Effect of Phospholipid Bilayer Phase Asymmetry on Phospholipase D Reaction-Induced Vesicle Rupture

Spherical phospholipid bilayers, vesicles, were formed with respect to phase of each layer via a double emulsion technique. At the outer layer of the vesicles, phospholipase D catalyzed for the conversion of phosphatidylcholine (PC) to phosphatidic acid (PA). The reaction caused by phospholipase D (PLD) induced a curvature change in the vesicles, which eventually led them to rupture. Response time from the PLD injection to the rupture was monitored for the phase of each layer by using fluorescence intensity changes of pH-sensitive dye encapsulated in the vesicles. It was found that low ionic strength and asymmetric phase retarded response time. The retardation seems to be related to the stability of the vesicles, which is due to the interaction between the lipid molecules. In the liquid phases of the outer lipid layers, the unexpected slow response time may be attributed either to the fast lateral diffusion, which relieves the curvature change of the vesicles, or to the low concentration of PCs, which are less for the reaction compared to the solid phase of the outer lipid layer, rather than the stability.     

Effect of Mixed-Phospholipid Layer on Phospholipase D Reaction-induced Vesicle Rupture

Spherical phospholipid bilayers, or vesicles, were prepared layer by layer using a double-emulsion technique, which allows the outer layer of the vesicles to be formed with two phospholipids that have different head groups: phosphatidylcholine (PC) and phosphatidylethanolamine. At the outer layer of the vesicles, the phospholipase D (PLD) catalyzed for the conversion of PC to phosphatidic acid. The reaction caused by PLD induced the curvature change of the vesicles, which eventually led to the rupture of the vesicles. Before the investigation, the ratio of dioleoylphosphatidylethanolamine to oleoylhydroxyphosphatidylethanolamine was found as a condition such that the vesicles made with the mixed lipids were as stable as those made with pure dioleoylphosphatidylcholine. Response time from the PLD injection to vesicle rupture was monitored by the composition of the outer layer by the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. The response time began to be slowed at approximately 30?% PC. The response times for the compositions were associated with the surface density of PC at the outer layer. These results also seem to be determined by the size of PLD, specifically the PLD active site.     

Exchange and interactions between lipid layers at the surface of a liposome solution

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. p] Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Exchange and interactions between lipid layers at the surface of a liposome solution.

Lipid organization and lipid transport processes occurring at the air-water interface of a liposome (lipid vesicle) solution are studied by conventional surface pressure-area measurements and interpreted by an adequate theory. At the interface of a dioleoyl phosphatidylcholine vesicle solution, used for demonstration, a well defined two layer structure selfassembles: vesicles disintegrate at the interface forming a surface-adsorbed lipid monolayer, which prevents further disintegration beyond about 1 dyne/cm surface pressure. A layer of vesicles now assembles in close association with the monolayer. This layer is in vesicle diffusion exchange with the solution and in lipid exchange with the monolayer. The lipid exchange occurs exclusively between the monolayer and the outer lipid layer of the vesicles; it is absent between outer and inner vesicle layers. Equilibration of the lipid density in the monolayer with that in the vesicle outer layer provides a coherent and quantitative explanation of the observed hysteresis effects and equilibrium states. The correspondence between monolayer and vesicle outer layer is traced down to equilibrium constants and rate constants and their dependences on surface pressure, vesicle size and concentration. Other alternate realizations of surface structure and exchange, including induced lipid flip-flop within vesicles or vesicle monolayer adhesion or fusion are potential applications of the proposed analysis.     

Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.     

Transmembrane movement of heme

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.     

Topological model of the division process of cellular and subcellular compartments

The application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for a formation of a topological model of membrane transformations during the division process of cellular and subcellular compartments, has been shown. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicle. The analysis shows eight succeeding topological stages of membrane transformations during the division process and these stages are characterised. It is concluded that there is a vectorial translocation of lipid molecules from the inner layer of the membrane bounding the vesicle before the division process to the outer layer of the membranes after the division process and there is no lipid translocation from the outer layer to the inner layers during the division process.     

Reorganizational dynamics of multilamellar lipid bilayer assemblies using continuously scanning Fourier transform infrared spectroscopic imaging

We employ an implementation of rapid-scan Fourier transform infrared (FT-IR) microspectroscopic imaging to acquire time-resolved images for assessing the non-repetitive reorganizational dynamics of aqueous dispersions of multilamellar lipid vesicles (MLVs) derived from distearoylphosphatidylcholine (DSPC). The spatially and temporally resolved images allow direct and simultaneous determinations of various physical and chemical properties of the MLVs, including the main thermal gel to liquid crystalline phase transition, comparisons of vesicle diffusion rates in both phases and the variation in lipid bilayer packing properties between the inner and outer lamellae defining the vesicle. Specifically, in the lipid liquid crystalline phase, the inner bilayers of the MLVs are more intermolecularly ordered than the outer regions, while the intramolecular acyl chain order/disorder parameters, reflecting the overall characteristics of the fluid phase, remain uniform across the vesicle diameter. In contrast, the lipid vesicle gel phase displays no intermolecular or intramolecular dependence as a function of distance from the MLV center.     

Application of (1)H and (31)P NMR to topological description of a model of biological membrane fusion: topological description of a model of biological membrane fusion

The process of biological membrane fusion can be analysed by topological methods. Mathematical analysis of the fusion process of vesicles indicated two significant facts: the formation of an inner, transient structure (hexagonal phase - H(II)) and a translocation of some lipids within the membrane. This shift had a vector character and only occurred from the outer to the inner layer. Model membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) was studied. (31)P- and (1)H-NMR methods were used to describe the process of fusion. (31)P-NMR spectra of multilamellar vesicles (MLV) were taken at various temperatures and concentrations of Ca(2+) ions (natural fusiogenic agent). A (31)P-NMR spectrum with the characteristic shape of the H(II) phase was obtained for the molar Ca(2+)/PS ratio of 2.0. During the study, (1)H-NMR and (31)P-NMR spectra for small unilamellar vesicle (SUV), which were dependent on time (concentration of Pr(3+) ions was constant), were also recorded. The presence of the paramagnetic Pr(3+) ions permits observation of separate signals from the hydrophilic part of the inner and outer lipid bilayers. The obtained results suggest that in the process of fusion translocation of phospholipid molecules takes place from the outer to the inner layer of the vesicle and size of the vesicles increase. The NMR study has showed that the intermediate state of the fusion process caused by Ca(2+) ions is the H(II) phase. The experimental results obtained are in agreement with the topological model as well.     

Topological analysis of the fusion process between cellular and subcellular compartments

The study presents an application of the theory of homeomorphic transformations of topological manifolds and the operation of the connected sum of manifolds for topological analysis of membrane transformations during the fusion process between cellular and subcellular compartments. The biological cell and the subcellular structures in the form of vesicles are modelled by an arrangement of two concentric spheres corresponding to the inner and outer layer of the membrane bounding the vesicles. The analysis shows eight succeeding topological stages of membrane transformations during the fusion process and these stages are characterized. It is concluded that there is a vectorial translocation of lipid molecules from the outer layers of the membranes before the fusion process to the internal layer of the membrane bounding the vesicle after the fusion process and there is no lipid translocation in the reverse direction.     

Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation

Most biomembranes have an asymmetric structure with regard to phospholipid distribution between the inner and outer leaflets of the lipid bilayers. Control of the asymmetric distribution plays a pivotal role in several cellular functions such as intracellular membrane fusion and cell division. The mechanism by which membrane asymmetry and its alteration function in these transformation processes is not yet clear. To understand the significance of membrane asymmetry on trafficking and metabolism of intracellular vesicular components, a system that experimentally reproduces the asymmetric nature of biomembranes is essential. Here, we succeeded in obtaining asymmetric vesicles by means of transphosphatidylation reactions with phospholipase D (PLD), which acts exclusively on phosphatidylcholine (PC) present in the outer leaflet of vesicles. By treating PC vesicles with PLD in the presence of 1.7 M serine and 0.3 M ethanolamine, we obtained asymmetric vesicles that are topologically similar to intracellular vesicles containing phosphatidylserine and phosphatidylethanolamine in the cytosolic leaflet. PLD and other unwanted compounds could be removed by trypsin digestion followed by dialysis. Our established technique has a great advantage over conventional methods in that asymmetric vesicles can be provided at high yield and high efficiency, which is requisite for most physicochemical assays.     

Enhancement of endocytosis due to aminophospholipid transport across the plasma membrane of living cells

Formation of intracellular vesicles is initiated by membranebudding. Here we test the hypothesis that the plasma membrane surfacearea asymmetry could be a driving force for vesicle formation duringendocytosis. The inner layer phospholipid number was therefore increased by adding exogenous aminophospholipids to living cells, whichwere then translocated from the outer to the inner layer of themembrane by the ubiquitous flippase. Addition of either phosphatidylserine or phosphatidylethanolamine led to an enhancement ofendocytosis, showing that the observed acceleration does not depend onthe lipid polar head group. Conversely, a closely related aminophospholipid that is not recognized by the flippase,lyso--phosphatidylserine, inhibited endocytosis, and similar resultswere obtained with a cholesterol derivative that also remains in theplasma membrane outer layer. Thus an increase of lipid concentration inthe inner layer enhanced internalization, whereas an increase of thelipid concentration in the outer layer inhibited internalization. These experiments suggest that transient asymmetries in lipid concentration might contribute to the formation of endocytic vesicles.     

Carbon-13 nuclear magnetic resonance study of spin labelled cholesteryl ester in model membranes

Carbon-13 NMR longitudinal relaxation times for unilamellar vesicles of egg phosphatidyl-choline (PC) in aqueous dispersion have been measured following the incorporation of spin labelled cholesteryl palmitate. The spin label induced relaxation rates. 1/T_1.5L, for fatty acyl chain carbons show that the C5 segment of the cholesteryl ester acyl chain is located near the C1 and C2 segments of the phospholipid acyl chains. A greater spin label induced enhancement of relaxation rate was observed for the inner vesicle layer than for the outer, and is attributed to a higher ester incorporation and/or tighter lipid packing in the inner layer.     

Detection of surface charge-related properties in model membrane systems by aqueous two-phase partition

Unilamellar vesicles composed of phosphatidylcholine (PC) and either phosphatidic acid (PA) or phosphatidylglycerol (PG) partition to the upper poly(ethylene glycol) (PEG)-rich phase of a charge-sensitive 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 7, consistent with the vesicles bearing a net negative charge. When prepared in the presence of a pH gradient (interior acidic), PC/PA vesicles exhibit an increased partition to the top PEG-rich phase, consistent with a redistribution of the PA from the inner to the outer monolayer of the vesicle bilayer. Conversely, when prepared in the presence of a pH gradient (interior basic), PC/PG vesicles exhibit a decreased top-phase partition, consistent with a redistribution of the PG from the outer to the inner monolayer of the vesicle bilayer. Unilamellar vesicles composed of PC and stearylamine partition to the lower dextran-rich phase of a 5%:5% (w/w) PEG 8000/Dextran T-500 phase system containing 10 mM sodium phosphate at pH 8.5, consistent with the vesicles bearing a net positive charge. When prepared in the presence of a pH gradient (interior acidic), conditions under which the stearylamine is trapped on the inner monolayer of the bilayer, the vesicles now partition predominantly to the interface in a manner similar to vesicles composed of PC alone. These results demonstrate that partitioning in aqueous two-phase polymer systems is a sensitive method for monitoring the asymmetry of charged lipids in model membrane systems and also suggests that partitioning in charge-sensitive systems depends only on the physical nature of the exterior surface of the membrane.     

Development of the pellicle and thecal plates following ecdysis in the dinoflagellateGlenodinium foliaceum

Summary The ultrastructure and development of the amphiesma of the dinoflagellateGlenodinium foliaceum was studied using conventional electron microscopy and immunocytochemistry. Ecdysis (shedding of the flagella, the outer two membranes of the cell, and the thecal plates) was induced by centrifugation. The cells were resuspended and the thickening of the pellicle and the development of the new thecal vesicles and plates was studied over a 9 h period. After ecdysis, the thin pellicle which underlay the thecal plates in the motile cells thickens to form a complex structure of four distinct layers: an outer layer of randomly oriented fibrils, a 50 nm layer of fibrils oriented perpendicular to the dense layer, the dense layer which has a trilaminate structure, and a wide inner homogeneous layer. The new thecal vesicles form in these pelliculate cells by the migration of electron translucent amphisomal vesicles over the layer of peripheral microtubules to a position directly under the plasmalemma. The thecal vesicles then flatten and elongate. A discontinuous pellicular layer appears within them. Subsequently, the thecal vesicles widen and are filled with a fibrillogranular substance overlying the pelliculate layer. The thecal plates form on top of this fibrillogranular material. By this time, most cells have escaped from the pellicle and are motile. At first, the outer thecal vesicle membrane is continuous with the inner thecal vesicle membrane at the sutures, but when this connection is broken, the dense pelliculate layers become continuous across the suture as does the inner thecal vesicle membrane. At ecdysis, this membrane becomes the new plasmalemma of the cell. Cells at each stage of pellicle thickening and thecal development were labelled with a polydonal antiserum raised against the 70 kDa epiplasmic protein ofEuglena acus. This antiserum labelled both the thecal plates of the motile cells and the inner homogeneous layer of the pellicle of ecdysed non-motile cells. No other amphiesmal structure was labelled, nor was any intracellular compartment.Abbreviations PBS phosphate-buffered saline - PIPES piperazine-N,N-bis[2-ethane sulfonic acid]     

Processive interfacial catalytic turnover by Bacillus cereus sphingomyelinase on sphingomyelin vesicles

Sphingomyelinase (SMase), a water-soluble enzyme from Bacillus cereus, is shown to bind with high affinity to vesicles of sphingomyelin (SM) but not to vesicles of phosphatidylcholine (PC). The reaction progress by SMase bound to SM vesicles occurs in the scooting mode with virtually infinite processivity of the successive interfacial turnover cycles. Three conditions for the microscopic steady state during the reaction progress at the interface are satisfied: the bound SMase does not leave the interface even after all the SM in the outer layer is converted to ceramide; the SMase-treated vesicles remain intact; and the ceramide product does not exchange with SM present in excess vesicles or in the inner layer of the hydrolyzed vesicle. Within these constraints, on accessibility and replenishment of the substrate, the extent of hydrolysis in the scooting mode reaction progress is a measure of the number of vesicles containing enzyme. The slope of the Poisson distribution plot, for the enzyme per vesicle versus the logarithm of the fraction of the total accessible substrate remaining unhydrolyzed in excess vesicles, shows that a single 32 kDa subunit of SMase is fully catalytically active. The maximum initial rate of hydrolysis, at the limit of the maximum possible substrate mol fraction, X(S)*=1, is 400 s(-1) in H(2)O and 220 s(-1) in D(2)O, which is consistent with the rate-limiting chemical step. The integrated reaction progress suggests that the ceramide product does not codisperse ideally on the hydrolyzed vesicles. Furthermore, complex reaction progress seen with covesicles of SM+PC are attributed to slow secondary changes in the partially hydrolyzed SM vesicles.     

Hydrolysis of phosphatidylcholine in phosphatidylcholine-cholate mixtures by porcine pancreatic phospholipase A2

Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate depletion becomes significant. The reaction can be divided phenomenologically into two phases: a latency phase where the amount of product formed is a square function of time (P(t) = At2) and a phase distinguished by a sudden increase in activity. The parameter A, which describes the activation rate of the enzyme during the initial phase in a quantitative fashion, increases with increasing [PLA2], decreasing [PC], decreasing vesicle size, and increasing relative cholate content of the vesicles. The effect of [PLA2] and [PC] on the hydrolysis reaction is similar to that found with pure DPPC unilamellar vesicles in their thermotropic phase transition region. The effect of cholate on the hydrolysis reaction is similar to that of temperature variation within the phase transition of temperature variation within the phase transition of DPPC. These results are consistent with our previously proposed model, which postulates that activation of PLA2 involves dimerization of the enzyme on the substrate surface and that the rate of activation is directly proportional to the magnitude of lipid structural fluctuations. It is suggested that large structural fluctuations, which exist in the pure lipid system in the phase transition range, are introduced into liquid crystalline vesicles by the presence of cholate and thus promote activation of the enzyme.     

Peroxidation and phospholipase A2 hydrolytic susceptibility of liposomes consisting of mixed species of phosphatidylcholine and phosphatidylethanolamine.

The relationship between lipid peroxidation and phospholipase A2 (PLA2) hydrolytic activity was studied using unilamellar vesicles (liposomes) as model membranes. Hydrolytic specificity was examined using vesicles prepared with pure bovine heart phosphatidylcholine (PC), bovine heart phosphatidylethanolamine (PE), or mixtures of these phospholipids, using two preparative procedures, i.e., sonication or extrusion. Lipid peroxidation was induced by incubating vesicles with cumene hydroperoxide and hematin at 37 degrees C. Determinations of the extent of peroxidation by means of diene conjugate content derived from second derivative spectra or by polarographic measurement of oxygen consumption rates provided a basis for comparing the extent of peroxidation of each phospholipid species to their subsequent hydrolysis by PLA2 (from Crotalus adamanteus). The extent of hydrolysis was determined through the release of arachidonic acid from either PC or PE. The PE distribution among the outer vs. inner leaflet of the membrane bilayer was nearly equal in sonicated vesicles, whereas most of the phospholipid was incorporated into the inner leaflet in extruded vesicles. The proportion of PE found in the inner leaflet progressively increased as the ratio of PE to PC increased in both sonicated and extruded vesicle preparations. Lipid peroxidation had no effect on PE distribution under the conditions examined. There was a clear preference for PC peroxidation for all vesicle compositions tested and PC was preferentially hydrolyzed by PLA2. This effect is proposed to result from a perturbation of membrane structure following peroxidation with assimilation of PC into PLA2-susceptible domains whereas PE peroxidation and hydrolysis is less affected in mixed PC/PE vesicles. Lipid peroxidation imposes an additional hydrolytic susceptibility over the effects exerted through the mixing of these phospholipids which is based on structural changes rather than formation of specific substrates for PLA2.     

锯缘青蟹精子发生的超微结构

采用透射电镜观察锯缘青蟹精子发生过程中超微结构的变化,结果表明：精原细胞椭圆形,染色质分布于核膜周围,胞质中具嵴少的线粒体,内质网小泡等。初级精母细胞染色质呈非浓缩状,胞质中具众 内质网小泡,特殊的膜系及晶格状结构。次级精母细胞核质间出现由内质小泡聚集成的腔。     

Salamander retina phospholipids and their localization by MALDI imaging mass spectrometry at cellular size resolution

Salamander large cells facilitated identification and localization of lipids by MALDI imaging mass spectrometry. Salamander retina lipid extract showed similarity with rodent retina lipid extract in phospholipid content and composition. Like rodent retina section, distinct layer distributions of phospholipids were observed in the salamander retina section. Phosphatidylcholines (PCs) composing saturated and monounsaturated fatty acids (PC 32:0, PC 32:1, and PC 34:1) were detected mainly in the outer and inner plexiform layers (OPL and IPL), whereas PCs containing polyunsaturated fatty acids (PC 36:4, PC 38:6, and PC 40:6) composed the inner segment (IS) and outer segment (OS). The presence of PCs containing polyunsaturated fatty acids in the OS layer implied that these phospholipids form flexible lipid bilayers, which facilitate phototransduction process occurring in the rhodopsin rich OS layer. Distinct distributions and relative signal intensities of phospholipids also indicated their relative abundance in a particular cell or a cell part. Using salamander large cells, a single cell level localization and identification of biomolecules could be achieved by MALDI imaging mass spectrometry.