Patterned domains of supported phospholipid bilayer using microcontact printing of Pll-g-PEG molecules.

In this work, we propose a reliable microcontact printing (µCP) process for generating Patterned Supported Phospholipids Bilayer (P-SPB) confined by Poly-L-(lysine)-grafted-polyethylene(glycol) (Pll-g-PEG) molecular barriers. The efficiency of Pll-g-PEG for inhibiting the fusion process of incubated liposome was first analyzed by Quartz Micro Balance (QCM) measurements. The quality and stability of Pll-g-PEG patterns were then both verified by fluorescence microscopy and Atomic Force Microscopy (AFM) in liquid media. The micro domains of P-SPB produced were stable in liquid environment during several weeks and also during AFM imaging. This exceptional stability is a clear improvement compared to previous studies involving proteins as confinement barriers.

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy.

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.

Deciphering the structure, growth and assembly of amyloid-like fibrils using high-speed atomic force microscopy.

Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to play a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins.

Surface topography of membrane domains.

Elucidating origin, composition, size, and lifetime of microdomains in biological membranes remains a major issue for the understanding of cell biology. For lipid domains, the lack of a direct access to the behaviour of samples at the mesoscopic scale has constituted for long a major obstacle to their characterization, even in simple model systems made of immiscible binary mixtures. By its capacity to image soft surfaces with a resolution that extends from the molecular to the microscopic level, in air as well as under liquid, atomic force microscopy (AFM) has filled this gap and has become an inescapable tool in the study of the surface topography of model membrane domains, the first essential step for the understanding of biomembranes organization. In this review we mainly focus on the type of information on lipid microdomains in model systems that only AFM can provide. We will also examine how AFM can contribute to understand data acquired by a variety of other techniques and present recent developments which might open new avenues in model and biomembrane AFM applications.

Organization of influenza A virus envelope at neutral and low pH.

Fusion of the influenza A H1N1 virus envelope with the endosomal membrane at low pH allows the intracellular delivery of the viral genome and plays an essential role in the infection process. Low pH induces an irreversible modification of the virus envelope, which has so far resisted 3D structural analysis, partly due to the virus pleiomorphy. This study showed that atomic force microscopy (AFM) in physiological buffer could be used to image the structural details of the virus envelope, both at neutral pH and after a low-pH treatment. At low and intermediate magnification, AFM of control virions confirmed both the pleiomorphy and the existence of zones devoid of glycoprotein spikes at the virus surface, as established by electron microscopy (EM). At higher magnification, the unique vertical resolution of the AFM in 3D topography demonstrated the lateral heterogeneity in spike distribution and strongly suggested that, at least locally, the spikes can be organized in an irregular honeycomb pattern. The surface honeycomb pattern was more easily detected due to an increase in spike height following low-pH treatment at low temperature, which probably prevented disruption of the organization. This enhanced contrast associated with low-pH treatment emphasized differences in the glycoprotein distribution between virions. It was concluded that, together with EM approaches, AFM may help to establish a correlation between surface structure and influenza virus infectivity/pathogenicity.

Contact-mode high-resolution high-speed atomic force microscopy movies of the purple membrane.

High-speed atomic force microscopy (HS-AFM) is becoming a reference tool for the study of dynamic biological processes. The spatial and time resolutions of HS-AFM are on the order of nanometers and milliseconds, respectively, and allow structural and functional characterization of biological processes at the single-molecule level. In this work we present contact-mode HS-AFM movies of purple membranes containing two-dimensional arrays of bacteriorhodopsin (bR). In high-resolution movies acquired at a 100 ms frame acquisition time, the substructure on individual bR trimers was visualized. In regions in between different bR arrays, dynamic topographies were observed and interpreted as motion of the bR trimers. Similarly, motion of bR monomers in the vicinity of lattice defects in the purple membrane was observed. Our findings indicate that the bR arrays are in a mobile association-dissociation equilibrium. HS-AFM on membranes provides novel perspectives for analyzing the membrane diffusion processes of nonlabeled molecules.

Contribution to the elucidation of the structure of the bacterial flagellum nano-motor through AFM imaging of the M-Ring.

The flagellar nano-motor of bacteria is one of the most interesting and amazing natural nano-machine. Despite its discovery 30 years ago, some details of its structure and mechanisms are not yet elucidated. Several studies have revealed some important aspects of its structure and numerous data are available today; however, the inner mechanisms of the nano-motor have not been yet resolved, partially due to the lack of information about the 3D assembly, shape and interactions of the different parts in experimental environment as close as possible as the native cellular conditions. We have developed an approach using atomic force microscopy imaging in liquid media, which allows us to study part of the motor in native liquid environment. In this work, we are interested in the FliG proteins, identified as the key functional proteins of this nano-machine. We report 3D images of their assembly on surfaces, which could be representative of the so-called M-ring part of the nano-motor. These images have been acquired on both mica surfaces and on supported bilayer membranes mimetics of E. coli native membrane. The systematic analysis of the shape and the size of different recorded assemblies made us believe that the FliG organization we observed could lead to a new model for the structure and mechanism of the flagellar nano-motor.

Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web.

Tetraspanins regulate cell migration, sperm-egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.

Temperature-dependent imaging of living cells by AFM.

Characterization of lateral organization of plasma membranes is a prerequisite to the understanding of membrane structure-function relationships in living cells. Lipid-lipid and lipid-protein interactions are responsible for the existence of various membrane microdomains involved in cell signalization and in numerous pathologies. Developing approaches for characterizing microdomains associate identification tools like recognition imaging with high-resolution topographical imaging. Membrane properties are markedly dependent on temperature. However, mesoscopic scale topographical information of cell surface in a temperature range covering most of cell biology experimentation is still lacking. In this work we have examined the possibility of imaging the temperature-dependent behavior of eukaryotic cells by atomic force microscopy (AFM). Our results establish that the surface of living CV1 kidney cells can be imaged by AFM, between 5 and 37 degrees C, both in contact and tapping modes. These first temperature-dependent data show that large cell structures appeared essentially stable at a microscopic scale. On the other hand, as shown by contact mode AFM, the surface was highly dynamic at a mesoscopic scale, with marked changes in apparent topography, friction, and deflection signals. When keeping the scanning conditions constant, a progressive loss in the image contrast was however observed, using tapping mode, on decreasing the temperature.

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM.

In plasma membranes, most glycosylphosphatidylinositol-anchored proteins (GPI proteins) would be associated with ordered microdomains enriched in sphingolipids and cholesterol. Debates on the composition and the nano- or mesoscales organization of these membrane domains are still opened. This complexity of biomembranes explains the use, in the recent years, of both model systems and atomic force microscopy (AFM) approaches to better characterize GPI proteins/membranes interactions. So far, the studies have mainly been focused on alkaline phosphatases of intestinal (BIAP) or placental (PLAP) origins reconstituted in model systems. The data show that GPI-anchored alkaline phosphatases (AP-GPI) molecules inserted in supported membranes can be easily imaged by AFM, in physiological buffer. They are generally observed in the most ordered domains of model membranes under phase separation, i.e. presenting both fluid and ordered domains. This direct access to the membrane structure at a mesoscopic scale allows establishing the GPI protein induced changes in microdomains size. It provides direct evidence for the temperature-dependent distribution of a GPI protein between fluid and ordered membrane domains. Origins of reported differences in the behavior of BIAP and PLAP are discussed. Finally, advantages and limits of AFM in the study of GPI proteins/membrane domains interactions are presented in this review.

Past, present and future of atomic force microscopy in life sciences and medicine.

To introduce this special issue of the Journal of Molecular Recognition dedicated to the applications of atomic force microscopy (AFM) in life sciences, this paper presents a short summary of the history of AFM in biology. Based on contributions from the first international conference of AFM in biological sciences and medicine (AFM BioMed Barcelona, 19-21 April 2007), we present and discuss recent progress made using AFM for studying cells and cellular interactions, probing single molecules, imaging biosurfaces at high resolution and investigating model membranes and their interactions. Future prospects in these different fields are also highlighted.

Temperature-dependent localization of GPI-anchored intestinal alkaline phosphatase in model rafts.

In plasma membranes, most of glycosylphosphatidylinositol (GPI)-anchored proteins would be associated with rafts, a category of ordered microdomains enriched in sphingolipids and cholesterol (Ch). They would be also concentrated in the detergent resistant membranes (DRMs), a plasma membrane fraction extracted at low temperature. Preferential localization of GPI-anchored proteins in these membrane domains is essentially governed by their high lipid order, as compared to their environment. Changes in the temperature are expected to modify the membrane lipid order, suggesting that they could affect the distribution of GPI-anchored proteins between membrane domains. Validity of this hypothesis was examined by investigating the temperature-dependent localization of the GPI-anchored bovine intestinal alkaline phophatase (BIAP) into model raft made of palmitoyloleoylphosphatidylcholine/sphingomyelin/cholesterol (POPC/SM/Chl) supported membranes. Atomic force microscopy (AFM) shows that the inserted BIAP is localized in the SM/Chl enriched ordered domains at low temperature. Above 30 degrees C, BIAP redistributes and is present in both the 'fluid' POPC enriched and the ordered SM/Chl domains. These data strongly suggest that in cells the composition of plasma membrane domains at low temperature differs from that at physiological temperature.

Remodeling of ordered membrane domains by GPI-anchored intestinal alkaline phosphatase.

Glycosylphosphatidyl-inositol (GPI)-anchored proteins preferentially localize in the most ordered regions of the cell plasma membrane. Acyl and alkyl chain composition of GPI anchors influence the association with the ordered domains. This suggests that, conversely, changes in the fluid and in the ordered domains lipid composition affect the interaction of GPI-anchored proteins with membrane microdomains. Validity of this hypothesis was examined by investigating the spontaneous insertion of the GPI-anchored intestinal alkaline phophatase (BIAP) into the solid (gel) phase domains of preformed supported membranes made of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC), DOPC/sphingomyelin (DOPC/SM), and palmitoyloleoylphosphatidylcholine/SM (POPC/SM). Atomic force microscopy (AFM) showed that BIAP inserted in the gel phases of the three mixtures. However, changes in the lipid composition of membranes had a marked effect on the protein containing bilayer topography. Moreover, BIAP insertion was associated with a net transfer of phospholipids from the fluid to the gel (DOPC/DPPC) or from the gel to the fluid (POPC/SM) phases. For DOPC/SM bilayers, transfer of lipids was dependent on the homogeneity of the gel SM phase. The data strongly suggest that BIAP interacts with the most ordered lipid species present in the gel phases of phase-separated membranes. They also suggest that GPI-anchored proteins might contribute to the selection of their own microdomain environment.

Continuous planar phospholipid bilayer supported on porous silicon thin film reflector.

Reconstituting artificial membranes for in vitro studies of cell barrier mechanisms and properties is of major interest in biology. Here, artificial membranes supported on porous silicon photonic crystal reflectors are prepared and investigated. The materials are of interest for label-free probing of supported membrane events such as protein binding, molecular recognition, and transport. The porous silicon substrates are prepared as multilayered films consisting of a periodically varying porosity, with pore dimensions of a few nanometers in size. Planar phospholipid bilayers are deposited on the topmost surface of the oxidized hydrophilic mesoporous silicon films. Atomic force microscopy provides evidence of continuous bilayer deposition at the surface, and optical measurements indicate that the lipids do not significantly infiltrate the porous region. The presence of the supported bilayer does not obstruct the optical spectrum from the porous silicon layer, suggesting that the composite structures can act as effective optical biosensors.

Influence of calcium on direct incorporation of membrane proteins into in-plane lipid bilayer.

Reconstitution of transmembrane proteins by direct incorporation into supported lipid bilayers (SLBs) is a new method to provide suitable samples for high-resolution atomic force microscopy (AFM) analysis of membrane proteins. First experiments have reported successful incorporation of proteins into detergent-destabilized SLBs. Here, we analyzed by AFM the incorporation of membrane proteins in the presence of calcium, a divalent cation functionally important for several membrane proteins. Using lipid-phase-separated membranes, we first show that calcium strongly stabilizes the SLBs decreasing the insertion of low cmc detergents, dodecyl-beta-maltoside, dodecyl-beta-thiomaltoside, and N-hexadecylphosphocholine (Fos-Choline-16) and further insertion of proteins. However, high yield of protein insertion is recovered in the presence of calcium by increasing the detergent concentration in the solution. These data revealed the importance of the calcium in the structure of SLBs and provided new insights into the mechanism of protein insertion into these model membranes.

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.

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.

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.

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.

[Atomic force microscopy: from cell imaging to molecular manipulation]. / La microscopie à force atomique, une technique de pointe appliquée à la biologie.

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.