Lipid selectivity in detergent extraction from bilayers.

Despite numerous studies on detergent-induced solubilization of membranes and on the underlying mechanisms associated with this process, very little is known regarding the selectivity of detergents for lipids during their extraction from membranes. To get insights about this phenomenon, solubilization of model bilayers prepared from binary lipid mixtures by different detergents was examined. Three commonly used detergents were used: the non-ionic Triton X-100 (TX), the negatively-charged sodium dodecylsulfate (SDS), and the positively-charged n-dodecyltrimethylammonium chloride (DTAC). Two model membranes were used in order to identify if specific intermolecular interactions can lead to lipid selectivity: bilayers made of a binary mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and of a binary mixture of POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG). Therefore, it was possible to describe systems presenting a combination of detergents bearing different charges with bilayers with different polymorphic propensities and charge. In conditions for which partial solubilization was observed, the composition of the extracted lipid phase was quantified with Liquid Chromatography coupled to Mass Spectrometry to elucidate whether a lipid selectivity occurred in the solubilization process. On one hand, it is found that repulsive or attractive electrostatic interactions did not lead to any lipid selectivity. On the other hand, POPE was systematically less extracted than POPC, regardless of the detergent nature. We propose that this lipid selectivity is inherent to the molecular shape of POPE unsuited for micelles curvature properties.

Very Long Chain Lipids Favor the Formation of a Homogeneous Phase in Stratum Corneum Model Membranes.

The stratum corneum (SC), the outermost layer of mammal epidermis, acts as a barrier dictating the rate of absorption of exogenous molecules through the skin, as well as to prevent excessive water loss from the body. The SC consists of protein-rich corneocytes embedded into a complex lipid mixture. The lipid fraction is mainly constituted of an equimolar mixture of ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol), forming a solid phase in the intracellular space; this lipid phase is supposed to play a fundamental role in the SC barrier function. An unusual characteristic of this biological membrane is that its lipids generally bear very long acyl chains, with the 24-carbon long ones being the most abundant. In this work, we used Raman microspectroscopy and infrared spectroscopy to study the influence of the acyl chain length on the lipid mixing properties in SC model membranes. Our results revealed that the combination of ceramides and FFA bearing a very long chain is required for the formation of homogeneous lipid mixtures, while lipids with shorter chains (16-carbon and 20-carbon atom long) lead to domains with micrometer dimensions. It is proposed that the biological machinery necessary for acyl chain elongation occurring at the mammalian skin level is required to inhibit lipid phase separation, a critical feature in the proper barrier functioning.

Evidence of hydrocarbon nanodrops in highly ordered stratum corneum model membranes.

The stratum corneum (SC), the top layer of skin, dictates the rate of both water loss through the skin and absorption of exogenous molecules into the body. The crystalline organization of the lipids in the SC is believed to be a key feature associated with the very limited permeability of the skin. In this work, we characterized the organization of SC lipid models that include, as in native SC, cholesterol, a series of FFAs (saturated with C16-C24 chains), as well as a ceramide bearing an oleate chain-linked to a very long saturated acyl chain [N-melissoyl-oleoyloxy hexacosanoyl-D-erythro-sphingosine (Cer EOS)]. The latter is reported to be essential for the native SC lipid organization. Our 2H-NMR, infrared, and Raman spectroscopy data reveal that Cer EOS leads to the formation of highly disordered liquid domains in a solid/crystalline matrix. The lipid organization imposes steric constraint on Cer EOS oleate chains in such a way that these hydrocarbon nanodroplets remain in the liquid state down to -30°C. These findings modify the structural description of the SC substantially and propose a novel role of Cer EOS, as this lipid is a strong modulator of SC solid/liquid balance.

Ceramide-C16 Is a Versatile Modulator of Phosphatidylethanolamine Polymorphism.

Ceramide-C16 (CerC16) is a sphingolipid associated with several diseases like diabetes, obesity, Parkinson disease, and certain types of cancers. As a consequence, research efforts are devoted to identify the impact of CerC16 on the behavior of membranes, and to understand how it is involved in these diseases. In this work, we investigated the impacts of CerC16 (up to 20 mol %) on the lipid polymorphism of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), using differential scanning calorimetry, and sequential 2H and 31P solid-state nuclear magnetic resonance spectroscopy. A partial phase diagram is proposed. The results indicate that the presence of CerC16 leads to an upshift of the temperature of the gel-to-liquid crystalline (Lß - Lα) phase transition, leading to a large Lß/Lα phase coexistence region where gel-phase domains contain ∼35 mol % CerC16. It also leads to a downshift of the temperature of the lamellar-to-inverted hexagonal (L - HII) phase transition of POPE. The opposite influence on the two-phase transitions of POPE brings a three-phase coexistence line when the two transitions overlap. The resulting HII phase can be ceramide enriched, coexisting with a Lα phase, or ceramide depleted, coexisting with a Lß phase, depending on the CerC16 proportions. The uncommon capability of CerC16 to modulate the membrane fluidity, its curvature propensity, and the membrane interface properties highlights its potential as a versatile messenger in cell membrane events.

Melittin-Induced Lipid Extraction Modulated by the Methylation Level of Phosphatidylcholine Headgroups.

Protein- and peptide-induced lipid extraction from membranes is a critical process for many biological events, including reverse cholesterol transport and sperm capacitation. In this work, we examine whether such processes could display specificity for some lipid species. Melittin, the main component of dry bee venom, was used as a model amphipathic α-helical peptide. We specifically determined the modulation of melittin-induced lipid extraction from membranes by the change of the methylation level of phospholipid headgroups. Phosphatidylcholine (PC) bilayers were demethylated either by substitution with phosphatidylethanolamine (PE) or chemically by using mono- and dimethylated PE. It is shown that demethylation reduces the association of melittin with membranes, likely because of the resulting tighter chain packing of the phospholipids, which reduces the capacity of the membranes to accommodate inserted melittin. This reduced binding of the peptide is accompanied by an inhibition of the lipid extraction caused by melittin. We demonstrate that melittin selectively extracts PC from PC/PE membranes. This selectivity is proposed to be a consequence of a PE depletion in the surroundings of bound melittin to minimize disruption of the interphospholipid interactions. The resulting PC-enriched vicinity of melittin would be responsible for the observed formation of PC-enriched lipid/peptide particles resulting from the lipid efflux. These findings reveal that modulating the methylation level of phospholipid headgroups is a simple way to control the specificity of lipid extraction from membranes by peptides/proteins and thereby modulate the lipid composition of the membranes.

Evolution and function of mammalian binder of sperm proteins.

Binder of sperm (BSP) proteins are ubiquitous among mammals and have been extensively investigated over the last three decades. They were first characterized in bull seminal plasma and have now been identified in more than 15 different mammalian species where they represent a superfamily. In addition to sharing a common structure, BSP proteins share many characteristics. They are expressed by seminal vesicles and epididymides, interact with similar ligands and bind to the outer leaflet of sperm membranes via an interaction with choline phospholipids. In addition to playing a major role in sperm capacitation, they are implicated as molecular chaperones in sperm motility and viability, in the formation of the oviductal sperm reservoir, in the regulation of cell volume and possibly in the interaction between sperm and oocytes, making them crucial multifunctional proteins. Furthermore, BSP proteins can bind to egg yolk low-density lipoproteins and milk components, an interaction important for the protection of sperm during semen preservation in liquid or frozen state. Our current knowledge of BSP proteins strongly emphasizes their fundamental importance in male fertility and in the optimization of semen preservation techniques. Much work is still ahead in order to fully understand all the mysteries of BSP proteins.

Interaction of milk proteins and Binder of Sperm (BSP) proteins from boar, stallion and ram semen.

BACKGROUND: Mammalian semen contains a family of closely related proteins known as Binder of SPerm (BSP proteins) that are added to sperm at ejaculation. BSP proteins extract lipids from the sperm membrane thereby extensively modifying its composition. These changes can ultimately be detrimental to sperm storage. We have demonstrated that bovine BSP proteins interact with major milk proteins and proposed that this interaction could be the basis of sperm protection by milk extenders. In the present study, we investigated if homologous BSP proteins present in boar, stallion and ram seminal plasma display a similar affinity for the milk proteins in order to assess whether the mechanism of sperm protection by milk for these species could be general. METHODS: Skim milk was incubated with seminal plasma proteins (boar, stallion and ram), chromatographed on a Sepharose CL-4B column and protein fractions were analyzed by immunoblotting. RESULTS: Boar, stallion and ram BSP proteins displayed affinity for a milk protein fraction (F1) mainly composed of α-lactalbumin, ß-lactoglobulin, and κ-casein. They also had affinity for another milk protein fraction (F2) composed mostly of casein micelles. However, stallion BSP showed higher affinity for the fraction (F1). CONCLUSIONS: These results further extend our view that the association of BSP proteins with milk proteins could be a general feature of the mechanism of mammalian sperm protection by milk to prevent detrimental effect of prolonged exposure of sperm to seminal plasma.

Chain Length of Free Fatty Acids Influences the Phase Behavior of Stratum Corneum Model Membranes.

The skin, the largest organ of the human body, forms a flexible interface between our internal and external environment that protects our organism from exogenous compounds as well as excessive water loss. The stratum corneum (SC), the outermost layer of mammal epidermis, is mainly responsible for the skin impermeability. The SC is formed by corneocytes embedded in a lipid matrix, which is mostly constituted of ceramides (Cer), free fatty acids (FFA), and cholesterol (Chol), organized in two coexisting crystalline lamellar phases. This arrangement of lipids is crucial to skin barrier function. The aim of this paper is to determine the impact of FFA chain length on the phase behavior of SC model lipid membranes using solid-state deuterium NMR and IR spectroscopy. We studied ternary mixtures of N-lignoceroyl-d-erythro-sphingosine (Cer24), cholesterol, and palmitic (FFA16) or lignoceric (FFA24) acid in an equimolar ratio. This proportion replicates the lipid composition found in the SC lipid matrix. Our studies revealed that the phase behavior of Cer24/FFA/Chol ternary mixtures is strongly affected by the length of the FFA. We found the formation of phase-separated crystalline lipid domains when using palmitic acid whereas the use of lignoceric acid results in a more homogeneous mixture. In addition, it was observed that mixtures with lignoceric acid form a gel phase, a very unusual feature for SC model mixtures.

SN-38 active loading in poly(lactic-co-glycolic acid) nanoparticles and assessment of their anticancer properties on COLO-205 human colon adenocarcinoma cells.

SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(D,L-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310 nm, a zeta potential of -9.69 mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.

Chemical and physical requirements for lipid extraction by bovine binder of sperm BSP1.

The bovine seminal plasma contains phosphocholine-binding proteins, which associate to sperm membranes upon ejaculation. These binder-of-sperm (BSP) proteins then induce a phospholipid and cholesterol efflux from these membranes. In this work, we determined physical and chemical parameters controlling this efflux by characterizing the lipid extraction induced by BSP1, the most abundant of BSP protein in bull seminal plasma, from model membranes with different composition. The model membranes were formed from binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) or cholesterol. The modulation of BSP1-induced lipid extraction from membranes by their chemical composition and their physical properties brings us to propose a 3-step extraction mechanism. First, the protein associates with membranes via specific binding to phosphocholine groups. Second, BSP1 penetrates in the membrane, essentially in the external lipid leaflet. Third, BSP1 molecules solubilize a lipid patch coming essentially from the outer lipid leaflet, without any lipid specificity, to ultimately form small lipid/protein auto-assemblies. The stoichiometry of these complexes corresponds to 10-15 lipids per protein. It is also shown that fluid-phase membranes are more prone to BSP1-induced lipid extraction than gel-phase ones. The inhibition of the lipid extraction in this case appears to be related to the inhibition of the protein penetration in the membrane (step 2) and not to the protein association with PC head groups (step 1). These findings contribute to our understanding of the mechanism by which BSP1 modify the lipid composition of sperm membranes, a key event in sperm capacitation.

Nonphospholipid fluid liposomes with switchable photocontrolled release.

We created novel nonphospholipid photosensitive liposomes from a mixture of a monoacylated azobenzene amphiphile (AzoC10N(+)) and cholesterol sulfate (Schol). This system belongs to the family of sterol-enriched nonphospholipid liposomes that were shown to form stable large unilamellar vesicles (LUVs) with enhanced impermeability. Fluid bilayers were successfully prepared from AzoC10N(+)/Schol (25/75 molar ratio) mixtures, and LUVs could be derived at room temperature using standard extrusion methods. The isomerization process of the bilayer-inserted AzoC10N(+) was characterized. Leakage from these liposomes could be induced by the photoconversion of AzoC10N(+) from its trans form to its cis form. This photocontrolled release from fluid liposomes contrasts with the case of phospholipid-based azo-containing liposomes, which are generally required to be in the gel phase to be photosensitive. It is proposed that the very high degree of conformational order of the monoalkylated amphiphile and the tight packing of the hydrophobic core of the AzoC10N(+)/Schol liposomes make them responsive to the presence of the bulky cis azo isomer. Interestingly, the liposome impermeability could be fully restored by the photoisomerization of the cis form back to the trans form, providing a sharp on-and-off control of payload release. In addition, these nonphospholipid liposomes display a very limited passive release. Therefore, it is shown that AzoC10N(+)/Schol LUVs can be used as nanocontainers, whose content can be released by light in a controlled and switchable manner.

Formation, stability, and pH sensitivity of free-floating, giant unilamellar vesicles using palmitic acid-cholesterol mixtures.

Despite the fact that palmitic acid (PA) and cholesterol (Chol) do not form fluid bilayers once hydrated individually, giant unilamellar vesicles (GUVs) were formed from a mixture of palmitic acid and cholesterol, 30/70 mol/mol. These free-floating GUVs were stable over weeks, did not aggregate and were shown to be highly stable in alkaline pH compared to conventional phospholipid-based GUVs. Acidic pH-triggered payload release from the GUVs was associated with the protonation state of palmitic acid that dictated the mixing lipid properties, thus affecting the stability of the fluid lamellar phase. The successful formation of PA-Chol GUVs reveals the possibility to create monoalkylated amphiphile-based GUVs with distinct pH stability/sensitivity.

Ethanol perturbs lipid organization in models of stratum corneum membranes: An investigation combining differential scanning calorimetry, infrared and (2)H NMR spectroscopy.

Ethanol is used in a variety of topical products. It is known to enhance the permeability of the skin by altering the ability of the stratum corneum (SC) intercellular membranes to form an effective barrier. In addition, ethanol and other alcohols are key components of antiseptic gels currently used for hand wash. Using infrared and deuterium NMR spectroscopy as well as calorimetry, we have investigated the effect of ethanol on a model membrane composed of lipids representing the three classes of SC lipids, an equimolar mixture of N-palmitoylsphingosine (ceramide), palmitic acid and cholesterol. Ethanol is found to influence the membrane in a dose dependent manner, disrupting packing and increasing lipid motion at low concentrations and selectively extracting lipids at moderate concentrations.

Characterization of the structure of human skin substitutes by infrared microspectroscopy.

The skin acts mainly as a protective barrier from the external environment, thanks to the stratum corneum which is the outermost layer of the skin. As in vitro tests on skin are essential to elaborate new drugs, the development of skin models closer to reality becomes essential. It is now possible to produce in vitro human skin substitutes through tissue engineering by using the self-assembly method developed by the Laboratoire d'Organogénèse Expérimentale. In the present work, infrared microspectroscopy imaging analyses were performed to get in-depth morpho-spectral characterization of the three characteristic layers of human skin substitutes and normal human skin, namely the stratum corneum, living epidermis, and dermis. An infrared spectral analysis of the skin is a powerful tool to gain information on the order and conformation of the lipid chains and the secondary structure of proteins. On one hand, the symmetric stretching mode of the lipid methylene groups (2,850 cm(-1)) is sensitive to the acyl chain conformational order. The evolution profile of the frequency of this vibrational mode throughout the epidermis suggests that lipids in the stratum corneum are more ordered than those in the living epidermis. On the other hand, the frequencies of the infrared components underneath the envelop of the amide I band provide information about the overall protein conformation. The analysis of this mode establishes that the proteins essentially adopt an α-helix conformation in the epidermis, probably associated with the presence of keratin, while modifications of the protein content are observed in the dermis (extracellular matrix made of collagen). Finally, the lipid organization, as well as the protein composition in the different layers, is similar for human skin substitutes and normal human skin, confirming that the substitutes reproduce essential features of real skin and are appropriate biomimetics.

Interactions between non-phospholipid liposomes containing cetylpyridinium chloride and biofilms of Streptococcus mutans: modulation of the adhesion and of the biodistribution.

Cetylpyridinium chloride (CPC) is a surfactant that binds strongly to bacteria and bacterial biofilms. In this study, fluorescence-based techniques were used to determine the penetration and adhesion of CPC when it was introduced in liposomes. In spite of a reduced adhesion as compared to pure CPC micelles, CPC-containing liposomes adhered significantly to the biofilms of Streptococcus mutans. In contrast, no binding was observed for liposomes that were composed of phosphatidylcholine-cholesterol. The influence of the charge of the liposome on its adhesion to biofilms was studied using cholesterol (Chol) and cholesterol sulfate (Schol). In spite of similar binding to the biofilms, positively charged CPC/Chol liposomes were located mainly in the core of the biofilm microcolonies, whereas the negatively charged CPC/Schol liposomes were mainly concentrated at their periphery. This effect may be attributed to the different availability of the CPC head group. In summary, this work demonstrates the high potential for tailoring drug nanovectors by modulating sterol selection in order to selectively target and bind biofilms.

Formation of pH-sensitive cationic liposomes from a binary mixture of monoalkylated primary amine and cholesterol.

It has been shown that mixtures of monoalkylated amphiphiles and sterols can form liquid-ordered (lo) lamellar phases. These bilayers can be extruded using conventional methods to obtain large unilamellar vesicles (LUVs) that have very low permeability and a specific response to a given stimulus. For example, pH variations can trigger the release from LUVs formed with palmitic acid and sterols. In the present work, the possibility to form non phospholipid liposomes with mixtures of stearylamine (SA) and cholesterol (Chol) was investigated. The phase behavior of these mixtures was characterized by differential scanning calorimetry, infrared, and (2)H NMR spectroscopy. It is found that this particular mixture can form a lo lamellar phase that is pH-sensitive as the system undergoes a transition from a lo phase to a solid state when pH is increased from 5.5 to 12. LUVs have been successfully extruded from equimolar SA/Chol mixtures. Release experiments as a function of time revealed the relatively low permeability of these systems. The fact that the stability of these liposomes is pH dependent implies that these LUVs display an interesting potential as new cationic carriers for pH-triggered release. This is the first report of non phospholipid liposomes with high sterol content combining an overall positive charge and pH-sensitivity.

Influence of the nature of the sterol on the behavior of palmitic acid/sterol mixtures and their derived liposomes.

The phase behavior of mixtures formed with palmitic acid (PA) and one of the following sterols (dihydrocholesterol, ergosterol, 7-dehydrocholesterol, stigmasterol and stigmastanol), in a PA/sterol molar ratio of 3/7, has been characterized by IR and (2)H NMR spectroscopy at different pH. Our study shows that it is possible to form liquid-ordered (lo) lamellar phases with these binary non-phospholipid mixtures. The characterization of alkyl chain dynamics of PA in these systems revealed the large ordering effect of the sterols. It was possible to extrude these systems, using standard extrusion techniques, to form large unilamellar vesicles (LUVs), except in the case of ergosterol-containing mixture. The resulting LUVs displayed a very limited passive permeability consistent with the high sterol concentration. In addition, the stability of these PA/sterol self-assembled bilayers was also found to be pH-sensitive, therefore, potentially useful as nanovectors. By examining different sterols, we could establish some correlations between the structure of these bilayers and their permeability properties. The structure of the side chain at C17 of the sterol appears to play a prime role in the mixing properties with fatty acid.

The major proteins of bovine seminal plasma interact with caseins and whey proteins of milk extender.

Milk has been used routinely as an extender for sperm preservation. Caseins, the major proteins in milk, are proposed to be the protective constituents of milk during sperm preservation. It is unclear whether the whey proteins in milk are also implicated in the protection of sperm. Our previous studies have shown that the major proteins of bovine seminal plasma (recently named as binder of sperm or BSP, which comprises BSP1, BSP3, and BSP5 proteins) mediate a continuous phospholipid and cholesterol efflux from the sperm plasma membrane that is detrimental for sperm preservation. In this study, we investigated whether the protective effect of milk could be due to an interaction between BSP proteins and milk proteins. The binding of BSP proteins to milk proteins was demonstrated by gel filtration chromatography. Milk was fractionated into three fractions: the first containing whey protein aggregates and kappa-casein, the second containing all milk proteins, and the third containing small peptides, salts, and sugars. BSP1 has a higher affinity for the milk proteins in the milk fractions as compared to BSP3 and BSP5. The binding of BSP proteins to milk proteins was further characterized by isothermal titration calorimetry. We demonstrated that BSP1 binds to caseins and the titration could be simulated with a Scatchard approach, leading to an affinity constant (K(a)) of 350 mM(-1) and a stoichiometric parameter for the association (n) of 4.5 BSP1 per casein. The association between BSP1 and alpha-lactalbumin was characterized by a K(a) of 240 mM(-1) and an n value of 0.8. These results indicate the existence of an interaction between BSP proteins and milk proteins that could be the origin of the protection of sperm during preservation in milk.

Comparison between nuclear magnetic resonance profiling and the source/sink approach for characterizing drug diffusion in hydrogel matrices.

In controlled drug delivery, the drug diffusion coefficient in a given matrix is a key factor for predicting its release rate. In this work, we compare (31)P nuclear magnetic resonance (NMR) profiling for obtaining the mutual-diffusion coefficient (D(m)(gel)) of a drug in hydrogel with results obtained from conventional source/sink experiments. Despite the fact that NMR profiling is a powerful approach for measuring transport properties, it is rarely used for characterizing drug diffusion in gel matrices in pharmaceutical sciences. This work provides an illustration of the applicability of this technique and highlights its advantages for studying drug release systems. The comparison with results obtained from the source/sink experiment clearly establishes the validity of the NMR profiling approach. Alendronate was used as a model drug while curdlan, a gel-forming bacterial polysaccharide, served as a model biomaterial. The determined (D(m)(gel)) value (5.6 ± 0.3 × 10(-10) m(2)/s) agrees with the one obtained from a conventional source/sink experiment (5.4 ± 0.5 × 10(-10) m(2)/s). In addition, the alendronate self-diffusion coefficients in solution (D(s)(sln)) and in the hydrogel (D(s)(gel)) were measured on the same system using pulse-field gradient (PFG) (31)P NMR. These supplementary parameters provided a more detailed characterization of the drug transport properties in the gel matrix.

Interaction of Tau construct K18 with model lipid membranes.

One of the hallmarks of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, resulting from the aggregation of the tubulin associated unit protein (Tau), which holds a vital role in maintaining neuron integrity in a healthy brain. The development of such aggregates and their deposition in the brain seem to correlate with the onset of neurodegeneration processes. The misfolding and subsequent aggregation of the protein into paired helical filaments that further form the tangles, lead to dysfunction of the protein with neuronal loss and cognitive decline. The aggregation of the protein then seems to be a causative factor of the neurodegeneration associated with AD. The hypothesis of an involvement of the membrane in modulating the misfolding and assembly of Tau into paired helical filaments attracts increasing interests. To provide more insight about how lipids can modulate the interactions with Tau, we have conducted a comprehensive Atomic Force Microscopy (AFM) study involving supported lipid bilayers of controlled compositions with the Tau microtubule-binding construct K18. Particularly, the effects of zwitterionic and negatively charged phospholipids on the interaction have been investigated. Deleterious solubilization effects have been evidenced on fluid zwitterionic membranes as well as an inability of K18 to fragment gel phases. The role of negative lipids in the aggregation of the peptide and the particular ability of phosphatidylinositol-4,5-bisphosphate (PIP2) in inducing K18 fibrillization on membranes are also reported.