perchlorate, was used to show that the tryptophan residues affected by ligand-induced conformational changes were in a hydrophobic region(s) of the enzyme. Using quenching of intrinsic tryptophan fluorescence, the enzyme was shown to penetrate the lipid bilayer to the C-16 position of labeled fatty acid probes. The association and subsequent penetration of the enzyme into the lipid bilayer was independent of divalent cations in these systems and had no significant effect on activator-independent substrate phosphorylation.