P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

P-glycoprotein inserted in planar lipid bilayers formed by liposomes opened on amorphous carbon and Langmuir-Blodgett monolayer

The insertion of proteins into planar lipid layers is of outstanding interest as the resulting films are suitable for the investigation of protein structure and aggregation in a lipid environment and/or the development of biotechnological applications as biosensors. In this study, purified P-glycoprotein (P-gp), a membrane drug pump, was incorporated in model membranes deposited on solid supports according to the method by Puu and Gustafson, Biochim. Biophys. Acta 1327 (1997) 149-161. The models were formed by a double lipid layer obtained by opening P-gp-containing liposomes onto two hydrophobic supports: amorphous carbon films and Langmuir-Blodgett (L-B) lipid monolayers, which were then observed by transmission electron microscopy and atomic force microscopy, respectively. Before the opening of liposomes, the P-gp structure and functionality were verified by circular dichroism spectroscopy and enzymatic assay. Our micrographs showed that liposomes containing P-gp fuse to the substrates more easily than plain liposomes, which keep their rounded shape. This suggests that the protein plays an essential role in the fusion of liposomes. To localize P-gp, the immunogold labeling of two externally exposed protein epitopes was carried out. Both imaging techniques confirmed that P-gp was successfully incorporated in the model membranes and that the two epitopes preserved the reactivity with specific mAbs, after sample preparation. Model membranes obtained on L-B monolayer incorporated few molecules with respect to those incorporated in the model membrane deposited onto amorphous carbon, probably because of the different mechanism of proteoliposome opening. Finally, all particles appeared as isolated units, suggesting that P-gp molecules were present as monomers.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

Defects in ordered aggregates of cardiolipin visualized by atomic force microscopy

The formation and the nature of defects in ordered aggregates of cardiolipin (tetra acyl diphosphatidylglycerol) supported on solid substrates have been investigated by atomic force microscopy (AFM). The experiments were performed on two model systems, i.e. three-dimensional liquid crystals dispersed in water and partially de-hydrated on a hydrophilic surface, and two-dimensional films of molecules self-assembled onto an isotropic hydrophobic surface. Defects were induced both by varying the preparation temperature and by treatment with specific chemicals known to modify the order parameters in natural and artificial membranes, specifically: 2,4-dinitro-phenol (DNP) and pentachloro-phenol (PCP). The effect of lipid oxidation on the nanocrystalline order was also investigated. The images obtained by AFM allow to characterize the type of defects and their local density at nanoscale level. They also provide additional information to differentiate the specific role of acyl chains and polar heads in the process of lipid self-organization.     

A complementary microscopy analysis of Sticholysin II crystals on lipid films: Atomic force and transmission electron characterizations

A new crystal form of the cytotoxin Sticholysin II (StnII) formed on lipid monolayers of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) has been characterized by transmission electron microscopy (TEM) and by tapping mode atomic force microscopy (AFM) under nearly physiological conditions. Both approaches show the existence of single- and double-layered 2D crystals possessing hexagonal symmetry and unit cell dimensions of a = b =10 nm and gamma = 120 degrees. However, single-layered StnII crystals could only be analysed by TEM and double-layered crystals by AFM. Considering the previously known atomic structure of native StnII and that of a tetrameric assembly, a model is proposed for this new crystal form in which StnII conserves its monomeric state upon interaction with the lipid monolayer. These results are in agreement with the existence of the so called M2 state of the actinoporins.     

α-Synuclein Senses Lipid Packing Defects and Induces Lateral Expansion of Lipids Leading to Membrane Remodeling

There is increasing evidence for the involvement of lipid membranes in both the functional and pathological properties of α-synuclein (α-Syn). Despite many investigations to characterize the binding of α-Syn to membranes, there is still a lack of understanding of the binding mode linking the properties of lipid membranes to α-Syn insertion into these dynamic structures. Using a combination of an optical biosensing technique and in situ atomic force microscopy, we show that the binding strength of α-Syn is related to the specificity of the lipid environment (the lipid chemistry and steric properties within a bilayer structure) and to the ability of the membranes to accommodate and remodel upon the interaction of α-Syn with lipid membranes. We show that this interaction results in the insertion of α-Syn into the region of the headgroups, inducing a lateral expansion of lipid molecules that can progress to further bilayer remodeling, such as membrane thinning and expansion of lipids out of the membrane plane. We provide new insights into the affinity of α-Syn for lipid packing defects found in vesicles of high curvature and in planar membranes with cone-shaped lipids and suggest a comprehensive model of the interaction between α-Syn and lipid bilayers. The ability of α-Syn to sense lipid packing defects and to remodel membrane structure supports its proposed role in vesicle trafficking.     

Characterization of cellulose whiskers and their nanocomposites by atomic force and electron microscopy

The aim of this work was to compare and explore electron microscopy and atomic force microscopy (AFM) for structure determination of cellulose whiskers and their nanocomposite with poly(lactic acid). From conventional bright-field transmission electron microscopy (TEM) it was possible to identify individual whiskers, which enabled determination of their sizes and shape. AFM overestimated the width of the whiskers due to the tip-broadening effect. Field emission scanning electron microscopy (FESEM) allowed for a quick examination giving an overview of the sample; however, the resolution was considered insufficient for detailed information. Ultramicrotomy of nanocomposite films at cryogenic temperatures enabled detailed inspection of the cellulose whiskers in the poly(lactic acid) matrix by AFM. FESEM applied on fractured surfaces allowed insight into the morphology of the nanocomposite, although rather restricted due to the metal coating and limited resolution. Detailed information was obtained from TEM; however, this technique required staining and suffered in general from limited contrast and beam sensitivity of the material.     

Atomic force microscopy of supported lipid bilayers

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h.     

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near‐native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4‐M23) was expressed in the X. laevis oocytes following their injection with AQP4‐M23 cRNA. AQP4‐M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4‐M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over‐expressed AQP4‐M23, the membranes from AQP4‐M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher‐order arrays of AQP4‐M23. In addition, but only infrequently, AQP4‐M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.     

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.     

Atomic force microscopy and electron microscopy analysis of retrovirus Gag proteins assembled in vitro on lipid bilayers

We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1, 2-di-O-hexadecyl-sn-glycero-3-(1'-2"-R-hydroxy-3'N-(5-amino-1-carboxy pentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-A distances and distances between dissimilar cage holes of 45.5 A. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-A distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.     

Visualization and characterization of prolamellar bodies with atomic force microscopy

Prolamellar bodies (PLBs) isolated from etiolated wheat seedlings were studied with the use of atomic force microscopy (AFM), transmission electron microscopy (TEM) and fluorescence spectroscopy. With AFM, PLBs were seen as spherical structures about 1–2 μm in diameter, more elastic than mica and poly-l-lysine substrate. TEM analyses confirmed that PLBs of wheat leaf etioplasts also had an average diameter of appr. 1 μm. Illumination induced the photoreduction of photoactive protochlorophyllide (Pchlide), i.e. Pchlide bound to protochlorophyllide oxidoreductase, which was shown in fluorescence spectra. The photoreduction was followed by the disruption of PLB structures, which started with the enlargement of PLB spheres and then their fragmentation into small balls as seen with AFM. Light-induced vesicle formation and the outgrowth of lamellar (pro)thylakoid membranes on the PLB surface were also confirmed by TEM analyses, and resulted in the apparent enlargement of the PLB diameter. The blue-shift of the fluorescence emission maximum of chlorophyllide observed for PLBs at room temperature after Pchlide photoreduction was completed within 25 min. However, structural changes in PLBs were still observed after the completion of the blue-shift. The incubation of PLBs in darkness with HgCl₂ also resulted in PLB enlargement and a loosening of their structure. AFM provides a unique opportunity to observe PLBs at a physiological temperature without the necessity of fixation.     

Atomic force microscopy for the study of membrane proteins

Fundamental biological processes such as cell-cell communication, signal transduction, molecular transport and energy conversion are performed by membrane proteins. These important proteins are studied best in their native environment, the lipid bilayer. The atomic force microscope (AFM) is the instrument of choice to determine the native surface structure, supramolecular organization, conformational changes and dynamics of membrane-embedded proteins under near-physiological conditions. In addition, membrane proteins are imaged at subnanometer resolution and at the single molecule level with the AFM. This review highlights the major advances and results achieved on reconstituted membrane proteins and native membranes as well as the recent developments of the AFM for imaging.     

Free-standing lipid films stabilized by Annexin-A5

Free-standing lipid bilayers in nano- and micro-pores are interesting membrane models and attractive for biotechnological applications. We describe here the controlled preparation of proteo-lipid mono- and bilayers using the Langmuir–Schaefer transfer or Langmuir–Blodgett technique, respectively on hydrophobic and hydrophilic surfaces. We demonstrate the formation of suspended proteo-lipid layers by Transmission Electron Microscopy (TEM) and in situ Atomic Force Microscopy (AFM) imaging. Using Annexin-A5 as a membrane-associated protein, continuous proteo-lipid mono- and bilayers were formed, which span pore arrays over areas of several square-micrometers. The 2D organization of proteins associated to lipid monolayer is well preserved during the transfer process and the protein association is Ca²⁺-dependent and therefore reversible. The simple formation and reliable transfer of stabilized free-standing lipid films is a first crucial step to create biomimetic membranes for biotechnological applications and membrane protein research.     

Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.     

Nanoscopic lipid domain dynamics revealed by atomic force microscopy

Intrinsic heterogeneities, represented as domain formations in biological membranes, are important to both the structure and function of the membranes. We observed domain formations in mixed lipid bilayers of dipalmitoylphosphatidylcholine (DPPC), dilauroylphosphatidylcholine (DLPC), and cholesterol (chol) in a fluid environment using an atomic force microscope (AFM). At room temperature, we demonstrated that both microscopic and nanoscopic domains coexist and the DPPC-rich domain is approximately 1.4 nm higher than the surrounding DLPC-rich membrane areas as a consequence of intrinsic phase differences. DPPC-rich microscopic domains became larger as DPPC concentration increased. In cholesterol-free mixtures, nanoscopic DPPC-rich domain sizes ranged from 26 to 46 nm depending on phospholipid concentration. Domain size varied between 33 and 48 nm in the presence of cholesterol (0 < or = [chol] < or = 40). The nanoscopic domains were markedly fragmented near [chol] = 0.135 and appeared to fuse more readily into microscopic domains at higher and lower [chol]. By phase balance analyses we demonstrated phase behavior differences between a free-vesicle GUV system studied by confocal light microscopy and a supported membrane system studied by AFM. We propose a new three-dimensional phase diagram elucidating the effects of a solid substrate support on lipid phase behavior relevant to complex membrane phase phenomena in biological systems.     

Two-chamber AFM: probing membrane proteins separating two aqueous compartments

Biological membranes compartmentalize and define physical borders of cells. They are crowded with membrane proteins that fulfill diverse crucial functions. About one-third of all genes in organisms code for, and the majority of drugs target, membrane proteins. To combine structure and function analysis of membrane proteins, we designed a two-chamber atomic force microscopy (AFM) setup that allows investigation of membranes spanned over nanowells, therefore separating two aqueous chambers. We imaged nonsupported surface layers (S layers) of Corynebacterium glutamicum at sufficient resolution to delineate a 15 A-wide protein pore. We probed the elastic and yield moduli of nonsupported membranes, giving access to the lateral interaction energy between proteins. We combined AFM and fluorescence microscopy to demonstrate the functionality of proteins in the setup by documenting proton pumping by Halobacterium salinarium purple membranes.     

Atomic force microscopy of pollen grains,cellulose microfibrils,and protoplasts

Summary Atomic force microscopy (AFM) holds unique prospects for biological microscopy, such as nanometer resolution and the possibility of measuring samples in (physiological) solutions. This article reports the results of an examination of various types of plant material with the AFM. AFM images of the surface of pollen grains ofKalanchoe blossfeldiana andZea mays were compared with field emission scanning electron microscope (FESEM) images. AFM reached the same resolutions as FESEM but did not provide an overall view of the pollen grains. Using AFM in torsion mode, however, it was possible to reveal differences in friction forces of the surface of the pollen grains. Cellulose microfibrils in the cell wall of root hairs ofRaphanus sativus andZ. mays were imaged using AFM and transmission electron microscopy (TEM). Imaging was performed on specimens from which the wall matrix had been extracted. The cell wall texture of the root hairs was depicted clearly with AFM and was similar to the texture known from TEM. It was not possible to resolve substructures in a single microfibril. Because the scanning tip damaged the fragile cells, it was not possible to obtain images of living protoplasts ofZ. mays, but images of fixed and dried protoplasts are shown. We demonstrate that AFM of plant cells reaches resolutions as obtained with FESEM and TEM, but obstacles still have to be overcome before imaging of living protoplasts in physiological conditions can be realized.Abbreviations AFM atomic force microscope - FESEM field emission scanning electron microscope - PyMS pyrolysis mass spectrometry - TEM transmission electron microscope     

Nanoscale analysis of supported lipid bilayers using atomic force microscopy

During the past 15 years, atomic force microscopy (AFM) has opened new opportunities for imaging supported lipid bilayers (SLBs) on the nanoscale. AFM offers a means to visualize the nanoscale structure of SLBs in physiological conditions. A unique feature of AFM is its ability to monitor dynamic events, like bilayer alteration, remodelling or digestion, upon incubation with various external agents such as drugs, detergents, proteins, peptides, nanoparticles, and solvents. Here, we survey recent progress made in the area.     

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.