An Efficient Method for Isolating and Purifying Nuclei from Mice Brain for Single-Molecule Imaging Using High-Speed Atomic Force Microscopy.

Nuclear pore complexes (NPCs) on the nuclear membrane surface have a crucial function in controlling the movement of small molecules and macromolecules between the cell nucleus and cytoplasm through their intricate core channel resembling a spiderweb with several layers. Currently, there are few methods available to accurately measure the dynamics of nuclear pores on the nuclear membranes at the nanoscale. The limitation of traditional optical imaging is due to diffraction, which prevents achieving the required resolution for observing a diverse array of organelles and proteins within cells. Super-resolution techniques have effectively addressed this constraint by enabling the observation of subcellular components on the nanoscale. Nevertheless, it is crucial to acknowledge that these methods often need the use of fixed samples. This also raises the question of how closely a static image represents the real intracellular dynamic system. High-speed atomic force microscopy (HS-AFM) is a unique technique used in the field of dynamic structural biology, enabling the study of individual molecules in motion close to their native states. Establishing a reliable and repeatable technique for imaging mammalian tissue at the nanoscale using HS-AFM remains challenging due to inadequate sample preparation. This study presents the rapid strainer microfiltration (RSM) protocol for directly preparing high-quality nuclei from the mouse brain. Subsequently, we promptly utilize HS-AFM real-time imaging and cinematography approaches to record the spatiotemporal of nuclear pore nano-dynamics from the mouse brain.

High-Speed Atomic Force Microscopy for Filming Protein Molecules in Dynamic Action.

Structural biology is currently undergoing a transformation into dynamic structural biology, which reveals the dynamic structure of proteins during their functional activity to better elucidate how they function. Among the various approaches in dynamic structural biology, high-speed atomic force microscopy (HS-AFM) is unique in the ability to film individual molecules in dynamic action, although only topographical information is acquirable. This review provides a guide to the use of HS-AFM for biomolecular imaging and showcases several examples, as well as providing information on up-to-date progress in HS-AFM technology. Finally, we discuss the future prospects of HS-AFM in the context of dynamic structural biology in the upcoming era. Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Nanoscopic Elucidation of Spontaneous Self-Assembly of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Open Reading Frame 6 (ORF6) Protein.

Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.

Protein dynamics by the combination of high-speed AFM and computational modeling.

High-speed atomic force microscopy (HS-AFM) allows direct observation of biological molecules in dynamic action. However, HS-AFM has no atomic resolution. This article reviews recent progress of computational methods to infer high-resolution information, including the construction of 3D atomistic structures, from experimentally acquired resolution-limited HS-AFM images.

Nanoscopic Assessment of Anti-SARS-CoV-2 Spike Neutralizing Antibody Using High-Speed AFM.

Anti-spike neutralizing antibodies (S NAbs) have been developed for prevention and treatment against COVID-19. The nanoscopic characterization of the dynamic interaction between spike proteins and S NAbs remains difficult. By using high-speed atomic force microscopy (HS-AFM), we elucidate the molecular property of an S NAb and its interaction with spike proteins. The S NAb appeared as monomers with a Y conformation at low density and formed hexameric oligomers at high density. The dynamic S NAb-spike protein interaction at RBD induces neither RBD opening nor S1 subunit shedding. Furthermore, the interaction was stable at endosomal pH. These findings indicated that the S NAb could have a negligible risk of antibody-dependent enhancement. Dynamic movement of spike proteins on small extracellular vesicles (S sEV) resembled that on SARS-CoV-2. The sensitivity of variant S sEVs to S NAb could be evaluated using HS-AFM. Altogether, we demonstrate a nanoscopic assessment platform for evaluating the binding property of S NAbs.

PQBP5/NOL10 maintains and anchors the nucleolus under physiological and osmotic stress conditions.

Polyglutamine binding protein 5 (PQBP5), also called nucleolar protein 10 (NOL10), binds to polyglutamine tract sequences and is expressed in the nucleolus. Using dynamic imaging of high-speed atomic force microscopy, we show that PQBP5/NOL10 is an intrinsically disordered protein. Super-resolution microscopy and correlative light and electron microscopy method show that PQBP5/NOL10 makes up the skeletal structure of the nucleolus, constituting the granule meshwork in the granular component area, which is distinct from other nucleolar substructures, such as the fibrillar center and dense fibrillar component. In contrast to other nucleolar proteins, which disperse to the nucleoplasm under osmotic stress conditions, PQBP5/NOL10 remains in the nucleolus and functions as an anchor for reassembly of other nucleolar proteins. Droplet and thermal shift assays show that the biophysical features of PQBP5/NOL10 remain stable under stress conditions, explaining the spatial role of this protein. PQBP5/NOL10 can be functionally depleted by sequestration with polyglutamine disease proteins in vitro and in vivo, leading to the pathological deformity or disappearance of the nucleolus. Taken together, these findings indicate that PQBP5/NOL10 is an essential protein needed to maintain the structure of the nucleolus.

Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II.

We report a case in which sub-stoichiometric binding of an actin-binding protein has profound structural and functional consequences, providing an insight into the fundamental properties of actin regulation. Rng2 is an IQGAP contained in contractile rings in the fission yeast Schizosaccharomyces pombe Here, we used high-speed atomic force microscopy and electron microscopy and found that sub-stoichiometric binding of the calponin-homology actin-binding domain of Rng2 (Rng2CHD) induces global structural changes in skeletal muscle actin filaments, including shortening of the filament helical pitch. Sub-stoichiometric binding of Rng2CHD also reduced the affinity between actin filaments and muscle myosin II carrying ADP and strongly inhibited the motility of actin filaments on myosin II in vitro. On skeletal muscle myosin II-coated surfaces, Rng2CHD stopped the actin movements at a binding ratio of 11%. Rng2CHD also inhibited actin movements on myosin II of the amoeba Dictyostelium, but in this case, by detaching actin filaments from myosin II-coated surfaces. Thus, sparsely bound Rng2CHD induces apparently cooperative structural changes in actin filaments and inhibits force generation by actomyosin II.

Technical advances in high-speed atomic force microscopy.

It has been 30 years since the outset of developing high-speed atomic force microscopy (HS-AFM), and 15 years have passed since its establishment in 2008. This advanced microscopy is capable of directly visualizing individual biological macromolecules in dynamic action and has been widely used to answer important questions that are inaccessible by other approaches. The number of publications on the bioapplications of HS-AFM has rapidly increased in recent years and has already exceeded 350. Although less visible than these biological studies, efforts have been made for further technical developments aimed at enhancing the fundamental performance of HS-AFM, such as imaging speed, low sample disturbance, and scan size, as well as expanding its functionalities, such as correlative microscopy, temperature control, buffer exchange, and sample manipulations. These techniques can expand the range of HS-AFM applications. After summarizing the key technologies underlying HS-AFM, this article focuses on recent technical advances and discusses next-generation HS-AFM.

Functional Implications of Dynamic Structures of Intrinsically Disordered Proteins Revealed by High-Speed AFM Imaging.

The unique functions of intrinsically disordered proteins (IDPs) depend on their dynamic protean structure that often eludes analysis. High-speed atomic force microscopy (HS-AFM) can conduct this difficult analysis by directly visualizing individual IDP molecules in dynamic motion at sub-molecular resolution. After brief descriptions of the microscopy technique, this review first shows that the intermittent tip-sample contact does not alter the dynamic structure of IDPs and then describes how the number of amino acids contained in a fully disordered region can be estimated from its HS-AFM images. Next, the functional relevance of a dumbbell-like structure that has often been observed on IDPs is discussed. Finally, the dynamic structural information of two measles virus IDPs acquired from their HS-AFM and NMR analyses is described together with its functional implications.

Development of hidden Markov modeling method for molecular orientations and structure estimation from high-speed atomic force microscopy time-series images.

High-speed atomic force microscopy (HS-AFM) is a powerful technique for capturing the time-resolved behavior of biomolecules. However, structural information in HS-AFM images is limited to the surface geometry of a sample molecule. Inferring latent three-dimensional structures from the surface geometry is thus important for getting more insights into conformational dynamics of a target biomolecule. Existing methods for estimating the structures are based on the rigid-body fitting of candidate structures to each frame of HS-AFM images. Here, we extend the existing frame-by-frame rigid-body fitting analysis to multiple frames to exploit orientational correlations of a sample molecule between adjacent frames in HS-AFM data due to the interaction with the stage. In the method, we treat HS-AFM data as time-series data, and they are analyzed with the hidden Markov modeling. Using simulated HS-AFM images of the taste receptor type 1 as a test case, the proposed method shows a more robust estimation of molecular orientations than the frame-by-frame analysis. The method is applicable in integrative modeling of conformational dynamics using HS-AFM data.

Spatiotemporal tracking of small extracellular vesicle nanotopology in response to physicochemical stresses revealed by HS-AFM.

Small extracellular vesicles (sEVs) play a crucial role in local and distant cell communication. The intrinsic properties of sEVs make them compatible biomaterials for drug delivery, vaccines, and theranostic nanoparticles. Although sEV proteomics have been robustly studied, a direct instantaneous assessment of sEV structure dynamics remains difficult. Here, we use the high-speed atomic force microscopy (HS-AFM) to evaluate nanotopological changes of sEVs with respect to different physicochemical stresses including thermal stress, pH, and osmotic stress. The sEV structure is severely altered at high-temperature, high-pH, or hypertonic conditions. Surprisingly, the spherical shape of the sEVs is maintained in acidic or hypotonic environments. Real-time observation by HS-AFM imaging reveals an irreversible structural change in the sEVs during transition of pH or osmolarity. HS-AFM imaging provides both qualitative and quantitative data at high spatiotemporal resolution (nanoscopic and millisecond levels). In summary, our study demonstrates the feasibility of HS-AFM for structural characterization and assessment of nanoparticles.

Guide to studying intrinsically disordered proteins by high-speed atomic force microscopy.

Intrinsically disordered proteins (IDPs) are partially or entirely disordered. Their intrinsically disordered regions (IDRs) dynamically explore a wide range of structural space by their highly flexible nature. Due to this distinct feature largely different from structured proteins, conventional structural analyses relying on ensemble averaging is unsuitable for characterizing the dynamic structure of IDPs. Therefore, single-molecule measurement tools have been desired in IDP studies. High-speed atomic force microscopy (HS-AFM) is a unique tool that allows us to directly visualize single biomolecules at 2-3 nm lateral and âˆ¼ 0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution under near physiological conditions, without any chemical labeling. HS-AFM has been successfully used not only to characterize the shape and motion of IDP molecules but also to visualize their function-related dynamics. In this article, after reviewing the principle and current performances of HS-AFM, we describe experimental considerations in the HS-AFM imaging of IDPs and methods to quantify molecular features from captured images. Finally, we outline recent HS-AFM imaging studies of IDPs.

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS.

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na+ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.

An ultrafast piezoelectric Z-scanner with a resonance frequency above 1.1 MHz for high-speed atomic force microscopy.

The Z-scanner is the major component limiting the speed performance of all current high-speed atomic force microscopy systems. Here, we present an ultrafast piezoelectric Z-scanner with a resonance frequency above 1.1 MHz, achieving a record response time of ∼0.14 µs, approximately twice as fast as conventional piezoelectric-based Z-scanners. In the mechanical design, a small piezo-stack is supported at its bottom four vertices on a cone-like hollow, allowing the resonance frequency of the Z-scanner to remain as high as that of the piezo in free vibration. Its maximum displacement, ∼190 nm at 50 V, is large enough for imaging bio-molecules. For imaging bio-molecules in a buffer solution, the upper half of the Z-scanner is wrapped in a thin film resistant to water and chemicals, providing an excellent waterproof and mechanical durability without lowering the resonance frequency. We demonstrate that this Z-scanner can observe actin filaments, fragile biological polymers, for more than five times longer than the conventional Z-scanner at a tip velocity of 800 µm/s.

Visualization of intrinsically disordered proteins by high-speed atomic force microscopy.

High-speed atomic force microscopy (HS-AFM) is a powerful tool established 13 years ago. This methodology can capture individual protein molecules carrying out functional activities under near-physiological conditions, without chemical labeling, at 2-3 nm lateral and ∼0.1 nm vertical spatial resolution, and at sub-100 ms temporal resolution. Although most biological HS-AFM studies thus far target structured proteins, HS-AFM is also ideally suited to study the dynamics of intrinsically disordered proteins. Here we review some of the dynamic structures and processes of intrinsically disordered proteins that have been unveiled by HS-AFM imaging.

Conformational Tuning of Amylin by Charged Styrene-Maleic-Acid Copolymers.

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene-maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in ß-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin's C terminus (residues 31-37) that belongs to one of the two ß-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.

Nano-scale physical properties characteristic to metastatic intestinal cancer cells identified by high-speed scanning ion conductance microscope.

Recent genetic studies have indicated relationships between gene mutations and colon cancer phenotypes. However, how physical properties of tumor cells are changed by genetic alterations has not been elucidated. We examined genotype-defined mouse intestinal tumor-derived cells using a high-speed scanning ion conductance microscope (HS-SICM) that can obtain high-resolution live images of nano-scale topography and stiffness. The tumor cells used in this study carried mutations in Apc (A), Kras (K), Tgfbr2 (T), Trp53 (P), and Fbxw7 (F) in various combinations. Notably, high-metastatic cancer-derived cells carrying AKT mutations (AKT, AKTP, and AKTPF) showed specific ridge-like morphology with active membrane volume change, which was not found in low-metastatic and adenoma-derived cells. Furthermore, the membrane was significantly softer in the metastatic AKT-type cancer cells than other genotype cells. Importantly, a principal component analysis using RNAseq data showed similar distributions of expression profiles and physical properties, indicating a link between genetic alterations and physical properties. Finally, the malignant cell-specific physical properties were confirmed by an HS-SICM using human colon cancer-derived cells. These results indicate that the HS-SICM analysis is useful as a novel diagnostic strategy for predicting the metastatic ability of cancer cells.

Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2 receptor and small extracellular vesicles.

SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2 (hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S, and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain (RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2 complex appears as an all-RBD up conformation. The complex acquires a distinct topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2 subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking the viral entry scenario. Altogether, our study provides a platform that is suitable for real-time visualization of various entry inhibitors, neutralizing antibodies, and sEV-based decoy in blocking viral entry. Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction with receptor ACE2 and sEV-based decoy in real time using HS-AFM.