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.     

Direct AFM observation of saposin C-induced membrane domains in lipid bilayers: from simple to complex lipid mixtures

Saposin C (Sap C) is a small glycoprotein required by glucosylceramidase (GCase) for hydrolysis of glucosylceramide to ceramide and glucose in lysosomes. The molecular mechanism underlying Sap C stimulation of the enzyme activation is not fully understood. Here, atomic force microscopy (AFM) has been used to study Sap C-membrane interactions under physiological conditions. First, to establish how Sap C-membrane interactions affect membrane structure, lipid bilayers containing zwitterionic and anionic phospholipids were used. It was observed that Sap C induced two types of membrane restructuring effects, i.e., the formation of patch-like domains and membrane destabilization. Bilayers underwent extensive structural reorganization. To validate the biological importance of the membrane restructuring effects, interaction of Sap C with lipid bilayers composed of cholesterol, sphingomyelin, and zwitterionic and anionic phospholipids were studied. Although similar membrane restructuring effects were observed, Sap C-membrane interactions, in this case, were remarkably modulated and their effects were restricted to a limited area. As a result, nanometer-sized domains were formed. The establishment of a model membrane system will allow us to further study the dynamics, structure and mechanism of the Sap C-associated membrane domains and to examine the important role that these domains may play in enzyme activation.     

Blistering of supported lipid membranes induced by Phospholipase D, as observed by real-time atomic force microscopy

Phospholipase D from Streptomyces chromofuscus (PLDSc) is a soluble enzyme known to be activated by the phosphatidic acid (PA)-calcium complexes. Despite the vast body of literature that has accumulated on this enzyme, the exact mechanism of activation remains poorly understood. In this work, we report the first observation of PLDSc activity in real time and at nanometer resolution using atomic force microscopy (AFM). AFM images of continuous and patchy dipalmitoylphosphatidylcholine (DPPC) bilayers were recorded, prior and after incubation with PLDSc. For continuous bilayers, the enzyme induced important morphological alterations; holes corresponding to the bilayer thickness were created, while an additional elevated phase, about 2.5 nm high, was observed. This bilayer blistering is believed to be due to the production of the negatively charged lipid PA that would cause localized repulsions between the bilayer and the underlying mica surface. By contrast, these elevated domains were not seen on patchy bilayers incubated with the enzyme. Instead, the shapes of DPPC patches were strongly deformed by enzyme activity and evolved into melted morphologies. These results point to the importance of lipid packing on PLD activity and illustrate the potential of AFM for visualizing remodeling enzymatic activities.     

PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP^C to scrapie isoform PrP^Sc). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126_CONH2 and PrP106-126_COOH) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126_COOH displayed a higher peptide-lipid binding affinity than PrP106-126_CONH2 (∼2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

Morphological changes induced in erythrocyte by amyloid beta peptide and glucose depletion: A combined atomic force microscopy and biochemical study

Circulating red blood cells (RBCs) undergo aging, a fundamental physiological phenomenon that regulates their turnover. We show that treatment with beta amyloid peptide 1–42 (Aβ) accelerates the occurrence of morphological and biochemical aging markers in human RBCs and influences the cell metabolism leading to intracellular ATP depletion. The morphological pattern has been monitored using Atomic Force Microscopy (AFM) imaging and measuring the RBCs' plasma membrane roughness employed as a morphological parameter capable to provide information on the structure and integrity of the membrane-skeleton. Results evidence that Aβ boosts the development of crenatures and proto-spicules simultaneously to acceleration in the weakening of the cell-cytoskeleton contacts and to the induction of peculiar nanoscale features on the cell membrane. Incubation in the presence of glucose can remove all but the latter Aβ-induced effects.Biochemical data demonstrate that contemporaneously to morphological and structural alterations, Aβ and glucose depletion trigger a complex signaling pathway involving caspase 3, protein kinase C (PKC) and nitric oxide derived metabolites.As a whole, the collected data revealed that, the damaging path induced by Aβ in RBC provide a sequence of morphological and functional intermediates following one another along RBC life span, including: (i) an acceleration in the development of shape alteration typically observed along the RBC's aging; (ii) the development of characteristic membrane features on the plasma membrane and (iii) triggering a complex signaling pathway involving caspase 3, PKC and nitric oxide derived metabolites.     

A novel in situ atomic force microscopy imaging technique to probe surface morphological features of starch granules

The application of atomic force microscopy (AFM) for observing iodine complexes in starch has been limited due to limitations including granular sample fixation techniques and possible unintended reactions with embedding materials such as epoxy resins or adhesives. In this paper, a new method is described that employs an optical microscopic technique to ensure that the tip of the AFM is scanning a specified granule without any probe-induced particle movement by the AFM probe motion. The direct sprinkling of samples on a two-sided adhesive tape allows investigations in an in situ environment of the un-embedded starch granule surface and thus provides high-resolution images of granule morphology and phase changes of starches in the presence of humidity and with iodine vapor. These observations demonstrate that this novel in situ AFM imaging technique allows us to visualize the hair-like structures on the surface of granular starches when starches are exposed to iodine vapor under humid environments. This study reveals that the hair-like extensions on the starch granule surfaces are strongly dependent on the organization of the glucan polymers within corn or potato starch.     

Single molecular dynamic interactions between glycophorin A and lectin as probed by atomic force microscopy

Glycophorin A (GpA) is one of the most abundant transmembrane proteins in human erythrocytes and its interaction with lectins has been studied as model systems for erythrocyte related biological processes. We performed a force measurement study using the force mode of atomic force microscopy (AFM) to investigate the single molecular level biophysical mechanisms involved in GpA-lectin interactions. GpA was mounted on a mica surface or natively presented on the erythrocyte membrane and probed with an AFM tip coated with the monomeric but multivalent Psathyrella velutina lectin (PVL) through covalent crosslinkers. A dynamic force spectroscopy study revealed similar interaction properties in both cases, with the unbinding force centering around 60 pN with a weak loading rate dependence. Hence we identified the presence of one energy barrier in the unbinding process. Force profile analysis showed that more than 70% of GpAs are free of cytoskeletal associations in agreement with previous reports.     

Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N-(5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device.     

Atomic force microscopy of cotton fiber cell wall surfaces in air and water: quantitative and qualitative aspects

Cotton (Gossypium hirsutum cv. MD51) fiber cell walls were analyzed with an atomic force microscope to determine the effect of chemical treatments on cell wall organization and topography. Analysis of fibers in either air or water and without any staining or coating produced high-resolution images of cell wall microstructure which could be used for detailed quantitative analysis. Treatment of fibers with 1% H₂O₂ had little effect on surface morphology. Alkali removed much of the cuticle, some primary wall components, and revealed mostly thin-diameter microfibrils. Acidic Updegraff reagent fragmented the fibers, removed much of the cuticle, and revealed mostly thick microfibrils. The surface roughness of fibers treated sequentially with alkali and acid was quantitatively distinguishable from all other fiber types based on the standard deviation of the height data, amplification of surface area, and integration of the scan line data. Analysis of the fractal dimension enabled untreated and peroxide-treated fibers to be clearly distinguished from the other fiber types. Segmentation of the fractal data revealed specific portions of the fractal dimension which were especially useful for defining the size of structures that differentiated fiber types. Areas containing microfibrils could be quantitatively differentiated from non-microfibrillar areas. In water, some alkali-treated fibers had microfibrils that were relatively small in diameter while others appeared to consist of crystalline arrays of smaller fibrils. Received: 10 December 1996 / Accepted: 29 January 1997     

Polymyxin B-lipid interactions in Langmuir-Blodgett monolayers of Escherichia coli lipids: a thermodynamic and atomic force microscopy study

The dramatically increased frequency of antibiotic resistance has led to intensive efforts towards developing new families of antibiotics. Membrane-active antibiotic peptides such as polymyxin B (PxB) hold promise as the next generation of antibiotics, since they rarely spur the evolution of resistance. At low concentrations in the membrane, PxB forms vesicle-vesicle contacts and induces lipid exchange without leakage or fusion, a phenomenon that can explain its specificity towards gram-negative bacteria by contact formation between the two phospholipids interfaces in the periplasmatic space. In this work, the interaction of PxB and the nonantibiotic derivative polymyxin B nonapeptide (PxB-NP) with monolayers of Escherichia coli membrane lipids (ECL) has been studied by thermodynamic and structural methods. PxB inserts itself into ECL monolayers as a conformation that forms intermembrane contacts with vesicles injected underneath, and induces lipid exchange when the monolayer surface pressure is set at 32 mN/m (membrane equivalence pressure) or net transfer vesicle-to-monolayer at lower surface pressures. Thermodynamic analysis of the compression isotherms of mixed monolayers indicates that PxB inserts into the monolayer with an expansion of the mean molecular area, implying that peptide and lipids form nonideal mixtures. At low concentrations, corresponding to the membrane-membrane contact form of PxB, the mixed monolayers present positive excess energy values (deltaGm(Ex)), and atomic force microscopy (AFM) imaging reveals structures of approximately 120-nm diameter that protrude from the lipid surface approximately 0.7 nm. At concentrations of PxB above 4 mol %, thermodynamic analysis gives a very high deltaGm(Ex), corresponding to nonfavorable interactions, and AFM images show round structures of 20-30 nm diameter. PxB-NP behaves in a totally different way, in agreement with its inability to form vesicle-vesicle contacts and its lack of antibiotic effect. These results are discussed in the light of the mechanism of action of PxB on the membrane of gram-negative bacteria.     

How to lose a kink and gain a helix: pH independent conformational changes of the fusion domains from influenza hemagglutinin in heterogeneous lipid bilayers

We have simulated two conformations of the fusion domain of influenza hemagglutinin (HA) within explicit water, salt, and heterogeneous lipid bilayers composed of POPC:POPG (4:1). Each conformation has seven different starting points in which the initial peptide structure is the same for each conformation, but the location across the membrane normal and lipid arrangement around the peptide are varied, giving a combined total simulation time of 140 ns. For the HA5 conformation (primary structure from recent NMR spectroscopy at pH = 5), the peptide exhibits a stable and less kinked structure in the lipid bilayer compared to that from the NMR studies. The relative fusogenic behavior of the different conformations has been investigated by calculation of the relative free energy of insertion into the hydrophobic region of lipid bilayer as a function of the depth of immersion. For the HA7 conformations (primary structure from recent NMR spectroscopy at pH = 7.4), while the N-terminal helix preserves its initial structure, the flexible C-terminal chain produces a transient helical motif inside the lipid bilayer. This conformational change is pH-independent, and is closely related to the peptide insertion into the lipid bilayer.     

Free energy of helical transmembrane peptide dimerization in OPLS-AA/Berger force field simulations: inaccuracy and implications for partner-specific Lennard-Jones parameters between peptides and lipids

Interactions between transmembrane (TM) peptides are important in biophysical chemistry, but there are few studies assessing atomistic simulation parameters concerning the energetics of interactions of TM helical peptides. Our potential of mean force analysis using OPLS-AA protein/Berger lipid force fields (FFs) shows that the dimerisation energy of (AALALAA)₃ helical peptides in the dioleoylphosphatidylcholine bilayer is ?4.4 kJ/mol, which was much smaller than the reported experimental value (?12.7 kJ/mol), thus calling for improvement of parameters of the combined FFs. As each of the FFs has been independently developed, we then tested the effects of downscaling the Lennard-Jones (LJ) energy terms between the OPLS-AA atoms and Berger lipid atoms, preserving the parameters within each FF. A 0.9-fold rescaling of the LJ energies was found to render the dimerisation energy close to the experimental value. Solvation of backbone atoms as well as side chain atoms in lipids is crucial for the TM helix interaction. In similar analyses, GROMOS 53A6 FF exhibited as weak dimerisation propensity (~?5.2 kJ/mol) as OPLS-AA/Berger, but CHARMM36 showed relatively accurate propensity (~?9.9 kJ/mol). Challenges and strategies in rendering the TM interaction energy realistic within the framework of current FFs are discussed.     

Structural evolution and membrane interactions of Alzheimer's amyloid‐beta peptide oligomers: New knowledge from single‐molecule fluorescence studies

Amyloid‐β peptide (Aβ) oligomers may represent the proximal neurotoxin in Alzheimer's disease. Single‐molecule microscopy (SMM) techniques have recently emerged as a method for overcoming the innate difficulties of working with amyloid‐β, including the peptide's low endogenous concentrations, the dynamic nature of its oligomeric states, and its heterogeneous and complex membrane interactions. SMM techniques have revealed that small oligomers of the peptide bind to model membranes and cells at low nanomolar‐to‐picomolar concentrations and diffuse at rates dependent on the membrane characteristics. These methods have also shown that oligomers grow or dissociate based on the presence of specific inhibitors or promoters and on the ratio of Aβ40 to Aβ42. Here, we discuss several types of single‐molecule imaging that have been applied to the study of Aβ oligomers and their membrane interactions. We also summarize some of the recent insights SMM has provided into oligomer behavior in solution, on planar lipid membranes, and on living cell membranes. A brief overview of the current limitations of the technique, including the lack of sensitive assays for Aβ‐induced toxicity, is included in hopes of inspiring future development in this area of research.     

Macromolecular interactions of the bacterial division FtsZ protein: from quantitative biochemistry and crowding to reconstructing minimal divisomes in the test tube

The division of Escherichia coli is an essential process strictly regulated in time and space. It requires the association of FtsZ with other proteins to assemble a dynamic ring during septation, forming part of the functionally active division machinery, the divisome. FtsZ reversibly interacts with FtsA and ZipA at the cytoplasmic membrane to form a proto-ring, the first molecular assembly of the divisome, which is ultimately joined by the rest of the division-specific proteins. In this review we summarize the quantitative approaches used to study the activity, interactions, and assembly properties of FtsZ under well-defined solution conditions, with the aim of furthering our understanding of how the behavior of FtsZ is controlled by nucleotides and physiological ligands. The modulation of the association and assembly properties of FtsZ by excluded-volume effects, reproducing in part the natural crowded environment in which this protein has evolved to function, will be described. The subsequent studies on the reactivity of FtsZ in membrane-like systems using biochemical, biophysical, and imaging technologies are reported. Finally, we discuss the experimental challenges to be met to achieve construction of the minimum protein set needed to initiate bacterial division, without cells, in a cell-like compartment. This integrated approach, combining quantitative and synthetic strategies, will help to support (or dismiss) conclusions already derived from cellular and molecular analysis and to complete our understanding on how bacterial division works.     

Structural model of ρ1 GABAC receptor based on evolutionary analysis: Testing of predicted protein–protein interactions involved in receptor assembly and function

The homopentameric ρ1 GABA_C receptor is a ligand‐gated ion channel with a binding pocket for γ‐aminobutyric acid (GABA) at the interfaces of N‐terminal extracellular domains. We combined evolutionary analysis, structural modeling, and experimental testing to study determinants of GABA_C receptor assembly and channel gating. We estimated the posterior probability of selection pressure at amino acid residue sites measured as ω‐values and built a comparative structural model, which identified several polar residues under strong selection pressure at the subunit interfaces that may form intersubunit hydrogen bonds or salt bridges. At three selected sites (R111, T151, and E55), mutations disrupting intersubunit interactions had strong effects on receptor folding, assembly, and function. We next examined the role of a predicted intersubunit salt bridge for residue pair R158–D204. The mutant R158D, where the positively charged residue is replaced by a negatively charged aspartate, yielded a partially degraded receptor and lacked membrane surface expression. The membrane surface expression was rescued by the double mutant R158D–D204R, where positive and negative charges are switched, although the mutant receptor was inactive. The single mutants R158A, D204R, and D204A exhibited diminished activities and altered kinetic profiles with fast recovery kinetics, suggesting that R158–D204 salt bridge perhaps stabilizes the open state of the GABA_C receptor. Our results emphasize the functional importance of highly conserved polar residues at the protein–protein interfaces in GABA_C ρ1 receptors and demonstrate how the integration of computational and experimental approaches can aid discovery of functionally important interactions.     

Aromaticity at position 39 in α‐synuclein: A modulator of amyloid fibril assembly and membrane‐bound conformations

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid‐binding features of αS through the design of site‐directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane‐bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death.Brief statement outlining significanceModulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane‐bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.