High-speed Atomic Force Microscopy Research Articles

Complement activation by IgG subclasses is governed by their ability to oligomerize upon antigen binding.

Complement activation through antibody-antigen complexes is crucial in various pathophysiological processes and utilized in immunotherapies to eliminate infectious agents, regulatory immune cells, or cancer cells. The tertiary structures of the four IgG antibody subclasses are largely comparable, with the most prominent difference being the hinge regions connecting the Fab and Fc domains, providing them with unique structural flexibility. Complement recruitment and activation depend strongly on IgG subclass, which is commonly rationalized by differences in hinge flexibility and the respective affinities for C1, the first component of the classical complement pathway. However, a unifying mechanism of how these different IgG subclass properties combine to modulate C1 activation has not yet been proposed. We here demonstrate that complement activation is determined by their varying ability to form IgG oligomers on antigenic surfaces large enough to multivalently bind and activate C1. We directly visualize the resulting IgG oligomer structures and characterize their distribution by means of high-speed atomic force microscopy, quantify their complement recruitment efficiency from quartz crystal microbalance experiments, and characterize their ability to activate complement on tumor cell lines as well as in vesicle-based complement lysis assays. We present a mechanistic model of the multivalent interactions that govern C1 binding to IgG oligomers and use it to extract kinetic rate constants from real-time interaction data from which we further calculate equilibrium dissociation constants. Together, we provide a comprehensive view on the parameters that govern complement activation by the different IgG subclasses, which may inform the design of future antibody therapies.

Single-Molecule Kinetic Observation of Antibody Interactions with Growing Amyloid β Fibrils.

Understanding the dynamic assembly process of amyloid β (Aβ) during fibril formation is essential for developing effective therapeutic strategies against Alzheimer's disease. Here, we employed high-speed atomic force microscopy to observe the growth of Aβ fibrils at the single-molecule level, focusing specifically on their interaction with anti-Aβ antibodies. Our findings show that fibril growth consists of intermittent periods of elongation and pausing, which are dictated by the alternating addition of Aβ monomers to protofilaments. We highlight the distinctive interaction of antibody 4396C, which specifically binds to the fibril ends in the paused state, suggesting a unique mechanism to hinder fibril elongation. Through real-time visualization of fibril growth and antibody interactions combined with molecular simulation, this study provides a refined understanding of Aβ assembly during fibril formation and suggests novel strategies for Alzheimer's therapy aimed at inhibiting the fibril elongation.

The basis of anti-Aβ antibody therapy: The toxicity of Aβ aggregates and the mechanism of action of anti-Aβ antibodies.

In the pathophysiology of Alzheimer's disease (AD), the amyloid hypothesis, which posits that amyloid β-protein (Aβ) abnormally aggregates and damages neurons with tau, has been proposed. It was originally thought that the accumulation of insoluble amyloid fibrils in the brain leads to AD-inducing neurotoxicity; however, in recent years, the positioning of early and intermediate aggregates has also been emphasized. In particular, following the positive results of phase 3 clinical trials of lecanemab and its approval in Japan and the United States, the pathology of protofibrils, which are the target molecules of lecanemab, has attracted attention. Using high-speed atomic force microscopy, we have previously reported that lecanemab, which has a high affinity for protofibrils, binds to and surrounds them. Donanemab, a recombinant monoclonal antibody that primarily targets fibrils composed of N3pG Aβ, has also attracted attention because of its efficacy in phase 3 clinical trials in patients with early stage AD.

CT584 Is Not a Protective Vaccine Antigen against Respiratory Chlamydial Challenge in Mice.

Background:Chlamydia trachomatis is the most prevalent bacterial sexually transmitted pathogen in humans worldwide. Since chlamydial infection is largely asymptomatic with the potential for serious complications, a preventative vaccine is likely the most viable long-term answer to this public health threat. Cell-free protein synthesis (CFPS) utilizes the cellular protein manufacturing machinery decoupled from the requirement for maintaining cellular viability, offering the potential for flexible, rapid, and decentralized production of recombinant protein vaccine antigens. Methods: Here, we use CFPS to produce the full-length putative chlamydial type three secretion system (T3SS) needle-tip protein, CT584, for evaluation as a vaccine antigen in mouse models. High-speed atomic force microscopy (HS-AFM) (RIBM, Tsukuba, Japan) imaging and computer simulations confirm that CFPS-produced CT584 retains a native-like structure prior to immunization. Female mice were primed with CT584 adjuvanted with CpG-1826 intranasally (i.n.) or CpG-1826 + Montanide ISA 720 intramuscularly (i.m.), followed four weeks later by an i.m. boost before respiratory challenge with 104 inclusion forming units (IFU) of Chlamydia muridarum. Results: Immunization with CT584 generated robust antibody responses but weak cell-mediated immunity and failed to protect against i.n. challenge as demonstrated by body weight loss, increased lung weights, and the presence of high numbers of IFUs in the lungs. Conclusion: While CT584 was not a protective vaccine candidate, the speed and flexibility with which CFPS can be used to produce other potential chlamydial antigens make it an attractive technique for antigen production.

Aggregation Characteristics of Biobased Anionic Surfactant, Hydroxy Alkane Sulfonate in Aqueous CaCl2 Solutions: Vesicle and Supported Bilayer Formation.

Vesicles are known to spontaneously adsorb onto solid-liquid interfaces and to form supported bilayers in aqueous solution. Cationic surfactants have typically been used to generate supported bilayers because solid surfaces in water are often negatively charged. The present study investigated the aggregation behavior of an anionic surfactant, hydroxy alkane sulfonate having a C18 alkyl chain (C18HAS) in aqueous CaCl2 solutions. These assessments were performed by acquiring data related to equilibrium surface tension, the solubilization of an oil-soluble dye, UV-visible transmittance, pyrene fluorescence and dynamic light scattering together with freeze-fracture transmission electron microscopy observations. The results suggest that C18HAS can form vesicles in aqueous CaCl2 solutions under certain surfactant concentrations. Specifically, this aggregation behavior is greatly affected by C18HAS/CaCl2 molar ratio. At the C18HAS/CaCl2 molar ratio is less than an equivalence point (that is, less than 2:1), phase separation occurs with the formation of a vesicle above solubility limit of the C18HAS Ca salt. On the other hand, in the case that the C18HAS/CaCl2 molar ratio is above an equivalence point (that is, above 2:1), the Na salt of C18HAS forms micelles above the critical micelle concentration (cmc), causing solubilization of vesicles. Analyses by high-speed atomic force microscopy demonstrated that the C18HAS vesicles can spontaneously form a supported bilayer on a negatively charged mica surfaces, similar to the behavior of cationic surfactant vesicles, even though C18HAS is an anionic surfactant. These results suggest that C18HAS could serve as a detergent component but also as a surface modifier when the C18HAS/CaCl2 molar ratio is optimized.

Modeling Conformational Transitions of Biomolecules from Atomic Force Microscopy Images using Normal Mode Analysis.

Observing a single biomolecule performing its function is fundamental in biophysics as it provides important information for elucidating the mechanism. High-speed atomic force microscopy (HS-AFM) is a unique and powerful technique that allows the observation of biomolecular motion in a near-native environment. However, the spatial resolution of HS-AFM is limited by the physical size of the cantilever tip, which restricts the ability to obtain atomic details of molecules. In this study, we propose a novel computational algorithm designed to derive atomistic models of conformational dynamics from AFM images. Our method uses normal-mode analysis to describe the expected motions of the molecule, allowing these motions to be represented with a limited number of coordinates. This approach mitigates the problem of overinterpretation inherent in the analysis of AFM images with limited resolution. We demonstrate the effectiveness of our algorithm, NMFF-AFM, using synthetic data sets for three proteins that undergo significant conformational changes. NMFF-AFM is a fast and user-friendly program that requires minimal setup and has the potential to be a valuable tool for biophysical studies using HS-AFM.

High-Speed Atomic Force Microscopy Reveals Fluctuations and Dimer Splitting of the N-Terminal Domain of GluA2 Ionotropic Glutamate Receptor-Auxiliary Subunit Complex.

α-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid glutamate receptors (AMPARs) enable rapid excitatory synaptic transmission by localizing to the postsynaptic density of glutamatergic spines. AMPARs possess large extracellular N-terminal domains (NTDs), which are crucial for AMPAR clustering at synaptic sites. However, the dynamics of NTDs and the molecular mechanism governing their synaptic clustering remain elusive. Here, we employed high-speed atomic force microscopy (HS-AFM) to directly visualize the conformational dynamics of NTDs in the GluA2 subunit complexed with TARP γ2 in lipid environments. HS-AFM videos of GluA2-γ2 in the resting and activated/open states revealed fluctuations in NTD dimers. Conversely, in the desensitized/closed state, the two NTD dimers adopted a separated conformation with less fluctuation. Notably, we observed individual NTD dimers transitioning into monomers, with extended monomeric states in the activated/open state. Molecular dynamics simulations provided further support, confirming the energetic stability of the monomeric NTD states within lipids. This NTD-dimer splitting resulted in subunit exchange between the receptors and increased the number of interaction sites with synaptic protein neuronal pentraxin 1 (NP1). Moreover, our HS-AFM studies revealed that NP1 forms a ring-shaped octamer through N-terminal disulfide bonds and binds to the tip of the NTD. These findings suggest a molecular mechanism in which NP1, upon forming an octamer, is secreted into the synaptic region and binds to the tip of the GluA2 NTD, thereby bridging and clustering multiple AMPARs. Thus, our findings illuminate the critical role of NTD dynamics in the synaptic clustering of AMPARs and contribute valuable insights into the fundamental processes of synaptic transmission.

Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes.

Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.

Membrane-Active Antibiotics Affect Domains in Bacterial Membranes as the First Step of Their Activity.

The need to combat antimicrobial resistance is becoming more and more pressing. Here we investigate the working mechanism of a small cationic agent, N-alkylamide 3d, by conventional and high-speed atomic force microscopy. We show that N-alkylamide 3d interacts with the membrane of Staphylococcus aureus, where it changes the organization and dynamics of lipid domains. After this initial step, supramolecular structures of the antimicrobial agent attach on top of the affected membrane gradually, covering it entirely. These results demonstrate that lateral domains in the bacterial membranes might be affected by small antimicrobial agents more often than anticipated. At the same time, we show a new dual-step activity of N-alkylamide 3d that not only destroys the lateral membrane organization but also effectively covers the whole membrane with aggregates. This final step could render the membrane inaccessible from the outside and possibly prevent signaling and waste disposal of living bacteria.

Functional role of carbohydrate-binding modules in multi-modular chitinase OfChtII

The primary distinction between insect and bacterial chitin degradation systems lies in the presence of a multi-modular endo-acting chitinase ChtII, in contrast to a processive exo-acting chitinase. Although the essential role of ChtII during insect development and its synergistic action with processive chitinase during chitin degradation have been established, the mechanistic understanding of how it deconstructs chitin remains largely elusive. Here OfChtII from the insect Ostrinia furnacalis was investigated employing comprehensive approaches encompassing biochemical and microscopic analyses. The results demonstrated that OfChtII truncations with more carbohydrate-binding modules (CBMs) exhibited enhanced hydrolysis activity, effectively yielding a greater proportion of fibrillary fractions from the compacted chitin substrate. At the single-molecule level, the CBMs in these OfChtII truncations have been shown to primarily facilitate chitin substrate association rather than dissociation. Furthermore, a greater number of CBMs was demonstrated to be essential for the enzyme to effectively bind to chitin substrates with high crystallinity. Through real-time imaging by high-speed atomic force microscopy, the OfChtII-B4C1 truncation with three CBMs was observed to shear chitin fibers, thereby generating fibrillary fragments and deconstructing the compacted chitin structure. This work pioneers in revealing the nanoscale mechanism of endo-acting multi-modular chitinase involved in chitin degradation, which provides an important reference for the rational design of chitinases or other glycoside hydrolases.

Deciphering the actin structure-dependent preferential cooperative binding of cofilin.

The mechanism underlying the preferential and cooperative binding of cofilin and the expansion of clusters toward the pointed-end side of actin filaments remains poorly understood. To address this, we conducted a principal component analysis based on available filamentous actin (F-actin) and C-actin (cofilins were excluded from cofilactin) structures and compared to monomeric G-actin. The results strongly suggest that C-actin, rather than F-ADP-actin, represented the favourable structure for binding preference of cofilin. High-speed atomic force microscopy explored that the shortened bare half helix adjacent to the cofilin clusters on the pointed end side included fewer actin protomers than normal helices. The mean axial distance (MAD) between two adjacent actin protomers along the same long-pitch strand within shortened bare half helices was longer (5.0-6.3 nm) than the MAD within typical helices (4.3-5.6 nm). The inhibition of torsional motion during helical twisting, achieved through stronger attachment to the lipid membrane, led to more pronounced inhibition of cofilin binding and cluster formation than the presence of inorganic phosphate (Pi) in solution. F-ADP-actin exhibited more naturally supertwisted half helices than F-ADP.Pi-actin, explaining how Pi inhibits cofilin binding to F-actin with variable helical twists. We propose that protomers within the shorter bare helical twists, either influenced by thermal fluctuation or induced allosterically by cofilin clusters, exhibit characteristics of C-actin-like structures with an elongated MAD, leading to preferential and cooperative binding of cofilin.

HS-AFM single-molecule structural biology uncovers basis of transporter wanderlust kinetics.

The Pyrococcus horikoshii amino acid transporter GltPh revealed, like other channels and transporters, activity mode switching, previously termed wanderlust kinetics. Unfortunately, to date, the basis of these activity fluctuations is not understood, probably due to a lack of experimental tools that directly access the structural features of transporters related to their instantaneous activity. Here, we take advantage of high-speed atomic force microscopy, unique in providing simultaneous structural and temporal resolution, to uncover the basis of kinetic mode switching in proteins. We developed membrane extension membrane protein reconstitution that allows the analysis of isolated molecules. Together with localization atomic force microscopy, principal component analysis and hidden Markov modeling, we could associate structural states to a functional timeline, allowing six structures to be solved from a single molecule, and an inward-facing state, IFSopen-1, to be determined as a kinetic dead-end in the conformational landscape. The approaches presented on GltPh are generally applicable and open possibilities for time-resolved dynamic single-molecule structural biology.

The cyanobacterial protein VIPP1 forms ESCRT-III-like structures on lipid bilayers.

The biogenesis and maintenance of thylakoid membranes require vesicle-inducing protein in plastids 1 (VIPP1). VIPP1 is a member of the endosomal sorting complex required for transport-III (ESCRT-III) superfamily, whose members form diverse filament-based supramolecular structures that facilitate membrane deformation and fission. VIPP1 cryo-electron microscopy (EM) structures in solution revealed helical rods and baskets of stacked rings, with amphipathic membrane-binding domains in the lumen. However, how VIPP1 interacts with membranes remains largely unknown. Here, using high-speed atomic force microscopy (HS-AFM), we show that VIPP1 assembles into right-handed chiral spirals and regular polygons on supported lipid bilayers via ESCRT-III-like filament assembly and dynamics. VIPP1 filaments grow clockwise into spirals through polymerization at a ring-shaped central polymerization hub, and into polygons through clockwise polymerization at the sector peripheries. Interestingly, VIPP1 initially forms Archimedean spirals, which upon maturation transform into logarithmic spirals through lateral annealing of strands to the outermore low-curvature spiral turns.

Linearizing AFM raster scans acquired using dual-stage lateral scanners

Dual-stage scanners can successfully increase the range of atomic force microscope (AFM) scanners in space and/or frequency by designing and constructing two nanopositioners with significant differences in static and dynamic characteristics. In such cases, the positioning signal has to be split to meet the displacement and frequency limitations of each stage. Without closed-loop displacement measurement sensors, linearizing images acquired using signals split in time and frequency can be challenging. A common method, often cumbersome and time consuming, uses inverse model-based feedforward compensation. In this work, we show that image-based linearization can be used to achieve acceptable results for raster scans acquired using dual-stage scanners. In particular, we apply the feedforward and image-based methods to our homemade dual-stage lateral scanner and high-speed AFM (HS-AFM) system. The acquired scans compare well for the two methods, indicating that image-based raster scan linearization can be used in place of inverse model-based feedforward approaches.

Mapping the Dynamics of Fluctuations and Defects in Confined Block Copolymer Films with High-Speed Atomic Force Microscopy

Mapping the Dynamics of Fluctuations and Defects in Confined Block Copolymer Films with High-Speed Atomic Force Microscopy

Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.

Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination

Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.

Conformational Dynamics of Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis.

Viral ribonucleoproteins (vRNPs) are the cornerstones of viral proliferation, as they form the macromolecular complexes that are responsible for the transcription and replication of most single-stranded RNA viruses. The influenza A virus (IAV) polymerase catalyzes RNA synthesis within the context of vRNPs where genomic viral RNA (vRNA) is packaged by the viral nucleoprotein (NP). We used high-speed atomic force microscopy and electron microscopy to study the conformational dynamics of individual IAV recombinant RNPs (rRNPs) during RNA synthesis. The rRNPs present an annular organization that allows for the real-time tracking of conformational changes in the NP-vRNA template caused by the advancing polymerase. We demonstrate that the rRNPs undergo a well-defined conformational cycle during RNA synthesis, which can be interpreted in light of previous transcription models. We also present initial estimations of the average RNA synthesis rate in the rRNP and its dependence on the nucleotide concentration and stability of the nascent RNA secondary structures. Furthermore, we provide evidence that rRNPs can perform consecutive cycles of RNA synthesis, accounting for their ability to recycle and generate multiple copies of RNA.

Microstructural analysis of ex-service neutron irradiated stainless steel nuclear fuel cladding by high-speed AFM

Fuel cladding in advanced gas-cooled nuclear reactors is made of an austenitic stainless steel referred to as 20Cr/25NiNb. This material can undergo microstructural changes in these extreme environments through a combination of thermal effects and radiation damage. In this work, the microstructures present in an ex-service irradiated 20Cr/25Ni-Nb sample are studied using correlated high-speed atomic force microscopy (HS-AFM) and electron microscopy techniques for the first time. Correlated topographic, crystallographic, and chemical information from the sample surface enabled identification of secondary phase precipitates (SPPs) including M23C6, sigma phase, NbC, and G phase. These SPPs can have adverse effects on the material properties. HS-AFM analysis showed surface textures unique to each SPP. Voids formed due to irradiation were also identified across the surface by HS-AFM. These voids were found to be larger in size and depth along grain boundaries. Further analysis identified a relationship between void size and frequency and grain boundary misorientation and SPP presence.

Nanoscale Structure, Interactions, and Dynamics of Centromere Nucleosomes.

Centromeres are specific segments of chromosomes comprising two types of nucleosomes: canonical nucleosomes containing an octamer of H2A, H2B, H3, and H4 histones and CENP-A nucleosomes in which H3 is replaced with its analogue CENP-A. This modification leads to a difference in DNA wrapping (∼121 bp), considerably less than 147 bp in canonical nucleosomes. We used atomic force microscopy (AFM) and high-speed AFM (HS-AFM) to characterize nanoscale features and dynamics for both types of nucleosomes. For both nucleosomes, spontaneous asymmetric unwrapping of DNA was observed, and this process occurs via a transient state with ∼100 bp DNA wrapped around the core, followed by a rapid dissociation of DNA. Additionally, HS-AFM revealed higher stability of CENP-A nucleosomes compared with H3 nucleosomes in which dissociation of the histone core occurs prior to the nucleosome dissociation. These results help elucidate the differences between these nucleosomes and the potential biological necessity for CENP-A nucleosomes.