Real-time Atomic Force Microscopy Imaging Research Articles

N-Formylation modifies membrane damage associated with PSMα3 interfacial fibrillation.

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins α3 (PSMα3) peptides, able to self-assemble into amyloid-like cross-α fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSMα3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining atomic force microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its interactions with model membranes of controlled lipid composition without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSMα3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains only in the fluid phase of membranes and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSMα3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSMα3 with membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.

Real-time microscopy of the relaxation of a glass

The understanding of the dynamics of a glass above its devitrification temperature remains incomplete. Here, we build a spatio-temporal map of the relaxation dynamics of a highly stable glass into its supercooled liquid using real-time atomic force microscopy imaging. This methodology enables direct visualization of the progression of the liquid phase and clarifies and quantifies the presence of localized fast mobility regions separated by giant length scales. Our data establish a clear correlation between dynamic length and time scales in glasses. This approach may also be applicable to unveil the microscopic structure and dynamics of other glass-forming systems with much shorter length and time scales, including liquid-cooled glasses.

Statistical Characterization of Heterogeneous Dissolution Rates of Calcite from In situ and Real-Time AFM Imaging

The evolution of the surface topography of a calcite crystal subject to dissolution is documented through in situ real-time imaging obtained via atomic force microscopy (AFM). The dissolution process takes place by exposing the crystal surface to deionized water. AFM data allow detection of nucleation and expansion of mono- and multilayer rhombic etch pits and are employed to estimate the spreading rate of these structures. Spatially heterogeneous distributions of local dissolution rate are evaluated from the difference between topographic measurements taken at prescribed time intervals. We rest on a stochastic framework of analysis viewing the dissolution rate as a generalized sub-Gaussian (GSG) spatially correlated random process. Our analysis yields: (i) a quantitative assessment of the temporal evolution of the statistics of the dissolution rates as well as their spatial increments; (ii) a characterization of the degree of spatial correlation of dissolution rates and of the way this is linked to the various mechanisms involved in the dissolution process and highlighted through the experimental evidences. Our results indicate that the parameters driving the statistics of the GSG distribution and the spreading rate of the multilayer pits display a similar trend in time, thus suggesting that the evolution of these structures imprints the statistical features of local dissolution rates.Article HighlightsWe investigate dynamics of dissolution patterns on a calcite crystal in contact with deionized water via AFM imagingTemporal behavior of parameters of our statistical model is consistent with surface pattern evolutionA nested model for the spatial correlation of rates embeds multiple mechanisms driving dissolution rate.

Quaternary Amine-Terminated Quantum Dots Induce Structural Changes to Supported Lipid Bilayers.

The cytoplasmic membrane represents an essential barrier between the cytoplasm and the environment external to cells. Interaction with nanomaterials can alter the integrity of the cytoplasmic membrane through the formation of holes and membrane thinning, which can ultimately lead to adverse biological impacts. Here we use supported lipid bilayers as experimental models for the cytoplasmic membrane to investigate the impact of quantum dots functionalized with the cationic polymer poly(diallyldimethylammonium chloride) (PDDA) on membrane structure. Using a quartz crystal microbalance with dissipation monitoring we show that the positively charged quantum dots attach to and induce structural rearrangement to zwitterionic bilayers in solely the liquid-disordered phase and in those containing phase-segregated liquid-ordered domains. Real-time atomic force microscopy imaging revealed that PDDA-coated quantum dots and, to a lesser extent, PDDA itself induced the disappearance of liquid-ordered domains. We hypothesize this effect is due to an increase in energy per unit area caused by collisions between PDDA-coated quantum dots at the membrane surface. This increase in free energy per area exceeds the approximate free-energy change associated with membrane mixing between the liquid-ordered and liquid-disordered phases and results in the destabilization of membrane domains.

Real-Time Atomic Force Microscopy Imaging of Block Copolymer Directed Self Assembly.

The kinetics of directed self-assembly of symmetric PS-b-PMMA diblock copolymer on chemically patterned templates were measured during in situ thermal annealing. Although these chemical guide patterns lead to well-aligned, defect-free lamellar patterns at thermodynamic equilibrium, in practice, challenges remain in understanding and optimizing the kinetic evolution for technological applications. High-speed, environmentally controlled atomic force microscopy imaging was used to track pattern evolution on the time scale of individual microdomain connections in real space and time, allowing the direct visualization of defect healing mechanisms. When we apply this highly general technique to films on chemically patterned substrates, we find that pattern alignment is mediated by a metastable nonbulk morphology unique to these samples, referred to as the "stitch" morphology. We observe diverse and anisotropic mechanisms for the conversion from this morphology to equilibrium lamellar stripes. Directed self-assembly on chemical templates is observed to follow exponential kinetics with an apparent energetic barrier of 360 ± 80 kJ/mol from 210-230 °C, a significant enhancement when compared with ordering rates on unpatterned substrates. Ultimately, from local imaging, we find that the presence of a chemical guiding field causes morphological ordering and lamellar alignment to occur irreversibly.

Real-time atomic force microscopy imaging of collagen fibril under ultraviolet irradiation

Morphological changes that occur on collagen fibril under ultraviolet (UV) irradiation were directly observed using real-time atomic force microscopy (AFM). By analyzing real-time AFM images as a function of UV irradiation time, we found that the gap region of a collagen fibril was more vulnerable to UV light exposure than was the overlap region. In addition, the degraded collagen fragments in the gap region relocated and began accumulating in the overlap region. Through real-time observation of morphological changes using AFM, we verified that collagen fibrils are directly degraded by UV irradiation. Our findings help reveal the mechanisms of skin aging.

Antimicrobial Protegrin-1 Forms Amyloid-Like Fibrils with Rapid Kinetics Suggesting a Functional Link

Protegrin-1 (PG-1) is an 18 residues long, cysteine-rich β-sheet antimicrobial peptide (AMP). PG-1 induces strong cytotoxic activities on cell membrane and acts as a potent antibiotic agent. Earlier we reported that its cytotoxicity is mediated by its channel-forming ability. In this study, we have examined the amyloidogenic fibril formation properties of PG-1 in comparison with a well-defined amyloid, the amyloid-β (Aβ1–42) peptide. We have used atomic force microscopy (AFM) and thioflavin-T staining to investigate the kinetics of PG-1 fibrils growth and molecular dynamics simulations to elucidate the underlying mechanism. AFM images of PG-1 on a highly hydrophilic surface (mica) show fibrils with morphological similarities to Aβ1–42 fibrils. Real-time AFM imaging of fibril growth suggests that PG-1 fibril growth follows a relatively fast kinetics compared to the Aβ1–42 fibrils. The AFM results are in close agreement with results from thioflavin-T staining data. Furthermore, the results indicate that PG-1 forms fibrils in solution. Significantly, in contrast, we do not detect fibrillar structures of PG-1 on an anionic lipid bilayer 2-dioleoyl-sn-glycero-3-phospho-L-serine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; only small PG-1 oligomers can be observed. Molecular dynamics simulations are able to identify the presence of these small oligomers on the membrane bilayer. Thus, our current results show that cytotoxic AMP PG-1 is amyloidogenic and capable of forming fibrils. Overall, comparing β-rich AMPs and amyloids such as Aβ, in addition to cytotoxicity and amyloidogenicity, they share a common structural motif, and are channel forming. These combined properties support a functional relationship between amyloidogenic peptides and β-sheet-rich cytolytic AMPs, suggesting that amyloids channels may have an antimicrobial function.

Real-time atomic force microscopy imaging of photoinduced surface deformation in AsxSe100−x chalcogenide films

We present direct measurements of the kinetics of surface relief gratings (SRGs) formation in amorphous AsxSe100−x (20≤xAs≤50) thin films. SRGs are induced in different holographic schemes of recording using near-band-gap light and their growth is further facilitated by illumination with an interference pattern and observed in real time by in situ atomic force microscopy. It is found that the kinetics of SRG formation depends upon film composition and incident light polarization. The light-stimulated vectorial surface deformations are maximized for Se-rich glasses and increase even further by additional illumination during recording.

Kinetics of interaction of HIV fusion protein (gp41) with lipid membranes studied by real-time AFM imaging

One of the most important steps in the process of viral infection is a fusion between cell membrane and virus, which is mediated by the viral envelope glycoprotein. The study of activity of the glycoprotein in the post-fusion state is important for understanding the progression of infection. Here we present a first real-time kinetic study of the activity of gp41 (the viral envelope glycoprotein of human immunodeficiency virus—HIV) and its two mutants in the post-fusion state with nanometer resolution by atomic force microscopy (AFM). Tracking the changes in the phosphatidylcholine (PC) and phosphatidylcholine–phosphatidylserine (PC:PS) membrane integrity over one hour by a set of AFM images revealed differences in the interaction of the three types of protein with zwitterionic and negatively charged membranes. A quantitative analysis of the slow kinetics of hole formation in the negatively charged lipid bilayer is presented. Specifically, analysis of the rate of roughness change for the three types of proteins suggests that they exhibit different types of kinetic behavior.

Atomic force microscopy and chemical force microscopy of microbial cells

Over the past years, atomic force microscopy (AFM) has emerged as a powerful tool for imaging the surface of microbial cells with nanometer resolution, and under physiological conditions. Moreover, chemical force microscopy (CFM) and single-molecule force spectroscopy have enabled researchers to map chemical groups and receptors on cell surfaces, providing valuable insight into their structure-function relationships. Here, we present protocols for analyzing spores of the pathogen Aspergillus fumigatus using real-time AFM imaging and CFM. We emphasize the use of porous polymer membranes for immobilizing single live cells, and the modification of gold-coated tips with alkanethiols for CFM measurements. We also discuss recording conditions and data interpretation, and provide recommendations for reliable experiments. For well-trained AFM users, the entire protocol can be completed in 2-3 d.

High-Resolution Cell Surface Dynamics of Germinating Aspergillus fumigatus Conidia

We used real-time atomic force microscopy with a temperature-controlled stage (37°C) to probe the structural and physicochemical dynamics of single Aspergillus fumigatus conidia during germination. Nanoscale topographic images of dormant spores revealed the presence of a layer of rodlets made of hydrophobins, in agreement with earlier electron microscopy observations. Within the 3-h germination period, progressive disruption of the rodlet layer was observed, revealing hydrophilic inner cell wall structures. Using adhesion force mapping with hydrophobic tips, these ultrastructural changes were shown to correlate with major differences in cell surface hydrophobicity. That is, the rodlet surface was uniformly hydrophobic due to the presence of hydrophobins, whereas the cell wall material appearing upon germination was purely hydrophilic. This study illustrates the potential of real-time atomic force microscopy imaging and force spectroscopy for tracking cell-surface dynamics.

Membrane Resistance to Triton X-100 Explored by Real-Time Atomic Force Microscopy

Lateral segregation of lipids and proteins in biological membranes leads to the formation of detergent-resistant domains, also called "rafts". Understanding the mechanisms governing the biomembrane's resistance to solubilization by detergents is crucial in biochemical research. Here, we used real-time atomic force microscopy (AFM) imaging to visualize the behavior of a model supported lipid bilayer in the presence of different Triton X-100 (TX-100) concentrations. Mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) supported bilayers were prepared by vesicle fusion. Real-time AFM imaging revealed that, at concentrations below the critical micelle concentration (CMC), TX-100 did not solubilize the bilayer, but the DPPC domains were eroded in a time-dependent manner. This effect was attributed to the DPPC molecular packing disorganization by the detergent starting from the DOPC/DPPC interface. Just above the CMC, the detergent led to a complete solubilization of the DOPC matrix, leaving the DPPC domains unaltered. At higher TX-100 concentrations, the DOPC was also immediately removed just after detergent addition, and the DPPC domains remaining on the mica surface appeared to be more swollen and were gradually solubilized. This progressive solubilization of the DPPC remaining phase did not start at the edge of the domains but from holes appearing and expanding at the center of DPPC patches. The swelling of the DPPC domains was directly correlated with TX-100 concentration above the CMC and with detergent intercalation between DPPC molecules. We are convinced that this approach will provide a key system to elucidate the physical mechanisms of membrane solubilization by nonionic detergents.

Nanoscale Modification of Supported Lipid Membranes: Synergetic Effect of Phospholipase D and Viral Fusion Peptides

Understanding the molecular bases of biomembrane fusion events is a challenging issue in current biomedical research in view of its involvement in controlling cellular functions and in mediating various important diseases. In this study, we used atomic force microscopy (AFM) to address the crucial question as to whether negatively curved lipids influence the ability of a viral fusion peptide to perturb the organization of supported lipid bilayers. To this end, an original approach was developed that makes use of an AFM tip functionalized with phospholipase D (PLD) enzymes to generate in situ small amounts of negatively curved phosphatidic acid (PA) in mixed dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers. Real-time AFM imaging revealed that this nanomodification dramatically enhanced subsequent interaction with the simian immunodeficiency virus (SIV) fusion peptide. At short incubation time, the SIV peptide induced a 1.9 nm thickness reduction of the DPPC domains, reflecting either interdigitation or fluidification of the lipids. At longer incubation time, these depressed domains transformed into elevated striated domains, protruding one to several nanometers above the bilayer surface. Two complementary experiments, i.e. addition of the peptide onto DOPC/DPPC/DOPA bilayers or onto DOPC/DPPC bilayers pretreated with a PLD solution, confirmed that both PA and SIV peptides are required to induce depressed and striated domains. Accordingly, this is the first time that a high-resolution imaging technique is used to demonstrate that negatively curved lipids affect the membrane activity of fusion peptides. We believe the nanoscale approach presented here, i.e. use of enzyme-functionalized AFM tips to modify lipid bilayers, will find exciting new applications in nanobiotechnology for the design of biomimetic surfaces.

Imaging real-time aggregation of amyloid beta protein (1–42) by atomic force microscopy

Amyloid beta protein (AβP) is the major fibrillar constituent of senile plaques. However, no causative role for AβP-fibers in Alzheimer’s disease (AD) pathology is established. Globular AβPs are continuously released during normal cellular metabolism at pico- to nano-molar concentration. We used atomic force microscopy (AFM) to examine aggregation of freshly prepared AβP 1–42 and to examine the role of AβP concentration, imaging medium (air, water, or PBS) and agonists/antagonists on AβP-fibrillogenesis. At even very high and non-physiological AβP concentrations, 24–48 h of real-time AFM imaging (a) in water show only multiple layers of globular aggregates and no fibrils and (b) in PBS show mainly the globular structures and some short fibrils. On-line addition of Zn, an agonist for AβP-fibrillogenesis, induced a slow but non-fibrillar aggregation of globular AβPs. EDTA, a chelator of Zn and calcium (a modulator of AβP-mediated toxicity) induced a reversible change in the Zn-mediated aggregation. These results strongly suggest that no AβP-fibers are formed for the physiologically relevant concentration and thus the plaque-associated fibers may not account for the AD pathophysiology.

Physiological role of gap-junctional hemichannels. Extracellular calcium-dependent isosmotic volume regulation.

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to </=1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and beta-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation.