Pressure tuning of the morphology of heterogeneous lipid vesicles: a two-photon-excitation fluorescence microscopy study

We used a technique that allows us to visualize local and morphological changes of the membrane of more component giant unilamellar vesicles due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/sphingomyelin/cholesterol as well as of the fluid mixture POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) by two-photon excitation fluorescence microscopy. The pressure-dependent experiments were carried out using a sample cell made from a thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to also follow the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is observed, accompanied by an increase of the average GP value, indicating an increasingly tighter packing of the lipids. Interestingly, the two systems studied show phenomena of budding and fission, and these at surprisingly low pressures of 200-300 bar. Moreover, these budding processes are not directly related to phase transitions to an overall ordered conformational state of the lipid membrane, which occur at much higher pressures. The topological changes of the lipid vesicles are irreversible and exhibit a different behavior depending on whether the pressure is increased or decreased. The results are discussed in light of the various contributions to the free energy functional of lipid vesicles. Finally, the biological relevance of these studies is highlighted.     

Detergents induce raft-like domains budding and fission from giant unilamellar heterogeneous vesicles: a direct microscopy observation

The effect of detergents on giant unilamellar vesicles (GUVs) composed of phosphatidylcholine, sphingomyelin and cholesterol and containing liquid-ordered phase (l(o)) domains was investigated. Such domains have been used as models for the lipid rafts present in biological membranes. The studied detergents included lyso-phosphatidylcholine, the product of phospholipase A2 activity, as well as Triton X-100 and Brij 98, i.e. detergents used to isolate lipid rafts as DRMs. Local external injection of each of the three detergents at subsolubilizing amounts promoted exclusion of l(o) domains from the GUV as small vesicles. The budding and fission processes associated with this vesiculation were interpreted as due to two distinct effects of the detergent. In this framework, the budding is caused by the initial incorporation of the detergent in the outer membrane leaflet which increases the spontaneous curvature of the bilayer. The fission is related to the inverted-cone molecular shape of the detergent which stabilizes positively curved structures, e.g. pores involved in vesicle separation. On the other hand, we observed in GUVs neither domain formation nor domain coalescence to be induced by the addition of detergents. This supports the idea that isolation of DRM from biological membranes by detergent-induced extraction is not an artifact. It is also suggested that the physico-chemical mechanisms involved in l(o) domain budding and fission might play a role in rafts-dependant endocytosis in cells.     

Adhesion of nanoparticles to vesicles: a Brownian dynamics simulation

We studied the interaction of bilayer vesicles and adhesive nanoparticles using a Brownian dynamics simulation. The nanoparticles are simple models of proteins or colloids. The adhering nanoparticle induces the morphological change of the vesicle: budding, formation of two vesicles in which only outer monolayers are connected, and fission. We also show that the nanoparticle promotes the fusion process: fusion-pore opening from a stalk intermediate, a neck-like structure that only connects outer monolayers of two vesicles. The nanoparticle bends the stalk, and induces the pore opening.     

Shape changes of giant liposomes induced by an asymmetric transmembrane distribution of phospholipids.

The influence of a phospholipid transmembrane redistribution on the shape of nonspherical flaccid vesicles was investigated at a fixed temperature by optical microscopy. In a first series of experiments, a transmembrane pH gradient was imposed on egg phosphatidylcholine (EPC)-egg phosphatidylglycerol (EPG) (100:1) giant vesicles. The delta pH induced an asymmetric distribution of EPG. Simultaneously, discoid vesicles were transformed into tubular or a series of connected small vesicles. The fraction of phospholipid transfer necessary for a shape change from discoid to two connected vesicles was of the order of 0.1% of the total phospholipids. Additional lipid redistribution was accompanied by a sequence of shape changes. In a second series of experiments, lyso phosphatidylcholine (L-PC) was added to, or subtracted from, the external leaflet of giant EPC vesicles. The addition of L-PC induced a change from discoid to a two-vesicle state without further evolution, suggesting that lipid transfer and lipid addition are not equivalent. L-PC depletion from the outer leaflet generated stomatocyte-like vesicles. Whenever possible, we have determined whether the giant vesicles undergoing shape changes were unilamellar or multilamellar by measuring the elastic area compressibility modulus, K, by the micropipette assay (Kwok and Evans, 1981). Shape transformations triggered by phospholipid modification of the most external bilayer were indeed influenced by the presence of other underlying membranes that played a role comparable to that of a passive cytoskeleton layer. It appears that in real cells, invaginations of the plasma membrane or budding of organelles could be triggered by a phospholipid transfer from one leaflet to the other caused, for instance, by the aminophospholipid translocase which is present in eukaryotic membranes.     

Rapid constriction of lipid bilayers by the mechanochemical enzyme dynamin

Dynamin, a large GTPase, is located at the necks of clathrin-coated pits where it facilitates the release of coated vesicles from the plasma membrane upon GTP binding, and hydrolysis. Previously, we have shown by negative stain electron microscopy that wild-type dynamin and a dynamin mutant lacking the C-terminal proline-rich domain, DeltaPRD, form protein-lipid tubes that constrict and vesiculate upon addition of GTP. Here, we show by time-resolved cryo-electron microscopy (cryo-EM) that DeltaPRD dynamin in the presence of GTP rapidly constricts the underlying lipid bilayer, and then gradually disassembles from the lipid. In agreement with the negative stain results, the dynamin tubes constrict from 50 to 40 nm, and their helical pitch decreases from approximately 13 to 9.4 nm. However, in contrast to the previous results, examination by cryo-EM shows that the lipid bilayer remains intact and small vesicles or fragments do not form upon GTP binding and hydrolysis. Therefore, the vesicle formation seen by negative stain may be due to the lack of mobility of the dynamin tubes on the grid during the GTP-induced conformational changes. Our results confirm that dynamin is a mechanochemical enzyme and suggest that during endocytosis dynamin is directly responsible for membrane constriction. In the cell, other proteins may enhance the activity of dynamin or the constraints induced by the surrounding coated pit and plasma membrane during constriction may cause the final membrane fission event.     

Shape transitions and shape stability of giant phospholipid vesicles in pure water induced by area-to-volume changes.

Shape transformations of vesicles of dimyristoylphosphatidylcholine (= DMPC) and palmitoyloleylphosphatidylcholine (= POPC) in ion-free water were induced by changing the area-to-volume ratio via temperature variations. Depending on the pretreatment we find several types of shape changes for DMPC (in pure water) at increasing area-to-volume ratio: (a) budding transitions leading to the formation of a chain of vesicles at further increase of the area-to-volume ratio, (b) discocyte-stomatocyte transitions, (c) reentrant dumbbell-pear-dumbbell transitions, and (d) spontaneous blebbing and/or tether formation of spherical vesicles. Beside these transitions a more exotic dumbbell-discocyte transition (e) was found which proceeded via local instabilities. Pears, discocytes, and stomatocytes are stable with respect to small temperature variations unless the excess area is close to values corresponding to limiting shapes of budded vesicles where temperature variations of less than or equal to 0.1 degree C lead to spontaneous budding to the inside or the outside. For POPC we observed only budding transitions to the inside leading either to chains of vesicles or to distributions of equally sized daughter vesicles protruding to the inside of the vesicle. Preliminary experiments concerning the effect of solutes are also reported. The first three types of shape transitions can be explained in terms of the bilayer coupling model assuming small differences in thermal expansivities of the two monolayers. This does not hold for the observed instabilities close to the limiting shapes.     

Osmotic shrinkage of giant egg-lecithin vesicles.

Osmotic shrinkage of giant egg-lecithin vesicles was observed by phase-contrast microscopy. The vesicles remained or became spherical when shrinking. Small and thick-walled vesicles formed visible fingers attached to the sphere. The water permeability of the single bilayer was found to be 41 micrometers/s. A variety of observations indicate that osmosis induces a parallel lipid flow between the monolayers of the bilayer, leading to a strong positive spontaneous curvature. They also suggest the formation of mostly submicroscopic daughter vesicles. The estimated coupling constant, 2 . 10(-6) mol/mol, is large enough to be biologically significant.     

Improving our picture of the plasma membrane: Rafts induce ordered domains in a simplified model cytoplasmic leaflet

By study of asymmetric membranes, models of the cell plasma membrane (PM) have improved, with more realistic properties of the asymmetric lipid composition of the membrane being explored. We used hemifusion of symmetric giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB) to engineer bilayer leaflets of different composition. During hemifusion, only the outer leaflets of GUV and SLB are connected, exchanging lipids by simple diffusion. aGUVs were detached from the SLB for study. In general these aGUVs are formed with one leaflet that phase-separates into Ld (liquid disordered) + Lo (liquid ordered) phases, and another leaflet with lipid composition that would form a single fluid phase in a symmetric bilayer. We observed that ordered phases of either Lo or Lβ (gel phase) induce an ordered domain in the apposed fluid leaflet that lacks high melting lipids. Results suggest both an inter-leaflet and an intra-leaflet redistribution of cholesterol. We used C-Laurdan spectral images to investigate the lipid packing/order of aGUVs, finding that cholesterol partitions into the induced ordered domains. We suggest this behavior to be commonplace, that when Ld + Lo phase separation occurs in a cell PM exoplasmic leaflet, an induced order domain forms in the cytoplasmic leaflet.     

An Asymmetry in Monolayer Tension Regulates Lipid Droplet Budding Direction

Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions. We thereupon determined that slight tension asymmetries between the membrane monolayers regulate the droplet budding side. A differential regulation of lipid or protein composition around a forming LD can generate a monolayer tension asymmetry that will determine the LD budding side. Our results offer, to our knowledge, new insights on how the proteins might regulate LD formation side by generating a monolayer tension asymmetry.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: Only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Interaction of a pseudosubstrate peptide of protein kinase C and its myristoylated form with lipid vesicles: only the myristoylated form translocates into the lipid bilayer

Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.     

Cholesterol esterase accelerates intestinal cholesterol absorption

Lipid bilayers of dimyristoyl phosphatidylcholine (DMPC) containing opioid peptide dynorphin A(1-17) are found to be spontaneously aligned to the applied magnetic field near at the phase transition temperature between the gel and liquid crystalline states (T(m)=24 degrees C), as examined by 31P NMR spectroscopy. The specific interaction between the peptide and lipid bilayer leading to this property was also examined by optical microscopy, light scattering, and potassium ion-selective electrode, together with a comparative study on dynorphin A(1-13). A substantial change in the light scattering intensity was noted for DMPC containing dynorphin A(1-17) near at T(m) but not for the system containing A(1-13). Besides, reversible change in morphology of bilayer, from small lipid particles to large vesicles, was observed by optical microscope at T(m). These results indicate that lysis and fusion of the lipid bilayers are induced by the presence of dynorphin A(1-17). It turned out that the bilayers are spontaneously aligned to the magnetic field above T(m) in parallel with the bilayer surface, because a single 31P NMR signal appeared at the perpendicular position of the 31P chemical shift tensor. In contrast, no such magnetic ordering was noted for DMPC bilayers containing dynorphin A(1-13). It was proved that DMPC bilayer in the presence of dynorphin A(1-17) forms vesicles above T(m), because leakage of potassium ion from the lipid bilayers was observed by potassium ion-selective electrode after adding Triton X-100. It is concluded that DMPC bilayer consists of elongated vesicles with the long axis parallel to the magnetic field, together with the data of microscopic observation of cylindrical shape of the vesicles. Further, the long axis is found to be at least five times longer than the short axis of the elongated vesicles in view of simulated 31P NMR lineshape.     

Interactions of the HIV-1 fusion peptide with large unilamellar vesicles and monolayers. A cryo-TEM and spectroscopic study

We have examined the interaction of the human immunodeficiency virustype 1 fusion peptide (23 amino acid residues) and of a Trp-containing analog with vesicles composed of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and cholesterol (molar ratio, 1:1:1). Both the native and the Trp-substituted peptides bound the vesicles to the same extent and induced intervesicular lipid mixing with comparable efficiency. Infrared reflection-absorption spectroscopy data are compatible with the adoption by the peptide of a main beta-sheet structure in a cospread lipid/peptide monolayer. Cryo-transmission electron microscopy observations of peptide-treated vesicles reveal the existence of a peculiar morphology consisting of membrane tubular elongations protruding from single vesicles. Tryptophan fluorescence quenching by brominated phospholipids and by water-soluble acrylamide further indicated that the peptide penetrated into the acyl chain region closer to the interface rather than into the bilayer core. We conclude that the differential partition and shallow penetration of the fusion peptide into the outer monolayer of a surface-constrained bilayer may account for the detected morphological effects. Such single monolayer-restricted interaction and its structural consequences are compatible with specific predictions of current theories on viral fusion.     

Asymmetric fusion between phospholipid vesicles and vesicles formed from synthetic di(n-alkyl)phosphates

We have investigated the fusion behavior of a mixed vesicle system consisting of vesicles prepared from the simple synthetic surfactants di(n-dodecyl)phosphate (DDP) or di(n-tetradecyl)phosphate (DTP) and vesicles prepared from the phospholipids phosphatidylserine (PS) or dioleoylphosphatidylcholine (DOPC). Fusion between the vesicles, induced by Ca2+, was determined by a resonance energy transfer assay for lipid mixing, sucrose density gradient analysis, and electron microscopy. We demonstrate that synthetic surfactant vesicles can specifically engage in asymmetric fusion events, provided that the incubation temperature is kept below the gel-liquid crystalline phase-transition temperature (Tc) of the synthetic amphiphile (29 and 48 degrees C for DDP and DTP, respectively) and that the physical state of the target membrane is fluid. Asymmetric fusion of DDP or DTP vesicles was most efficient with PS vesicles, but it also occurred with zwitterionic PC vesicles. In the latter case, fusion proceeded spontaneously, but the process was markedly accelerated upon addition of Ca2+. Furthermore, in contrast to a massive transformation of bilayer into nonbilayer hexagonal HII tubular structures, as occurs upon symmetric Ca(2+)-induced fusion of DDP vesicles, asymmetric fusion with phospholipid bilayers predominantly leads to the formation of larger vesicles. This indicates that both PS and DOPC stabilize the DDP bilayer structure in the fusion product.     

Interaction of negatively charged liposomes with nuclear membranes: adsorption, lipid mixing and lysis of the vesicles

Fluorescence energy transfer studies reveal that negatively charged lipid vesicles interact with nuclei from mouse liver cells. This interaction was observed with charged lipid vesicles composed of PA or PS but not with the uncharged PC or PE:PC vesicles. The vesicles were prepared by bath sonication and contained either a fluorescent marker in the lipid bilayer or in the vesicular interior. The negatively charged vesicles showed an adsorption to the nuclear membrane visible by fluorescence microscopy. The results obtained by resonance energy transfer experiments are interpreted in terms of a mixing of the lipids from the vesicles with the nuclear membrane. Encapsulation studies documented a staining of the nuclei only if the dye molecules of high or low molecular weight were encapsulated inside negatively charged vesicles. As consequence of the vesicle-nuclei interaction morphological changes on the nuclear surface became visible.     

Interactions between cholesterol and lipids in bilayer membranes. Role of lipid headgroup and hydrocarbon chain-backbone linkage

We have employed four lipids in the present study, of which two are cationic and two bear phosphatidylcholine (PC) headgroups. Unlike dipalmitoylphosphatidylcholine, the other lipids employed herein do not have any ester linkage between the hydrocarbon chains and the respective lipid backbones. Small unilamellar vesicles formed from each of the PC and cationic lipids with or without varying amounts of cholesterol have been examined using the steady-state fluorescence anisotropy method as a function of temperature. The anisotropy data clearly indicate that the order in the lipid bilayer packing is strongly affected upon inclusion of cholesterol. This effect is similar irrespective of the electrostatic character of the lipid employed. The influence of cholesterol inclusion on multi-lamellar lipid dispersions has also been examined by 1H-nuclear magnetic resonance spectroscopy above the phase transition temperatures. With all the lipids, the line widths of (CH2)n protons of hydrocarbon chains in the NMR spectra respond to the addition of cholesterol to membranes. The influence on the bilayer widths of various lipids upon inclusion of cholesterol was determined from X-ray diffraction studies of the cast films of the lipid-cholesterol coaggregates in water. The effect of cholesterol on the efflux rates of entrapped carboxyfluorescein (CF) from the phospholipid vesicles was determined. Upon incremental incorporation of cholesterol into the phospholipid vesicles, the CF leakage rates were progressively reduced. Independent experiments measuring transmembrane OH- ion permeation rates from cholesterol-doped cationic lipid vesicles using entrapped dye riboflavin also demonstrated that the addition of cholesterol into the cationic lipid vesicles reduced the leakage rates irrespective of lipid molecular structure. It was found that the cholesterol induced changes on the membrane properties such as lipid order, linewidth broadening, efflux rates, bilayer widths, etc., did not depend on the ability of the lipids to participate in the hydrogen bonding interactions with the 3beta-OH of cholesterol. These findings emphasize the importance of hydrophobic interaction between lipid and cholesterol and demonstrate that it is not necessary to explain the observed cholesterol induced effects on the basis of the presence of hydrogen bonding between the 3beta-OH of cholesterol and the lipid chain-backbone linkage region or headgroup region.     

Chirality-induced budding: a raft-mediated mechanism for endocytosis and morphology of caveolae?

The formation of transport carriers (spherical vesicles and tubules) involves membrane budding, growth, and ultimately fission. We propose a mechanism of membrane budding, wherein the tilt and chirality of constituent molecules, confined to a patch of area A, induces buds of approximately 50-100 nm that are comparable to vesicles involved in endocytosis. Because such chiral and tilted lipid molecules are likely to exist in "rafts", we suggest the involvement of this mechanism in generating membrane buds in the clathrin and dynamin-independent, raft-component mediated endocytosis of glycosylphosphatidylinositol-anchored proteins. We argue that caveolae, permanent cell surface structures with characteristic morphology and enriched in raft constituents, are also likely to be formed by this mechanism. Thus, molecular chirality and tilt, and its expression over large spatial scales may be a common organizing principle in membrane budding of transport carriers.     

Vesicle diffusion close to a membrane: intermembrane interactions measured with fluorescence correlation spectroscopy

The protein machinery controlling membrane fusion (or fission) has been well studied; however, the role of vesicle diffusion near membranes in these critical processes remains unclear. We experimentally and theoretically investigated the dynamics of small vesicles (∼50 nm in diameter) that are diffusing near supported planar bilayers acting as “target” membranes. Using total internal reflection-fluorescence correlation spectroscopy, we examined the validity of theoretical analyses of vesicle-membrane interactions. Vesicles were hindered by hydrodynamic drag as a function of their proximity to the planar bilayer. The population distributions and diffusion kinetics of the vesicles were further affected by changing the ionic strength and pH of the buffer, as well as the lipid composition of the planar membrane. Effective surface charges on neutral bilayers were also analyzed by comparing experimental and theoretical data, and we show the possibility that vesicle dynamics can be modified by surface charge redistribution of the planar bilayer. Based on these results, we hypothesize that the dynamics of small vesicles, diffusing close to biomembranes, may be spatially restricted by altering local physiological conditions (e.g., salt concentration, lipid composition, and pH), which may represent an additional mechanism for controlling fusion (or fission) dynamics.     

Ripples and the formation of anisotropic lipid domains: imaging two-component supported double bilayers by atomic force microscopy

Direct visualization of the fluid-phase/ordered-phase domain structure in mica-supported bilayers composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphocholine mixtures is performed with atomic force microscopy. The system studied is a double bilayer supported on a mica surface in which the top bilayer (which is not in direct contact with the mica) is visualized as a function of temperature. Because the top bilayer is not as restricted by the interactions with the surface as single supported bilayers, its behavior is more similar to a free-standing bilayer. Intriguing straight-edged anisotropic fluid-phase domains were observed in the fluid-phase/ordered-phase coexistence temperature range, which resemble the fluid-phase/ordered-phase domain patterns observed in giant unilamellar vesicles composed of such phospholipid mixtures. With the high resolution provided by atomic force microscopy, we investigated the origin of these anisotropic lipid domain patterns, and found that ripple phase formation is directly responsible for the anisotropic nature of these domains. The nucleation and growth of fluid-phase domains are found to be directed by the presence of ripples. In particular, the fluid-phase domains elongate parallel to the ripples. The results show that ripple phase formation may have implications for domain formation in biological systems.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.