Chemistry and biology of N-(7-nitrobenz-2-oxa-l,3-diazol- 4-yl)-labeled lipids" fluorescent probes of biological and model membranes-Chattopadhyay 1990

Chemistry and Physics of Lipids, 53 (1990) 1--15 1 Elsevier Scientific Publishers Ireland Ltd.

Review Article

Chemistry and biology of N-(7-nitrobenz-2-oxa-l,3-diazol- 4-yl)-labeled lipids" fluorescent probes of biological and

model membranes

Amitabha Chat topadhyay*

Department of Biochemistry and Biophysics, University of California, Davis, California 95616 (U.S.A.)

(Received June 6th, 1989; revised and accepted July 26th, 1989)

Lipids that are covalently labeled with the 7-nitrobenz-2-oxa-l,3-diazol-4-yl (NBD) group are widely used as fluorescent analogues of native lipids in model and biological membranes to study a variety of processes. The fluorescent NBD group may be attached either to the polar or the apolar regions of a wide variety of lipid molecules. Synthetic routes for preparing the lipids, and spectroscopic and ionization properties of these probes are reviewed in this report. The orientation of various NBD-labeled lipids in membranes, as indicated by the location of the NBD group, is also discussed. The NBD group is uncharged at neutral pH in membranes, but loops up to the surface if attached to acyl chains of phospholipids. These lipids find applications in a variety of membrane-related studies which include membrane fusion, lipid motion and dynamics, organization of lipids and proteins in membranes, intracellular lipid transfer, and bilayer to hexagonal phase transition in liposomes. Use of NBD-labeled lipids as analogues of natural lipids is critically evaluated.

Keywords: NBD-labeled lipid; model membrane; fluorescence; resonance energy transfer; location; ionization.

Introduction

In 1968, Ghosh and Whitehouse reported a new reagent, 4-chloro-7-nitrobenz-2-oxa- l ,3- diazole, which reacts with amino groups to form stable, highly f luorescent compounds [1]. They named this new reagent "NBD chlor ide" (see Fig. 1). It was later reported that NBD chloride also reacts with thioi groups to give stable f luorescent derivatives [2,3]. Since then, NBD chloride has been widely used as a reagent for introducing a f luorescent group into proteins [4 6]. In addition, compounds that are closely

*Present address: Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

related to NBD chloride have been used in studies of protein structure and conformational changes [7]. Recently, NBD fluoride has been introduced as a substitute for NBD chloride for protein labeling and other applications [8]. NBD fluoride is more reactive and has fewer side reactions than NBD chloride [9---13]. Both these compounds have been used for chromatographic detection of amino acids [14 18]. NBD fluoride has also been used to f luorescently label colcemid to give NBD-colcemid, which is used as a probe for studying the interactions of colcemid with cytoskeletal e lements [19].

Another important application of the 7-nitrobenz-2-oxa-l ,3-diazol-4-yl (NBD) group, one that is the focus of this review, is its increasing

0009-3084/90/$03.50 1990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

(a )

(b)

CI

N% NBD Chlor ide

O I

. r c -o -c~ R~- C--O--CH O

"1 I I O C!"12-- O-- P-- O-- CH2- CH,.- N H

NBD- PE ~)" "~1~O N%

O !

,c. o NH o c~-o-~-oH

6 - NBD - PA NO 2 O

I R~- C -o - ICH 2

NH O

NO 2

. .p- -(c~-c.-.-ch

NBD - Cho les tero l

OH I

Hc- CH--CH-- (CHpl~-CH 3 I

HC - NH -- C - (CI..~=- NH I II ' :~ l

NBD - Ceramide NO2

C H 2 - O- P - O - Cl-L- Cl-L- N;.- Ct-L I ' ~ I '= O" CH 3

12 - NBD - PC

Fig. 1. Chemical structures of (a) NBD chloride and (b) various types of NBD-labeled lipids.

use to monitor the properties of biological and model membranes. Various phospholipids and cholesterol analogues have been synthesized with the NBD group attached to the polar head group or to the non-polar fatty acyl chain of the lipid [20--25]. These lipids are used as fluorescent analogues of native lipids in biological and model membranes to study a variety of processes. These processes include spontaneous and protein-mediated transfer of lipids between vesicles and between vesicles and cell membranes [26-- 38]; membrane fusion, aggregation, and lateral

phase separation by resonance energy transfer and related methods [39---97]; lateral mobility of lipids, proteins, and probes in membranes by fluorescence recovery after photobleaching [98--- 141]; intracellular lipid transport and lipid metabolism in living cells [142--158]; bilayer to hexagonal phase transition in liposomes [159--- 162]; and spatial organization and distribution of lipids, proteins, and probes in biological membranes [163--166]. In addition, NBD-labeled lipids have been used to study lipid monolayers [167,168], the Golgi complex [169,170,204], lipid

asymmetry in membranes [171,172,201], membrane fluidity in normal and diseased cells [173,174], hydrolysis of phosphatidylcholines mediated by protein kinase C [175], develop- ment of fetal lung maturity [176--179], and transfer of free fatty acids between vesicles [180]. They have also been used as substrates in es- timating enzyme activity [181--186], and as fluorescent tracers to detect antibody-mediated agglutination of liposomes with membrane proteins and incorporation of membrane proteins into liposomes [187]. NBD-labeled sterols have been used for labeling low density lipoproteins [25,188---190], and to study Mycoplasma pul- monis-mediated hemolysis [191]. NBD-labeled phosphatidylserines have been used as inhibitors of secretion and phospholipase A 2 in intact mast cells [192,193] and as an activator of human platelets [194,195]. Molecules that are structural- ly similar to these NBD lipids have also been used as photoaffinity-labeling phospholipids [196]. Chemical structures of some commonly used NBD-iabeled lipids are shown in Fig. 1.

In view of the diverse nature of the areas of interest in which these lipids are often used (from chemistry to cell biology), a comprehen- sive review of these lipids would be very useful. The objective of this review is to bring together certain important aspects and applications of NBD-labeled lipids. This review by no means provides an exhaustive account of the biophysi- cal, biochemical, clinical and cell biological studies in which various NBD-labeled lipids have been used as probes of biological and model membranes. Rather, the review attempts to ex- amine these iipids from their initial synthesis a decade back, and follows their increasing use in studying membrane phenomena. The spectroscopic and ionization properties of these lipids and their orientation in membranes are reviewed. Applications of these lipids in studies of membrane fusion, lipid transport and other membrane phenomena are also discussed. The synthesis and chemical, biochemical, and phar- macological properties of compounds having a similar heterocyclic ring system like NBD (ben- zofurazans) have been reviewed [197,198].

Chemical synthesis

Phosphatidylethanolamine (PE) derivatives in which the NBD group is attached to the headgroup of a PE molecule are the most extensively used NBD-labeled lipids and will be termed NBD-PE in this review, irrespective of the fatty acyl chains they contain. NBD-PE was one of the earliest NBD-labeled lipids to be synthesized by alkylation of the free amino group of PE NBD chloride [21,199]. Although in the initial synthesis by Monti et al. egg PE was used [21,199], this synthetic route has been utilized to make PE derivatives containing the NBD group with different fatty acyl chains. A dilauroyl PE derivative has been synthesized with the NBD group attached to the head group [28]. A variety of NBD-PE lipids is now commercially available with dioleoyl, dipalmitoyl and dimyristoyl fatty acyl chains. Phosphatidylcholines (PC) with the NBD group attached to the 2-position fatty acyl chain have also been synthesized either by alkylation of amino fatty acids with NBD chloride followed by esterification of lyso PC [20] or by treatment of tert-butoxycarbonyl PC with NBD chloride under acidic conditions [26]. Phos- phatidylcholines with NBD groups attached at the 6 and 12 positions of the fatty acyl chains (6- and 12-NBD-PC, respectively) have been synthesized by the above mentioned procedures [20,26]. One problem of acylation of NBD- labeled fatty acids is that the product obtained is impure and often contains an appreciable amount (about 20%) of the undesirable mixed chain isomer (i.e., the final product having the NBD label at the 1-position rather than at the 2-position fatty acyl chain) due to vigorous acylation conditions [22]. A method using reverse phase high performance liquid chromatography has been developed to separate and purify these isomers [200]. Alternatively, a synthetic route using a protective group avoids this problem and yields products with a high degree of isomeric purity [23]. NBD-labeled phospholipids having similar overall structure but with different head- groups have been synthesized by utilizing phospholipase D catalyzed headgroup exchange on

6-NBD-PC [142,147,148,171,200~202]. It is to be noted that in these molecules the NBD labels are attached to the terminal carbon atom of the hydrocarbon chain. However, PC with NBD labels attached to the methyl-terminal half of one acyl chain have been recently synthesized [24]. In these lipids, the NBD group is attached to a methylene carbon other than the terminal carbon atom. This was achieved by first synthe- sizing the NBD-labeled fatty acid from the corresponding hydroxy fatty acid with NBD chloride, followed by coupling with lyso PC to give the corresponding phospholipid. Besides phospholipids, a number of NBD-labeled sphingo- lipids have been synthesized by Pagano and coworkers from the corresponding long chain bases (sphingosines) and NBD-labeled fatty acids by oxidation-reduction condensation with tri- phenylphosphine and 2,2'-dipyridyl disulfide, or by reaction of the long chain base with the N-hydroxysuccinimidyl ester of the NBD-labeled fatty acid [203,204].

Two principal types of NBD-iabeled steroids have been synthesized and used in membrane studies. In one of these, the NBD group occurs as part of the flexible hydrocarbon chain [25,205]. The location of the NBD group in such compounds in membrane bilayers has been determined and will be discussed later in this review. In the other class, a cholesterol nucleus is attached via a hydrophilic spacer to the NBD group [206]. The hydrophilic spacer group increases solubility in water.

Spectroscopic properties

The NBD-labeled lipids are practically non- fluorescent in aqueous suspensions, but are highly fluorescent in organic solvents, or at low concentrations (1 mol% or less) in membranes. The quantum yields of NBD-PE in ethanol and in model membranes of dioleoyl PC are 0.39 and 0.32, respectively, relative to quinine sulfate (D.E. Wolf and A.P. Winiski, personal communications). These lipids are characterized by absorption spectra and fluorescence excitation spectra having maxima around 340 and 460 nm in ethanolic solution [21], and with a molar

absorption coefficient (~) of 20,000---25,000 M -I .cm -1 for the NBD group at 460 nm ([19], A.P. Winiski, personal communications). The exact position of the maxima depends on the specific lipid used [20,21,25]. These lipids exhibit a good Stoke's shift. When excited at a wavelength around 450 nm, their fluorescence emission spectra consist of a broad band with a maximum of fluorescence emission anywhere between 490 and 550 nm, depending on the nature of the environment around the fluorophore [20,21,102,207,208]. Thus, NBD fluorescence is very sensitive to its environment and this can be utilized to estimate its location in membranes [207]. As is generally the ease, a blue shift (i.e., a shift towards lower wavelength) in wavelength maximum is observed with decreasing polarity of the medium. This is also accompanied by an increase in fluorescence intensity and lifetime [208]. The lifetimes of dilauroyl and dimyristoyi NBD-PE in egg PC liposomes have been determined from fluorescence decay times and have been found to be 6--8 ns [208]. However, in addition to being polarity dependent, the rela- tionship between the emission maxima and the properties of the medium is complicated and could depend on factors such as the ability of the solvent to form hydrogen bonds. Also, formation of lipid aggregates in very non-polar solvents makes interpretation of spectral data more dif- ficult [207].

Fluorescence and absorbance of NBD-labeled phospholipids in membranes are sensitive to pH [207,209]. In general, there is a decrease in intensity with increase in pH with a midpoint around 11.5. There is also a blue shift in the wavelength of the absorbance maximum. The significance of these spectral changes in relation to the ionization properties of the NBD group in membranes wil be discussed later.

The fluorescence of the NBD-labeled lipids in membranes is quenched at higher concentrations due to self quenching [26,164]. This type of quenching is very sensitive to surface density of lipids in the membrane. Thus, at concentrations greater than 50 mol%, the fluorescence of 6- NBD-PC, 12-NBD-PC and NBD-PE are more than 98% quenched [26,164]. This has been el-

fectively utilized to study Ca2+-induced lateral phase separation in liposomes containing phos- phatidylserine [164]. During such phase separa- tions, the NBD-labeled lipids get concentrated in restricted domains of the bilayer. This increase in the local concentration of the probe in the membrane results in self quenching. This method has also been used to study phase separation of membrane lipids induced by integral membrane proteins [163]. The fluorescence of the NBD group can also be quenched by the aqueous quencher cobaltous ion (Co2+). The paramagnetic Co 2 is soluble in water and is an efficient quencher of NBD fluorescence, probably by a dynamic mechanism [210,211]. Another paramagnetic ion, the cupric (Cu 2) ion, has also been used to quench NBD fluorescence [102,103]. In addition, the fluorescence of NBD- PE in lipid bilayers can be quenched by charged, water soluble spin labels (nitroxide) such as tem- pamine. Such quenching has been used to estimate the electrostatic surface potentials of liposomes [212]. The quenching data for aqueous quenchers can be analyzed in terms of Stern- Volmer analysis [213]. The fluorescence of the membrane embedded NBD group can be effec- tively quenched by membrane bound quenchers. Thus, fluorescence of NBD-labeled lipids in liposomes can be quenched by spin-labeled phospholipids having a nitroxide group on the fatty acyl chain [214,215]. Quenching of this type occurring in membranes is predominantly static in nature [216].

One of the most useful properties of NBD- labeled lipids is their ability to be used as resonance energy transfer donors or acceptors in membranes in conjunction with lipids (or lipid analogues) that are labeled with other probes such as anthroyloxy [39,40], rhodamine [41] or bimane [55]. Resonance energy transfer offers a conven- ient and sensitive way to monitor membrane fusion, aggregation and spontaneous intracellular (or intravesicular) transfer of lipids, and it has been extensively utilized to study these effects (see below). Such transfer of energy in model membranes and in living cells can be visual- ized by resonance energy transfer microscopy, as demonstrated by Uster and Pagano [217].

Ionization properties

Since NBD-labeled lipids are often used as analogues of natural lipids in membranes, the charge of the NBD group under physiological conditions is of concern. The conformation and organization of NBD-labeled lipids in membranes could depend on its ionization state. The ionization properties of the NBD group in aqueous solution were investigated by Meyers et al. [218] using a series of NBD analogues of acetyl- choline. Based on a sharp change in fluorescence with pH and other NMR evidence, they concluded that in solution the NBD group is neutral at pH 7, but probably undergoes deprotonation of its amino group at higher pH, giving the NBD group a negative charge. However, these authors did not provide any direct evidence demonstrat- ing deprotonation at high pH. A different pic- ture emerges from other reports that conclude that at high pH the NBD group forms a "Meisenheimer adduct" [219] due to addition of a hydroxide ion at one of the electrophilic carbon atoms in the NBD ring [220,221]. This is further supported by the observation that NBD chloride, which does not have any amino group (and thus is incapable of deprotonation), exhibits a pK a around pH 9.8 [222]. These two alternate schemes for the NBD group at high pH are shown in Fig. 2.

The ionization properties of NBD-labeled lipids in model membranes were studied in detail by Chattopadhyay and London using fluorescence, absorbance and electrophoretic mobility measurements [207,209]. For NBD-PE and 6- and 12-NBD-PC in model membranes, an apparent ionization near pH 11.5 involving the NBD group was detected both by a decrease in fluorescence intensity and a blue shift in the wavelength of the absorbance maximum. These changes in fluorescence and absorbance were reversible. In addition, analysis of zeta potentials obtained from electrophoretic mobility measurements in model membranes indicated that the NBD group was uncharged at neutral pH. However, at higher pH (greater than 11) the NBD group was negatively charged. The ionization behavior of the NBD group on NBD-labeled lipids was quite

eNR NR

NHR

pH>11 '~ NHR

Fig. 2. Two alternate reaction schemes for the NBD moiety at high pH (R denotes rest of the molecule besides NBD): (a) deprotonation of the amino group and (b) formation of "Meisenheimer adduct" due to addition of hydroxide ion at one of the electrophilic carbon atoms in NBD ring. Notice that irrespective of the actual mechanism, the net charge will be the same in either case.

similar to that of aqueous solutions of soluble NBD compounds [218], except that the pK a for the phospholipids was about 1.5 pH units higher than in aqueous solution. Considering that these lipids are in membranes and not in aqueous solutions, this shift in pK a is not surprising. There are other examples of such pKa shifts in going from an aqueous to a membrane-like environment [223,224]. It is still not very clear whether these changes around pH 11.5 are due to deprotonation [218] or hydroxylation [220,221], since both could give rise to a negative charge on the NBD group, which was detected by zeta potential measurements. However, it was pointed out that the equilibria involved in reversible deprotonation and hydroxylation were oper- ationally indistinguishable [207]. Another lipid used in the ionization studies had a methylated NBD group placed in the flexible "tail" of cholesterol (NBD-cholesterol). A small irrevers- ible and time-dependent change in absorbance was observed for NBD-cholesterol at a much higher pH (greater than 12). An even larger pH shift than for NBD-labeled phospholipids was observed between the apparent high pH ioniza-

tion of water soluble methylated NBD compounds and that of NBD-cholesterol incorporated in model membranes (A. Chattopadhyay and E. London, unpublished observations). This is consistent with the observation that the NBD group of NBD-cholesterol is buried more deeply than that of the NBD-labeled phospholpids (see below). However, interpretation of the results with methylated NBD compounds was complicated by the fact that the changes were irrevers- ible. It is possible that these compounds rear- range irreversibly after formation of the Meisenheimer adduct.

Location and orientation in membranes

The membrane penetration depths of NBD groups for different types of NBD-labeled lipids in model membranes have been studied by Chat- topadhyay and London by a novel fluorescence quenching method [214,215]. In these studies, the fluorescence of the NBD group in membranes was quenched by a spin-labeled phospholipid. Since the extent of quenching is dependent on the distance between the fluorophore and the quencher, variation of the attachment site of the spin-label (nitroxide) group in the acyl chain of the phospholipid gave different amounts of quenching for a specific fluorophore. The method involves determination of the parallax in the apparent location of fluorophores detected when quenching by phospholipids spin-labeled at two different depths is compared. From these types of quenching data, membrane penetration depths of the NBD moiety were analyzed using a hard sphere-like static quenching model that is appropriate for quenching occurring in membranes [214,215]. The lipids used in these studies were head group-labeled NBD-PE, acyl chain- labeled 6- and 12-NBD-PC and NBD-cholesterol in which the NBD group was at the flexible "tail" of cholesterol. Analysis of quenching data indicated that the NBD group in head group- labeled NBD-PE was at the polar region of the membrane and that an NBD label on the "tail" of NBD-cholesteroi was deeply buried (see Fig. 3). However, NBD labels placed at the end of fatty acyl chains of PC (6-NBD-PC and 12-NBD-

NBD -

CHOLESTEROL NBD - PE

~" ~ O- O" fl N - I

o / o / .o o Q. I i

o :

o

6 - NBD - PC 12 - NBD - PC

- - " o

,)

Fig. 3. A schematic diagram of half of the membrane bilayer showing the orientation and location of NBD-labeled lipids as measured by differential spin-label quenching (adapted with permission from Ref. 215, copyright (1989), American Chemical Society). The horizontal line at the bottom indicates the center of the bilayer.

PC) also appeared to be near the polar region. In fact, there was no significant difference between the penetration depths for 6-NBD-PC and 12-NBD-PC. This implied that the NBD groups in 6- and 12-NBD-PC were looping up to the surface in model membranes. These results were further confirmed by investigation of the spectroscopic and ionization characteristics of these lipids in membranes as determined by fluorescence, absorbance and electrophoretic mobility measurements [207]. Based on: (i) similarity of pK a values of NBD-PE and NBD-PC, (ii) quenching by aqueous quencher Co 2+ which measures the degree of exposure of the NBD group, and (iii) comparison of the position of fluorescence emission maxima in membranes and in solvents of varying polarity, it was demonstrated that the NBD group of NBD-PC and NBD-PE were in a polar region of the membrane bilayer and that it was deeply buried in the case of NBD-cholesterol. These results were in good agreement with the direct spin-label quenching measurements of NBD group depth [215].

Since the NBD group was not charged at neutral pH (as shown by electrophoretic mobility measurements, and discussed above), the polarity of the oxygen- and nitrogen-rich NBD group

most probably resulted in looping back to the surface when NBD groups were attached to acyl chains. Such looping up of other spectroscopic probes in micellar and vesicular environments has been demonstrated by Balasubramanian and coworkers [225---228]. In the case of NBD- cholesterol, either the rigidity of the sterol rings or the reduction of hydrophilicity due to the methyl group attached to NBD are possible reasons accounting for its deeper location in the membrane. The rigidity of the sterol ring is a particularly appealing one since cholesterol labeled at the "tail" with other polar probes have been shown to have an orientation perpen- dicular to the membrane bilayer and the polar group resides deep in the membrane instead of looping up [229--231]. In fact, besides sterol rings this stereochemical rigidity can also be imposed by other chemical structures. For exam- ple, the presence of the rigid isoprenoid side chain has been shown to prevent the NBD group from looping up in a fluorescent (NBD) derivative of ubiquinone in membranes [102,103]. It is also interesting to note that the NBD group is methylated in this NBD derivative of ubiquinone, just like NBD-cholesterol, which also has a deep location.

In a recent paper, based on analysis of energy transfer results with N-(lissamine rhodamine B sulfonyl) phosphatidylethanolamine (Rh-PE), Connor and Schroit [171] have concluded that the NBD group on 6-NBD-PC is buried deep in the membrane. However, they assumed that an average NBD-Rh distance could be applied to a random lateral distribution of 6-NBD-PC and Rh-PE. This is not strictly true in membranes. In addition, their triangulation analysis assumed that the distance between two sites is given by the difference of their distances from a third site. This is an incorrect assumption and is true only when all the three sites are along a straight line. This leads to a large underestimation of the distance between NBD groups in opposite leaflets.

The looping up of the NBD group in acyl chain labeled phospholipids could have significant implications in cases where the acyl chain conformation is important. Recently, Pagano and Martin synthesized a series of NBD-labeled- N-acylsphingosines for studying intracellular transport of lipids [204]. When the half-times for spontaneous transfer of N-acyI-D-erythrosphing- osines containing different NBD fatty acids were determined, a surprising result was obtained: the half-times for the D and L isomers of N-(a-NBD- aminohexanoyl)sphingosine were significantly higher than those for the other derivatives. One explanation for these slower transfer rates, as pointed out by these authors, was that the hexa- noic acid side chain might intercalate better into the membrane when the NBD group was close to the polar region of the N-acylsphingosine than when it was at the end of the fatty acyl chain. In other words, the tendency of the NBD group to loop back to the surface, when attached to ends of flexible acyl chains in lipids, results in faster spontaneous transfer of lipid monomers between membranes.

Applications in membrane studies

The range of applications of NBD-iabeled lipids in membrane related studies has been mentioned earlier. The basis of some of the applications are outlined below.

(a) Membrane fusion. Membrane fusion is a very important and widely studied process in model and biological membranes. Fusion events are known to take place during fertilization, myogenesis, virus infection, phagocytosis, endo- cytosis, exocytosis and extracellular release of neurotransmitters, hormones and enzymes. Dur- ing membrane fusion, the internal aqueous compartments of the two cells (or vesicles) coalesce accompanied by an intermixing of membrane lipids. For any event to be classified as membrane fusion, these two criteria must be met. Several assays have been developed for monitoring these two events (for reviews, see Refs. 232--234). Fluorescence resonance energy transfer has been used extensively to quantify membrane fusion [39,41]. One of the most popular assays for lipid mixing during membrane fusion is based on resonance energy transfer between NBD-PE as the donor molecule and Rh-PE as the acceptor [41]. These lipids have been shown to be non-exchangeable between phospholipid vesicles, even when the vesicles are aggregated [31,235]. There are two versions of this assay. In one version [42,236], the two fluorescent probes are incorporated into separate populations of vesicles. Fusion of the vesicles results in energy transfer between the donor and the acceptor. In the other version of this assay [41,237], both probes are incorporated in the membrane bilayer of one population of vesicles, called "labeled" vesicles. These vesicles are mixed with a population of "unlabeled" vesicles. Fusion results in a decrease of surface density of the probes, and thus in a reduction of the energy transfer ef- ficiency, since resonance energy transfer depends on the distance between fluorophores. This leads to an increase in the donor (NBD) fluorescence, which is monitored continuously as a measure of fusion. The latter version has the advantage that any possible energy transfer due to aggregation of separately labeled vesicles is eliminated. Also, this approach is especially suitable for monitoring fusion of liposomes with biological membranes.

(b) Intracellular and intervesicular lipid transport. Spontaneous, protein-mediated and vesicle- mediated transfer of lipid molecules through the

aqueous phase between two vesicles or cells offers a way for two physically separated membranes to interact with each other. This could be especially relevant in sorting and translocation of newly synthesized lipid molecules from their sites of synthesis within cells to other intracellular compartments where they are required for membrane assembly. Thus, it is an important biological process responsible for membrane assembly and control of membrane composition (for reviews, see Refs. 145,146,238). Pagano and coworkers have developed an elegant method using NBD-labeled lipids in liposomes to study this cell biological problem [26,27]. In this method, fluorescence resonance energy transfer is followed between an acyl chain-labeled NBD-lipid (e.g., 12-NBD-PC) and Rh-PE, in which the fluorescent rhodamine group is attached to the head group of PE. Both probes are incorporated in one population of vesicles, called "donor" vesicles. The NBD fluorescence remains quenched due to energy transfer to the neighbouring rhodamine groups. These are then mixed with an unlabeled population of vesicles, called "acceptor" vesicles. This causes an immediate and continuous increase of NBD fluorescence due to spontaneous transfer of the NBD-labeled lipid from donor to acceptor vesicles. Rh-PE is not transferred under these conditions [172]. By continuously monitoring NBD fluorescence intensity, the rate of transfer and equilibrium distribution of the NBD-labeled lipid can be determined. Such transfer involves migration of phospholipid molecules from the outer leaflet only and this has been utilized to generate asymmetric membrane vesicles [172]. The half-times for equilibration of NBD-labeled lipids between liposomes are on the time scale of minutes. Lipids with shorter fatty acyl chains have higher water solubility and thus lower half-times for transfer [28,36,208]. A kinetic model based on transfer of soluble lipid monomers has been developed [26,27]. According to this model, the transfer of lipids in liposomes occurs by dissocia- tion of lipid monomers from the donor membrane, diffusion through the aqueous phase, followed by association with the acceptor membrane. This property is very useful in metabolic

and transport studies with living cells. It allows incorporation of exogeneous NBD-labeled lipids into cellular membranes by incubating the cells with liposomes containing NBD-labeled lipids. Once the NBD-labeled lipids get incorporated into the cell, they can be observed by fluorescence microscopy. This permits observation of intracellular lipid transport in living cells, which can then be correlated with metabolism [145,146].

(c) Bilayer to hexagonal phase transition in liposomes. Certain lipids form non-bilayer structures such as the inverted hexagonal (Hlx) phase when dispersed in water (for reviews, see Refs. 239---241). The involvement of the non-bilayer phase in membrane processes such as membrane fusion and transbilayer transport has been sug- gested [239--241]. The non-bilayer phase is usu- ally detected by 31p NMR, X-ray diffraction, differential scanning calorimetry, freeze fracture electron microscopy and infrared spectroscopy [241--246]. However, none of these techniques is suitable to detect a bilayer to hexagonal transition in dilute membrane suspensions. An assay based on environmental sensitivity of NBD-PE fluorescence has recently been developed to detect bilayer to hexagonal phase transition at low lipid concentrations [159,160]. This assay is based on the observation that the fluorescence quantum yield of NBD-PE, when incorporated into liposomes containing lipids that undergo a bilayer to hexagonal transition, increases due to the change in local environment caused by the transition around the fluorophore [159]. Thus, there is an increase in fluorescence as the lipids go from a lamellar to a hexagonal phase. This method has been used to study the formation of hexagonal phase for liposomes containing phosphatidylethanolamine and cardiolipin using various agents that are known to promote the bilayer to hexagonal transition [160--162].

Conclusions

NBD-labeled lipids are currently one of the most widely used fluorescent lipid analogues in membrane studies at various levels of organization. From the initial synthesis of the NBD

10

group [1], it took almost ten years for the synthesis of the NBD-labeled lipids [20,21,199]. In the decade that followed, these lipids found wide- spread applications in a variety of studies. This is because of their chemical stability, suitable fluorescence properties (good spectral overlap with other fhorophores like rhodamine allowing efficient energy transfer, minimal interference with other biological fluorophores, self quenching at high concentrations and environmental sensitivity), rapid incorporation into living cells and ease of synthesis. Fundamental aspects of these lipids along with their applications have been brought together in this review.

One concern with NBD-labeled lipids, especially in cellular studies involving fluorescence microscopy, is that during continuous monitoring these lipids get photobleached, preventing re- peated exposure of the same cell [217]. Although this could be avoided by using low light level detector technology and other techniques [217], fluorescent lipids that are more photostable have been synthesized recently. These lipids contain borondipyrromethene (Bodipy) as the fluorophore [247,248].

A general concern in many studies using NBD-labeled lipids is to what extent they mimic the behavior of native lipids in membranes. As discussed by Pagano and Sleight [145], the metabolism and intracellular translocation of NBD- labeled lipids refect the behavior of endogenous lipids quite well. Also, the NBD group is not charged at neutral pH [207]. This means the actual charge on NBD-labeled lipids labeled in their acyi chains will be the same as the corresponding endogenous lipids under physiological conditions. However, for those lipids that are labeled in their acyl chains, the NBD group loops back to the polar region of the membrane, as detected by differential spin-label quenching and other studies, giving a perturbed acyl chain conformation [207,214,215]. This means that these lipids should be used as analogues of natural lipids with caution, especially if the acyl chain conformation is important. While this looping back is due to the polar nature of the NBD group, it is this polarity that helps rapid incorporation of the probe into living cells. Moreover,

the enzymes involved in metabolism, translocation and flip-flop for various lipid species main- tain their function in the presence of exogenous- ly added NBD-lipids. In addition, in many applications (like membrane fusion studies) the exact location of the NBD group in the membrane may not be very crucial. Therefore, use of NBD- labeled lipids as fluorescent analogues of native lipids is justified in these cases. Thus, in spite of possible complications due to probe effects, NBD-labeled lipids, in general, have served as reasonably good analogues to study a variety of membrane- and lipid-related cellular processes.

Acknowledgements

I would like to express my gratitude to Dr. Erwin London, in whose laboratory I worked with the NBD-labeled lipids, for going over the manuscript carefully and providing helpful com- ments and suggestions. I would also like to express my thanks and appreciation to Drs. An- thony Winiski, Mark McNamee and David Deamer for reading the manuscript critically and for providing valuable suggestions. Thanks are due to Allen Plummer for his help with the figures.

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Documents

Chemistry and biology of N-(7-nitrobenz-2-oxa-l,3-diazol- 4-yl)-labeled lipids" fluorescent probes of biological and model membranes-Chattopadhyay 1990