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Christian Le Grimellec

Publications and source records attributed to Christian Le Grimellec.

17 recordsLinked to original sources
PubMed2006

High-resolution AFM of membrane proteins directly incorporated at high density in planar lipid bilayer.

The heterologous expression and purification of membrane proteins represent major limitations for their functional and structural analysis. Here we describe a new method of incorporation of transmembrane proteins in planar lipid bilayer starting from 1 pmol of solubilized proteins. The principle relies on the direct incorporation of solubilized proteins into a preformed planar lipid bilayer destabilized by dodecyl-beta-maltoside or dodecyl-beta-thiomaltoside, two detergents widely used in membrane biochemistry. Successful incorporations are reported at 20 degrees C and at 4 degrees C with three bacterial photosynthetic multi-subunit membrane proteins. Height measurements by atomic force microscopy (AFM) of the extramembraneous domains protruding from the bilayer demonstrate that proteins are unidirectionally incorporated within the lipid bilayer through their more hydrophobic domains. Proteins are incorporated at high density into the bilayer and on incubation diffuse and segregate into protein close-packing areas. The high protein density allows high-resolution AFM topographs to be recorded and protein subunits organization delineated. This approach provides an alternative experimental platform to the classical methods of two-dimensional crystallization of membrane proteins for the structural analysis by AFM. Furthermore, the versatility and simplicity of the method are important intrinsic properties for the conception of biosensors and nanobiomaterials involving membrane proteins.

Elasticity↗
PubMed2005

Coexistence of a two-states organization for a cell-penetrating peptide in lipid bilayer.

Primary amphipathic cell-penetrating peptides transport cargoes across cell membranes with high efficiency and low lytic activity. These primary amphipathic peptides were previously shown to form aggregates or supramolecular structures in mixed lipid-peptide monolayers, but their behavior in lipid bilayers remains to be characterized. Using atomic force microscopy, we have examined the interactions of P(alpha), a primary amphipathic cell-penetrating peptide which remains alpha-helical whatever the environment, with dipalmitoylphosphatidylcholine (DPPC) bilayers. Addition of P(alpha) at concentrations up to 5 mol % markedly modified the supported bilayers topography. Long and thin filaments lying flat at the membrane surface coexisted with deeply embedded peptides which induced a local thinning of the bilayer. On the other hand, addition of P(alpha) only exerted very limited effects on the corresponding liposome's bilayer physical state, as estimated from differential scanning calorimetry and diphenylhexatriene fluorescence anisotropy experiments. The use of a gel-fluid phase separated supported bilayers made of a dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine mixture confirmed both the existence of long filaments, which at low peptide concentration were preferentially localized in the fluid phase domains and the membrane disorganizing effects of 5 mol % P(alpha). The simultaneous two-states organization of P(alpha), at the membrane surface and deeply embedded in the bilayer, may be involved in the transmembrane carrier function of this primary amphipathic peptide.

Lipid Bilayers↗
PubMed2005

Fusion peptide-phospholipid noncovalent interactions as observed by nanoelectrospray FTICR-MS.

Viral infections are propagated by the fusing of the viral membrane with a host cell membrane. Initiation of the fusion process occurs upon perturbation of the membrane of the cell under attack by a subunit of the viral protein known as a fusion peptide. Fusion peptides must insert into the lipid-rich host cell membrane to initiate rupture and merging of the two entities, but much remains unknown about the details of the fusion process. We present detailed electrospray mass spectrometry studies of binding specificities of model fusion peptides P294 and P326 with cell membrane phospholipids, i.e., phosphatidylcholines (PCs, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) and phosphatidylglycerols (PGs, such as 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPG)). The fusion peptides clearly bind more strongly to negatively charged DMPG than to zwitterionic DMPC. Detected binding between P294/P326 and PC/PG in 100% aqueous solution was disrupted by addition of methanol, which is known to weaken hydrophobic interactions; a higher percentage of methanol was needed to destroy a stronger initial binding. Further increases in the methanol volume fraction generally resulted in a reappearance of peptide-lipid binding, with binding strength quotients of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-dilauroyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DLPG)-peptide complexes rising more steeply than those of DMPC/DMPG-peptide complexes. Compared to fusion peptides P294 and P326, a hydrophilic peptide, fibrinopeptide B, showed much weaker affinity for zwitterionic DMPC, but had moderate binding affinity to negatively charged DMPG in 100% aqueous solutions. However, upon progressive addition of methanol, this hydrophilic peptide showed only a minor initial decrease in binding to DMPG before the detected binding eventually increased. These results contrast with those obtained for the hydrophobic peptides, and offer corroborative evidence that hydrophobic interactions play a key role in the mass spectrometrically observed binding between fusion peptides and phospholipids. Because the rate of viral infection has been found to be pH-dependent, the effect of initial solution pH on peptide-lipid binding was also studied. As the pH was lowered, P326-DMPC binding had a steep and immediate weakening, whereas the P294-DMPC binding was slightly strengthened at pH 3.7 and then gradually weakened with a further decrease in pH. Both P326 and P294 exhibited affinities toward unsaturated lipids; (18:1)PC bound slightly more strongly to P294 than (18:3)PC. These experiments offer further evidence of the ability of electrospray mass spectrometry to provide binding information concerning noncovalent interactions that were established principally by the hydrophobic effect in solution.

Amino Acid Sequence↗
PubMed2004

Interaction of primary amphipathic cell-penetrating peptides with phospholipid-supported monolayers.

The mesoscopic organization adopted by two primary amphipathic peptides, P(beta) and P(alpha), in Langmuir-Blodgett (LB) films made of either the pure peptide or peptide-phospholipid mixtures was examined by atomic force microscopy. P(beta), a potent cell-penetrating peptide (CPP), and P(alpha) mainly differ by their conformational states, predominantly a beta-sheet for P(beta) and an alpha-helix for P(alpha), as determined by Fourier transform infrared spectroscopy. LB films of pure peptide, transferred significantly below their collapse pressure, were characterized by the presence of supramolecular structures, globular aggregates for P(beta) and filaments for P(alpha), inserted into the monomolecular film. In mixed peptide-phospholipid films, similar structures could be observed, as a function of the phospholipid headgroup and acyl chain saturation. They often coexisted with a liquid-expanded phase composed of miscible peptide-lipid. These data strongly suggest that primary amphipathic CPP and antimicrobial peptides may share, to some extent, common mechanisms of interaction with membranes.

Membranes, Artificial↗
PubMed2004

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide-cargo complex.

Bilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide-cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32).

Amino Acid Sequence↗
PubMed2004

Primary amphipathic cell-penetrating peptides: structural requirements and interactions with model membranes.

To identify rules for the design of efficient cell-penetrating peptides that deliver therapeutic agents into subcellular compartments, we compared the properties of two closely related primary amphipathic peptides that mainly differ by their conformational state. On the basis of a peptide Pbeta that is nonstructured in water and that promotes efficient cellular uptake of nucleic acids through noncovalent association, we have designed a peptide [Palpha] that is predicted to adopt a helical conformation. We show that [Pbeta] undergoes a lipid-induced conformational transition into a sheet structure, while [Palpha] remains helical. Penetration experiments show that both peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that both peptides interact with phospholipids in the liquid expanded and liquid condensed states. AFM observations reveal that the peptides strongly disrupt the lipid organization of the monolayers and that the conformational state can influence the uptake by model membranes.

Adsorption↗
PubMed2004

Use of cyclodextrin for AFM monitoring of model raft formation.

The lipid rafts membrane microdomains, enriched in sphingolipids and cholesterol, are implicated in numerous functions of biological membranes. Using atomic force microscopy, we have examined the effects of cholesterol-loaded methyl-beta-cyclodextrin (MbetaCD-Chl) addition to liquid disordered (l(d))-gel phase separated dioleoylphosphatidylcholine (DOPC)/sphingomyelin (SM) and 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SM supported bilayers. We observed that incubation with MbetaCD-Chl led to the disappearance of domains with the formation of a homogeneously flat bilayer, most likely in the liquid-ordered (l(o)) state. However, intermediate stages differed with the passage through the coexistence of l(o)-l(d) phases for DOPC/SM samples and of l(o)-gel phases for POPC/SM bilayers. Thus, gel phase SM domains surrounded by a l(o) matrix rich in cholesterol and POPC could be observed just before reaching the uniform l(o) state. This suggests that raft formation in biological membranes could occur not only via liquid-liquid but also via gel-liquid immiscibility. The data also demonstrate that MbetaCD-Chl as well as the unloaded cyclodextrin MbetaCD make holes and preferentially extract SM in supported bilayers. This strongly suggests that interpretation of MbetaCD and MbetaCD-Chl effects on cell membranes only in terms of cholesterol movements have to be treated with caution.

Cholesterol↗
PubMed2004

Temperature dependence of the surface topography in dimyristoylphosphatidylcholine/distearoylphosphatidylcholine multibilayers.

Simple lipid binary systems are intensively used to understand the formation of domains in biological membranes. The size of individual domains present in the gel/fluid coexistence region of single supported bilayers, determined by atomic force microscopy (AFM), generally exceeds by two to three orders of magnitude that estimated from multibilayers membranes by indirect spectroscopic methods. In this article, the topography of equimolar dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) multibilayers, made of two superimposed bilayers supported on mica surface, was studied by AFM in a temperature range from room temperature to 45 degrees C. In the gel/fluid phase coexistence region the size of domains, between approximately 100 nm and a few micrometers, was of the same order for the first bilayer facing the mica and the top bilayer facing the buffer. The gel to fluid phase separation temperature of the first bilayer, however, could be increased by up to 8 degrees C, most likely as a function of the buffer layer thickness that separated it from the mica. Topography of the top bilayer revealed the presence of lipids in ripple phase up to 38-40 degrees C. Above this temperature, a pattern characteristic of the coexistence of fluid and gel domains was observed. These data show that difference in the size of lipid domains given by AFM and spectroscopy can hardly be attributed to the use of multibilayers models in spectroscopy experiments. They also provide a direct evidence for metastable ripple phase transformation into a gel/fluid phase separated structure upon heating.

Dimyristoylphosphatidylcholine↗
PubMed2004

Interactions of the human calcitonin fragment 9-32 with phospholipids: a monolayer study.

Human calcitonin and its C-terminal fragment 9-32 (hCT(9-32)) administered in a spray translocate into respiratory nasal epithelium with an effect similar to intravenous injection. hCT(9-32) is an efficient carrier to transfer the green fluorescent protein into excised bovine nasal mucosa. To understand the translocation of hCT(9-32) across plasma membranes, we investigated its interactions with phospholipids and its interfacial structure using model lipid monolayers. A combination of physicochemical methods was applied including surface tension measurements on adsorbed and spread monolayers at the air-water interface, Fourier transform infrared, circular dichroism, and atomic force microscopy on Langmuir-Blodgett monolayers. The results disclose that hCT(9-32) preferentially interacts with negatively charged phospholipids and does not insert spontaneously into lipid monolayers. This supports a nonreceptor-mediated endocytic internalization pathway as previously suggested. Structural studies revealed a random coil conformation of hCT(9-32) in solution, transforming to alpha-helices when the peptide is localized at lipid-free or lipid-containing air-water interfaces. Atomic force microscopy studies of monolayers of the peptide alone or mixed with dioleoylphosphatidylcholine revealed that hCT(9-32) forms filaments rolled into spirals. In contrast, when interacting with dioleoylphosphatidylglycerol, hCT(9-32) does not adopt filamentous structures. A molecular model and packing is proposed for the spiral-forming hCT(9-32).

Calcitonin↗
PubMed2004

[Atomic force microscopy: from cell imaging to molecular manipulation].

The atomic force microscope (AFM) allows to explore the surface of biological samples bathed in physiological solutions, with vertical and horizontal resolutions ranging from nanometers to angströms. Complex biological structures as well as single molecules can be observed and recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra and intermolecular forces from single molecules are also presented.

Animals↗
PubMed2003

AFM imaging of lipid domains in model membranes.

Characterization of the two-dimensional organization of biological membranes is one of the most important issues that remains to be achieved in order to understand their structure-function relationships. According to the current view, biological membranes would be organized in in-plane functional microdomains. At least for one category of them, called rafts, the lateral segregation would be driven by lipid-lipid interactions. Basic questions like the size, the kinetics of formation, or the transbilayer organization of lipid microdomains are still a matter of debate, even in model membranes. Because of its capacity to image structures with a resolution that extends from the molecular to the microscopic level, atomic force microscopy (AFM) is a useful tool for probing the mesoscopic lateral organization of lipid mixtures. This paper reviews AFM studies on lateral lipid domains induced by lipid-lipid interactions in model membranes.

Animals↗
PubMed2003

[Atomic force microscopy: from cellular imaging to molecular manipulation].

Using a sharp tip attached at the end of a soft cantilever as a probe, the atomic force microscope (AFM) explores the surface topography of biological samples bathed in physiological solutions. In the last few years, the AFM has gained popularity among biologists. This has been obtained through the improvement of the equipment and imaging techniques as well as through the development of new non-imaging applications. Biological imaging has to face a main difficulty that is the softness and the dynamics of most biological materials. Progress in understanding the AFM tip-biological samples interactions provided spectacular results in different biological fields. Recent examples of the possibilities offered by the AFM in the imaging of intact cells, isolated membranes, membrane model systems and single molecules at work are discussed in this review. Applications where the AFM tip is used as a nanotool to manipulate biomolecules and to determine intra- and intermolecular forces from single molecules are also presented.

Animals↗
PubMed2002

High-resolution three-dimensional imaging of the lateral plasma membrane of cochlear outer hair cells by atomic force microscopy.

The outer hair cells (OHCs) from the mammalian organ of Corti are assumed to enhance the sensitivity and the selectivity of the cochlea via an electromotile response to sound stimulation. These OHC mechanical changes feed energy back into the cochlea before completion of the transduction process by inner hair cells. OHC electromotility is thought to depend on specific transmembrane motor proteins. Electron microscopy has been used previously to image the OHC lateral plasma membrane, where voltage sensors and motors are located. A very specific and regular organization of membrane particles has been described, together with an equally specific submembraneous meshwork of cytoskeleton anchored to the plasma membrane. To confirm and extend these observations, we have used, for the first time on the OHC lateral wall, atomic force microscopy (AFM). As a result of an improved tapping mode technique as well as the unique ultrastructural organization of the OHC plasma membrane, we have obtained high-resolution three-dimensional (3D) images of a markedly enhanced quality, allowing high-resolution 3D imaging. Tapping-mode AFM confirmed the presence of regularly aligned particles (presumably transmembrane proteins) on both faces of the OHC plasma membrane. It also revealed the presence of markedly different membrane domains, smooth and undulating. The differences between these zones probably are due to local differences in cytoskeleton-membrane interactions. Moreover, 3D reconstructions allowed us to distinguish between globular and pore-like particles, a distinction that may be of great functional significance.

Animals↗
PubMed2002

Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin-dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl-) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl- lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.

Alkaline Phosphatase↗
PubMed2001

Cholesterol is not crucial for the existence of microdomains in kidney brush-border membrane models.

The external membrane leaflet plays a key role in the organization of the cell plasma membrane as a mosaic of ordered microdomains enriched in sphingolipids and cholesterol and of fluid domains. In this study, the thermotropic behavior and the topology of bilayers made of a phosphatidylcholine/sphingomyelin mixture, which mimicks the lipid composition of the external leaflet of renal brush-border membranes, were examined by differential scanning calorimetry and atomic force microscopy. In the absence of cholesterol, a broad phase separation process occurred where ordered gel phase domains of size varying from the mesoscopic to the microscopic scale, enriched in sphingomyelin, occupied half of the bilayer surface at room temperature. Increasing amounts of cholesterol progressively decreased the enthalpy of the transition and modified the topology of membranes domains up to a concentration of 33 mol % for which no membrane domains were detected. These results strongly suggest that, in membranes highly enriched in sphingolipids like renal and intestinal brush borders, there is a threshold close to the physiological concentration above which cholesterol acts as a suppressor rather than as a promoter of membrane domains. They also suggest that cholesterol depletion does not abolish the lateral heterogenity in brush-border membranes.

Animals↗