Gel Domains Research Articles

Gel Domains in the Plasma Membrane of Saccharomyces cerevisiae: HIGHLY ORDERED, ERGOSTEROL-FREE, AND SPHINGOLIPID-ENRICHED LIPID RAFTS

The plasma membrane of Saccharomyces cerevisiae was studied using the probes trans-parinaric acid and diphenylhexatriene. Diphenylhexatriene anisotropy is a good reporter of global membrane order. The fluorescence lifetimes of trans-parinaric acid are particularly sensitive to the presence and nature of ordered domains, but thus far they have not been measured in yeast cells. A long lifetime typical of the gel phase (>30 ns) was found in wild-type (WT) cells from two different genetic backgrounds, at 24 and 30 °C, providing the first direct evidence for the presence of gel domains in living cells. To understand their nature and location, the study of WT cells was extended to spheroplasts, the isolated plasma membrane, and liposomes from total lipid and plasma membrane lipid extracts (with or without ergosterol extraction by cyclodextrin). It is concluded that the plasma membrane is mostly constituted by ordered domains and that the gel domains found in living cells are predominantly at the plasma membrane and are formed by lipids. To understand their composition, strains with mutations in sphingolipid and ergosterol metabolism and in the glycosylphosphatidylinositol anchor remodeling pathway were also studied. The results strongly indicate that the gel domains are not ergosterol-enriched lipid rafts; they are mainly composed of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins, suggesting an important role in membrane traffic and signaling, and interactions with the cell wall. The abundance of the sphingolipid-enriched gel domains was inversely related to the cellular membrane system global order, suggesting their involvement in the regulation of membrane properties.

A computational Study of Gel Nucleation in Lipid Bilayer

Even though registration and anti-registration in lipid bilayers have been observed on the level of individual lipids experimentally in a nearest-neighbor recognition analysis and computationally in coarse-grained (CG) simulations, the switching mechanism from one matching to another between two leaflets remains unclear. In mean field analysis, coupling between domains was described by a composition-dependent surface tension or, in the case of small compositional changes, by a single coupling constant induced mainly by dynamic chain interdigitation, but the theory has not been tested. Here, we aim to provide a quantitative analysis of the coupling between gel domains at the early stage of nucleation by combining forward flux sampling (FFS) with molecular dynamics simulations of lipid bilayer membranes with a systematic solvent-free CG model. The combination of both techniques enables us to sample the rare events of gel domain nucleation in the lipid bilayers at moderate computational expense, while still preserving chemical specificity and quantitative accuracy. The FFS is implemented to capture the growing and shrinking scenarios of a gel nucleus in a bilayer. We first study gel nucleation in a fluid DPPC bilayer membrane to understand the coupling between domains independent of composition change. Afterwards, we study gel nucleation in a DPPC-DOPC binary membrane, which adds the complication of a composition-dependent surface tension of the gel domain. The free-energy of gel nucleation is computed as a function of gel domain size, and both intra- and inter-leaflet coupling between domains are analyzed.

Dihydrosphingomyelin Impairs HIV-1 infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (ld) and the liquid-ordered (lo); the putative presence of gel domains (so) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid lo domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Band, target, and onion patterns in Co(OH)2Liesegang systems

The study of morphology and shape development has gained considerable interest in certain sciences, notably biology and geology. Liesegang experiments producing Co(OH)2 stratification are performed here, in one, two, and three dimensions for comparison of the pattern morphologies. We obtain well-resolved bands in one dimension, target patterns (rings) in two dimensions, and onion patterns (spherical shells) in three dimensions. The morphological characteristics of the various patterns (spacing coefficients, rate of growth of ring spacing with distance) were measured. The spacing ratio of the strata in the different spatial dimensions was found to be anticorrelated with the surface-to-volume ratio of the gel domain. Some studies featuring the importance of morphology in Liesegang systems are briefly surveyed.

Ethanol effects on binary and ternary supported lipid bilayers with gel/fluid domains and lipid rafts

Ethanol–lipid bilayer interactions have been a recurrent theme in membrane biophysics, due to their contribution to the understanding of membrane structure and dynamics. The main purpose of this study was to assess the interplay between membrane lateral heterogeneity and ethanol effects. This was achieved by in situ atomic force microscopy, following the changes induced by sequential ethanol additions on supported lipid bilayers formed in the absence of alcohol. Binary phospholipid mixtures with a single gel phase, dipalmitoylphosphatidylcholine (DPPC)/cholesterol, gel/fluid phase coexistence DPPC/dioleoylphosphatidylcholine (DOPC), and ternary lipid mixtures containing cholesterol, mimicking lipid rafts (DOPC/DPPC/cholesterol and DOPC/sphingomyelin/cholesterol), i.e., with liquid ordered/liquid disordered (ld/lo) phase separation, were investigated. For all compositions studied, and in two different solid supports, mica and silicon, domain formation or rearrangement accompanied by lipid bilayer thinning and expansion was observed. In the case of gel/fluid coexistence, low ethanol concentrations lead to a marked thinning of the fluid but not of the gel domains. In the case of ld/lo all the bilayer thins simultaneously by a similar extent. In both cases, only the more disordered phase expanded significantly, indicating that ethanol increases the proportion of disordered domains. Water/bilayer interfacial tension variation and freezing point depression, inducing acyl chain disordering (including opening and looping), tilting, and interdigitation, are probably the main cause for the observed changes. The results presented herein demonstrate that ethanol influences the bilayer properties according to membrane lateral organization.

Physicochemical studies on the interaction of serum albumin with pulmonary surfactant extract in films and bulk bilayer phase

Functionality, structure and composition of the adsorbed films of bovine lipid extract surfactant (BLES), in the absence and presence of bovine serum albumin (BSA), at the air-buffer interface was characterized through surface tension, atomic force microscopy and time of flight secondary ion mass spectrometric methods. Gel and fluid domains of BLES films were found to be altered significantly in the presence of BSA. Differential scanning calorimetric studies on BLES dispersions in presence of BSA revealed that the perturbations of the lipid bilayer structures were significant only at higher amount of BSA. FTIR studies on the BLES dispersions in buffer solution revealed that BSA could affect the lipid head-group hydrations in bilayer as well as the methylene and methyl vibration modes of fatty acyl chains of the phospholipids present in BLES. Serum albumin could perturb the film structure at pathophysiological concentration while higher amount of BSA was required in perturbing the bilayer structures. The studies suggest a connected perturbed bilayer to monolayer transition model for surfactant inactivation at the alveolar-air interface in dysfunctional surfactants.

Determination of Phase Transition Temperatures for Atomistic Models of Lipids from Temperature-Dependent Stripe Domain Growth Kinetics

Many lipid bilayers undergo a reversible order-disorder transition between the gel and liquid crystalline (LC) phases at a main phase transition temperature T(m) that is an important characteristic property of the lipid. Although T(m) should serve as a useful standard for validation and calibration of simulation models of lipid bilayers, its evaluation within simulations is difficult due to the slow kinetics of the gel-LC transition, especially near T(m). A stripe growth strategy for calculating T(m), which aims to bypass the slowest steps in this transition, has been applied to dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine bilayers represented with a commonly used united-atom force field. The strategy consists of initial preparation of a bilayer containing gel and LC domains arranged as parallel stripes, observation of the direction and rate of domain growth over a range of temperatures, and fitting rates to an Arrhenius-like equation for their temperature dependence that crosses zero at T(m). Calculated T(m)'s for both lipids are 5-6 degrees lower than their experimental values, in much closer agreement with experiment than suggested by recent simulations that simulate heating and cooling of bilayer patches. The stripe growth method also yields rates of phase front propagation that are in order-of-magnitude agreement with experimental estimates, as well as insight into glycerol backbone disordering at the LC-gel interface.

Effect of sphingosine on domain morphology in giant vesicles

Sphingosine is a bioactive molecule which is known to participate in the regulation of a number of cellular processes such as apoptosis, cell differentiation, growth, etc. Sphingosine was observed to exhibit different domain morphology depending on the surrounding lipid matrix in biomimetic systems such as giant vesicles. Our current results showed that in a glycerophospholipid matrix sphingosine segregated in gel leaf-like domains whereas cholesterol presence increased its miscibility by melting gel domains in a concentration-dependent manner. Sphingosine and cholesterol did not form merging liquid domains on the micron scale as observed for sphingomyelin and cholesterol. However, we were able to visualize that sphingosine appears as a stabilizer and amplifier of domains in liquid-ordered phase by increasing the temperature of their formation and fraction. These results imply that sphingosine acts as a modulator of the lipid domain formation and thus it could exert its biological role, not only through direct binding to proteins, but also indirectly by influencing their sorting in membranes and modulating the processes of signal transduction.

Comparison of the liquid-ordered bilayer phases containing cholesterol or 7-dehydrocholesterol in modeling Smith-Lemli-Opitz syndrome

The phase behavior of egg sphingomyelin (ESM) mixtures with cholesterol or 7-dehydrocholesterol (7-DHC) has been investigated by independent methods: fluorescence microscopy, X-ray diffraction, and electron spin resonance spectroscopy. In giant vesicles, cholesterol-enriched domains appeared as large and clearly delineated domains assigned to a liquid-ordered (Lo) phase. The domains containing 7-DHC were smaller and had more diffuse boundaries. Separation of a gel phase assigned by X-ray examination to pure sphingomyelin domains coexisting with sterol-enriched domains was observed at temperatures less than 38 degrees C in binary mixtures containing 10-mol% sterol. At higher sterol concentrations, the coexistence of liquid-ordered and liquid-disordered phases was evidenced in the temperature range 20 degrees -50 degrees C. Calculated electron density profiles indicated the location of 7-DHC was more loosely defined than cholesterol, which is localized precisely at a particular depth along the bilayer normal. ESR spectra of spin-labeled fatty acid partitioned in the liquid-ordered component showed a similar, high degree of order for both sterols in the center of the bilayer, but it was higher in the coexisting disordered phase for 7-DHC. The differences detected in the models of the lipid membrane matrix are said to initiate the deleterious consequences of the Smith-Lemli-Opitz syndrome.

Dihydrosphingomyelin Impairs HIV-1 Infection by Rigidifying Liquid-Ordered Membrane Domains

The lateral organization of lipids in cell membranes is thought to regulate numerous cell processes. Most studies focus on the coexistence of two fluid phases, the liquid crystalline (l(d)) and the liquid-ordered (l(o)); the putative presence of gel domains (s(o)) is not usually taken into account. We show that in phospholipid:sphingolipid:cholesterol mixtures, in which sphingomyelin (SM) promoted fluid l(o) domains, dihydrosphingomyelin (DHSM) tended to form rigid domains. Genetic and pharmacological blockade of the dihydroceramide desaturase (Des1), which replaced SM with DHSM in cultured cells, inhibited cell infection by replication-competent and -deficient HIV-1. Increased DHSM levels gave rise to more rigid membranes, resistant to the insertion of the gp41 fusion peptide, thus inhibiting viral-cell membrane fusion. These results clarify the function of dihydrosphingolipids in biological membranes and identify Des1 as a potential target in HIV-1 infection.

Influence of Lipid Heterogeneity and Phase Behavior on Phospholipase A2 Action at the Single Molecule Level

We monitored the action of phospholipase A2 (PLA2) on L- and D-dipalmitoyl-phosphatidylcholine (DPPC) Langmuir monolayers by mounting a Langmuir-trough on a wide-field fluorescence microscope with single molecule sensitivity. This made it possible to directly visualize the activity and diffusion behavior of single PLA2 molecules in a heterogeneous lipid environment during active hydrolysis. The experiments showed that enzyme molecules adsorbed and interacted almost exclusively with the fluid region of the DPPC monolayers. Domains of gel state L-DPPC were degraded exclusively from the gel-fluid interface where the buildup of negatively charged hydrolysis products, fatty acid salts, led to changes in the mobility of PLA2. The mobility of individual enzymes on the monolayers was characterized by single particle tracking. Diffusion coefficients of enzymes adsorbed to the fluid interface were between 3.2 μm2/s on the L-DPPC and 4.9 μm2/s on the D-DPPC monolayers. In regions enriched with hydrolysis products, the diffusion dropped to ≈0.2 μm2/s. In addition, slower normal and anomalous diffusion modes were seen at the L-DPPC gel domain boundaries where hydrolysis took place. The average residence times of the enzyme in the fluid regions of the monolayer and on the product domain were between ≈30 and 220 ms. At the gel domains it was below the experimental time resolution, i.e., enzymes were simply reflected from the gel domains back into solution.

The Nonsteroidal Anti-Inflammatory Drug Indomethacin Induces Heterogeneity in Lipid Membranes: Potential Implication for Its Diverse Biological Action

BackgroundThe nonsteroidal anti-inflammatory drug (NSAID), indomethacin (Indo), has a large number of divergent biological effects, the molecular mechanism(s) for which have yet to be fully elucidated. Interestingly, Indo is highly amphiphilic and associates strongly with lipid membranes, which influence localization, structure and function of membrane-associating proteins and actively regulate cell signaling events. Thus, it is possible that Indo regulates diverse cell functions by altering micro-environments within the membrane. Here we explored the effect of Indo on the nature of the segregated domains in a mixed model membrane composed of dipalmitoyl phosphatidyl-choline (di16∶0 PC, or DPPC) and dioleoyl phosphatidyl-choline (di18∶1 PC or DOPC) and cholesterol that mimics biomembranes.Methodology/Principal FindingsUsing a series of fluorescent probes in a fluorescence resonance energy transfer (FRET) study, we found that Indo induced separation between gel domains and fluid domains in the mixed model membrane, possibly by enhancing the formation of gel-phase domains. This effect originated from the ability of Indo to specifically target the ordered domains in the mixed membrane. These findings were further confirmed by measuring the ability of Indo to affect the fluidity-dependent fluorescence quenching and the level of detergent resistance of membranes.Conclusion/SignificanceBecause the tested lipids are the main lipid constituents in cell membranes, the observed formation of gel phase domains induced by Indo potentially occurs in biomembranes. This marked Indo-induced change in phase behavior potentially alters membrane protein functions, which contribute to the wide variety of biological activities of Indo and other NSAIDs.

Texture of Membrane Gel Domains

In this work1 we investigate the texture of gel (g) domains in supported binary lipid membranes. Lateral organization of lipid bilayer membranes is a topic of fundamental and biological importance. Whereas questions related to the size and composition of fluid domains are well studied, the possibility of texture in condensed solid/gel domains has received limited attention. Gel domains are expected to be prominent in skin membranes and in ceramide domains during apoptosis. When using polarized light for two-photon excitation of the lipid probe Laurdan, the emission intensity is highly sensitive to the angle between the polarization and the tilt orientation of lipid acyl chains. By imaging the intensity variations as a function of the polarization angle, we map the lateral variations of the lipid tilt within domains. Results reveal that gel domains are composed of distinct subdomains with different lipid tilt directions. Vortex structures centered at the domain core can be observed. Texture patterns of the same type have historically been associated with the presence of hexatic order in monolayers. The hexatic phase is an intermediate phase between the crystal and fluid states, having short range positional order and long range orientation order. The present results provide some support for the notion that hexatic order may persist in bilayers. Using the generalized polarization (GP) function of Laurdan, we demonstrate that although gel domains have heterogeneous texture, the membrane phase state is uniform within domains.[1] U. Bernchou et. al. ‘Texture of Lipid Bilayer Domains’. J. Am. Chem. Soc., 2009. DOI: 10.1021/ja903375m

Cholesterol-Rich Fluid Membranes Solubilize Ceramide Gel Domains. Implications for the Organization of Mammalian Membranes

A uniquely sensitive method for ceramide-domain detection allowed us to study in detail cholesterol-ceramide interactions in lipid bilayers with low (physiological) ceramide (Cer) concentrations, and ranging from low or no cholesterol (Chol) (a situation similar to intracellular membranes, such as endoplasmic reticulum) to high Chol, (similar to mammalian plasma membrane). Fluorescence spectroscopy and microscopy experiments were conducted showing that for low Chol amounts Cer segregates into gel domains that disappear upon increasing Chol levels. This was observed in raft (sphingomyelin/Chol-containing) and non-raft (sphingomyelin-absent) membranes, i.e. mimicking different types of cell membranes. Chol-Cer interactions have been described mainly as raft sphingomyelin-dependent. In this work, sphingomyelin independence is demonstrated. Moreover, we show that Cer-rich domains re-appear when either Chol is converted by cholesterol oxidase to cholestenone, or temperature is decreased. The inability of cholestenone-rich membranes to dissolve Cer-gel domains shows that the cholesterol ordering and packing properties are fundamental to the mixing process. Cer solubility is dependent on the average gel-fluid transition temperature of the remaining membrane lipids, and is higher in Chol-rich fluid membranes than in Chol-poor ones. We also show that the solubility of Chol in Cer domains is low. The results are rationalized by a ternary phospholipid/ ceramide/ cholesterol phase diagram, providing the framework for a better understanding of biochemical phenomena modulated by Chol-Cer interactions such as cholesterol oxidase activity, lipoprotein metabolism and lipid targeting in cancer therapy. It also suggests that the lipid compositions of different organelles are such that ceramide gel domains are not formed, unless a stress or pathological situation occurs.Further details in Castro, BM, Silva, LC, Fedorov, A, de Almeida, RFM, and Prieto, M (2009). J. Biol. Chem.284 (5), 22978-22987.FCT (Portugal) is acknowledged for research grants and scholarships.

Ceramide Gel Domain Formation in a Phospholipid Bilayer: The Impact of Ceramide Acyl Chain

Nowadays, it is recognize the critical role of Ceramide (Cer) in signalling pathways that leads to cell proliferation, apoptosis, growth and inflammation. It is believed that the biological action of Cer depends largelly on its biophysical properties, e.g. the formation of Cer platforms in cell membranes was suggested to lead to the activation of apoptotic signaling pathways. Moreover, it has been proposed that Cer containing different fatty acids have distinct impact upon cell physiology. Regarding these observations, the biophysical behaviour of long and saturated (C16:0, C18:0) and very long and unsaturated (C24:1) acyl chain Cer in a fluid membrane (palmitoyloleoylphosphatidylcholine (POPC)) was studied and compared.The application of i) fluorescent spectroscopy and ii) confocal fluorescence microscopy showed that Cer acyl chain is the limiting factor on Cer/fluid phospholipd interaction. For instance, at room temperature (24oC) at least 10% of C24:1 Cer are needed to observe lateral segregation, in contrast only 4% of C16:0 and C18:0 Cer are required for microdomains formation. From confocal fluorescent microscopy complex tubular structure, known as cochleate, were only observed for bilayers composed of small amounts of C24:1 Cer (20-30%). Moreover the used of fluorescence correlation spectroscopy for studding N-rhodamine-dipalmitoylphosphatidylethanolamine diffusion in giant unilamellar vesicles (Cer/POPC), revealed a higher capacity of C16:0 and C18:0 for ordering fluid areas surrounding Cer platforms. Altogether, these observations support the suggestion that each Cer are involved in distinct cellular processes and membrane physical properties are a key mechanism for cell metabolism regulation.

Lipid Raft Composition Modulates Sphingomyelinase Activity and Ceramide-Induced Membrane Physical Alterations

Lipid rafts and ceramide (Cer)-platforms are membrane domains that play an important role in several biological processes. Cer-platforms are commonly formed in the plasma membrane by the action of sphingomyelinase (SMase) upon hydrolysis of sphingomyelin (SM) within lipid rafts. The interplay between SMase activity, initial membrane properties (i.e., phase behaviour and lipid lateral organization) and lipid composition, amount of product (Cer) generated, and how it modulates membrane properties was studied using fluorescence methodologies in model membranes. The activity of SMase was evaluated by following the hydrolysis of radioactive SM. It was observed that: i) enzyme activity and extent of hydrolysis is strongly dependent on membrane physical properties but not on substrate content, being higher in raft-like mixtures, i.e., those mixtures with ld-lo (liquid disordered-liquid ordered) phase separation, and ii) Cer-induced alterations are also dependent on membrane composition, namely on cholesterol (Chol) content. In the lowest-Chol range, Cer segregates together with SM into small, ∼8.5 nm, Cer/SM-gel domains. Upon increasing Chol, the ability of Cer to recruit SM and to form gel domains strongly decreases. In the high Chol range, a Chol-enriched/SM-depleted lo phase predominates. Together, these data suggest that in biological membranes Chol in particular, and raft domains in general, play an important role in the modulation of SMase activity and in the regulation of membrane physical properties by restraining Cer-induced alterations.

AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers

Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies.

Phase equilibria in DOPC/DPPC: Conversion from gel to subgel in two component mixtures

Biological membranes contain a mixture of phospholipids with varying degrees of hydrocarbon chain unsaturation. Mixtures of long chain saturated and unsaturated lipids with cholesterol have attracted a lot of attention because of the formation of two coexisting fluid bilayer phases in such systems over a broad range of temperature and composition. Interpretation of the phase behavior of such ternary mixtures must be based on a thorough understanding of the phase behavior of the binary mixtures formed with the same components. This article describes the phase behavior of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with 1,2-di-d(31)-palmitoyl-sn-glycero-3-phosphocholine (DPPC) between -20 and 50 degrees C. Particular attention has been paid to the phase coexistence below about 16 degrees C where the subgel phase appears. The changes in the shape of the spectrum (and its spectral moments) during the slow transformation process leads to the conclusion that below 16 degrees C the gel phase is metastable and the gel component of the two-phase mixture slowly transforms to the subgel phase with a slightly different composition. This results in a line of three-phase coexistence near 16 degrees C. Analysis of the transformation of the metastable gel domains into the subgel phase using the nucleation and growth model shows that the subgel domain growth is a two dimensional process.

Texture of Lipid Bilayer Domains

We investigate the texture of gel (g) domains in binary lipid membranes composed of the phospholipids DPPC and DOPC. Lateral organization of lipid bilayer membranes is a topic of fundamental and biological importance. Whereas questions related to size and composition of fluid membrane domain are well studied, the possibility of texture in gel domains has so far not been examined. When using polarized light for two-photon excitation of the fluorescent lipid probe Laurdan, the emission intensity is highly sensitive to the angle between the polarization and the tilt orientation of lipid acyl chains. By imaging the intensity variations as a function of the polarization angle, we map the lateral variations of the lipid tilt within domains. Results reveal that gel domains are composed of subdomains with different lipid tilt directions. We have applied a Fourier decomposition method as a convenient way to analyze the angular intensity variations. Texture patterns of the same type have been associated with the presence of hexatic order in monolayers. The present results provide some support for the notion that hexatic order may persist in bilayers. Laurdan exhibits an emission spectral shift which correlates with the phase state of the membrane. This is quantified by the generalized polarization (GP) function, and we demonstrate that a GP analysis can be performed on supported membranes. The results show that although the gel domains have heterogeneous texture, the membrane phase state does not show spatial variation within each domain.

Cytochrome c interaction with neutral lipid membranes: influence of lipid packing and protein charges

The interaction of cytochrome c (cyt c) with fluid/gel neutral supported lipid membranes was investigated by time-lapse atomic force microscopy (AFM). AFM revealed the random formation of depressed areas in fluid membranes promoted by cyt c. These depressions corresponded to the desorption of fluid bilayer patches induced by cyt c. By contrast, the gel domains were never desorbed but they were progressively thickened in the presence of the protein. These results suggest that cyt c molecules might intercalate between the mica and the lipid bilayer. Although the interaction of cyt c with the mica surface is likely to be an artifact, this work is the first direct observation of cyt c ability to cross membranes. Furthermore, our data show that the net positive charge of cyt c molecules plays a pivotal role but it is not the sole factor responsible for cyt c insertion in the membrane.