High-Speed Atomic Force Microscopy Reveals the Nucleosome Sliding and DNA Unwrapping/Wrapping Dynamics of Tail-less Nucleosomes.

Each nucleosome contains four types of histone proteins, each with a histone tail. These tails are essential for the epigenetic regulation of gene expression through post-translational modifications (PTMs). However, their influence on nucleosome dynamics at the single-molecule level remains undetermined. Here, we employed high-speed atomic force microscopy to visualize nucleosome dynamics in the absence of the N-terminal tail of each histone or all of the N-terminal tails. Loss of all tails stripped 6.7 base pairs of the nucleosome from the histone core, and the DNA entry-exit angle expanded by 18° from that of wild-type nucleosomes. Tail-less nucleosomes, particularly those without H2B and H3 tails, showed a 10-fold increase in dynamics, such as nucleosome sliding and DNA unwrapping/wrapping, within 0.3 s, emphasizing their role in histone-DNA interactions. Our findings illustrate that N-terminal histone tails stabilize the nucleosome structure, suggesting that histone tail PTMs modulate nucleosome dynamics.

Organ-Specific Gene Expression Control Using DNA Origami-Based Nanodevices.

Nanodevices that function in specific organs or cells are one of the ultimate goals of synthetic biology. The recent progress in DNA nanotechnology such as DNA origami has allowed us to construct nanodevices to deliver a payload (e.g., drug) to the tumor. However, delivery to specific organs remains difficult due to the fragility of the DNA nanostructure and the low targeting capability of the DNA nanostructure. Here, we constructed tough DNA origami that allowed us to encapsulate the DNA origami into lipid-based nanoparticles (LNPs) under harsh conditions (low pH), harnessing organ-specific delivery of the gene of interest (GOI). We found that DNA origami-encapsulated LNPs can increase the functionality of payload GOIs (mRNA and siRNA) inside mouse organs through the contribution from different LNP structures revealed by cryogenic electron microscope (Cryo-EM). These data should be the basis for future organ-specific gene expression control using DNA origami nanodevices.

Kinetochore-microtubule attachment in human cells is regulated by the interaction of a conserved motif of Ska1 with EB1.

The kinetochore establishes the linkage between chromosomes and the spindle microtubule plus ends during mitosis. In vertebrates, the spindle-kinetochore-associated (Ska1,2,3) complex stabilizes kinetochore attachment with the microtubule plus ends, but how Ska is recruited to and stabilized at the kinetochore-microtubule interface is not understood. Here, our results show that interaction of Ska1 with the general microtubule plus end-associated protein EB1 through a conserved motif regulates Ska recruitment to kinetochores in human cells. Ska1 forms a stable complex with EB1 via interaction with the motif in its N-terminal disordered loop region. Disruption of this interaction either by deleting or mutating the motif disrupts Ska complex recruitment to kinetochores and induces chromosome alignment defects, but it does not affect Ska complex assembly. Atomic-force microscopy imaging revealed that Ska1 is anchored to the C-terminal region of the EB1 dimer through its loop and thereby promotes formation of extended structures. Furthermore, our NMR data showed that the Ska1 motif binds to the residues in EB1 that are the binding sites of other plus end targeting proteins that are recruited to microtubules by EB1 through a similar conserved motif. Collectively, our results demonstrate that EB1-mediated Ska1 recruitment onto the microtubule serves as a general mechanism for the formation of vertebrate kinetochore-microtubule attachments and metaphase chromosome alignment.

Structural Dynamics of Amyloid-ß Protofibrils and Actions of Anti-Amyloid-ß Antibodies as Observed by High-Speed Atomic Force Microscopy.

Amyloid-ß (Aß) aggregation intermediates, including oligomers and protofibrils (PFs), have attracted attention as neurotoxic aggregates in Alzheimer's disease. However, due to the complexity of the aggregation pathway, the structural dynamics of aggregation intermediates and how drugs act on them have not been clarified. Here we used high-speed atomic force microscopy to observe the structural dynamics of Aß42 PF at the single-molecule level and the effect of lecanemab, an anti-Aß PF antibody with the positive results from Phase 3 Clarity AD. PF was found to be a curved nodal structure with stable binding angle between individual nodes. PF was also a dynamic structure that associates with other PF molecules and undergoes intramolecular cleavage. Lecanemab remained stable in binding to PFs and to globular oligomers, inhibiting the formation of large aggregates. These results provide direct evidence for a mechanism by which antibody drugs interfere with the Aß aggregation process.

High-Speed Atomic Force Microscopy Reveals Spontaneous Nucleosome Sliding of H2A.Z at the Subsecond Time Scale.

Nucleosome dynamics, such as nucleosome sliding and DNA unwrapping, are important for gene regulation in eukaryotic chromatin. H2A.Z, a variant of histone H2A that is highly evolutionarily conserved, participates in gene regulation by forming unstable multipositioned nucleosomes in vivo and in vitro. However, the subsecond dynamics of this unstable nucleosome have not been directly visualized under physiological conditions. Here, we used high-speed atomic force microscopy (HS-AFM) to directly visualize the subsecond dynamics of human H2A.Z.1-nucleosomes. HS-AFM videos show nucleosome sliding along 4 nm of DNA within 0.3 s in any direction. This sliding was also visualized in an H2A.Z.1 mutant, in which the C-terminal half was replaced by the corresponding canonical H2A amino acids, indicating that the interaction between the N-terminal region of H2A.Z.1 and the DNA is responsible for nucleosome sliding. These results may reveal the relationship between nucleosome dynamics and gene regulation by histone H2A.Z.

Atomic-Level Viscosity Distribution in the Hydration Layer.

The viscosity of solvation structures is crucial for the development of energy-efficient biofunctional and electrochemical devices. Elucidating their subnanoscale distributions can cause the formation of a sustainable energy society. Here, we visualize the site-specific three-dimensional damping distribution on a CaCO_{3} surface composed of binary ion species using ultra-low-noise frequency modulation atomic force microscopy. With the support from molecular dynamics simulation, we found a strikingly large damping at the calcium sites, which demonstrates the capability of this methodology to visualize atomic-scale viscosity in the hydration layers. Our finding will expedite the evolutions of various functional devices.

Atomic-Scale 3D Local Hydration Structures Influenced by Water-Restricting Dimensions.

Hydration structures at solid-liquid interfaces mediate between the atomic-level surface structures and macroscopic functionalities in various physical, chemical, and biological processes. Atomic-scale local hydration measurements have been enabled by ultralow noise three-dimensional (3D) frequency-modulation atomic force microscopy. However, for their application to complicated surface structures, e.g., biomolecular devices, understanding the relationship between the hydration and surface structures is necessary. Herein, we present a systematic study based on the concept of the structural dimensionality, which is crucial in various scientific fields. We performed 3D measurements and molecular dynamics simulations with silicate surfaces that allow for 0, 1, and 2 degrees of freedom to water molecules. Consequently, we found that the 3D hydration structures reflect the structural dimensions and the hydration contrasts decrease with increasing dimension due to the enlarged water self-diffusion coefficient and increased embedded hydration layers. Our results provide guidelines for the analysis of complicated hydration structures, which will be exploited in extensive fields.

Combined Experimental and Simulation Study of Amplitude Modulation Atomic Force Microscopy Measurements of Self-Assembled Monolayers in Water.

Atomic force microscopy (AFM) can be used to measure surface properties at the nanoscale. However, interpretation of measurements from amplitude modulation AFM (AM-AFM) in liquid is not straightforward due to the interactions between the AFM tip, the surface being imaged, and the water. In this work, amplitude-distance measurements and molecular dynamics simulations of AM-AFM were employed to study the effect of surface chemistry on the amplitude of tip oscillation in water. The sample surfaces consisted of self-assembled monolayers where the hydrophilicity or hydrophobicity was determined by the terminal group of the alkanethiols. Analysis showed that surface chemical composition influences the hydration structure near the interface which affects the forces experienced by the tip and in turn changes the amplitude profile. This observation could aid our understanding of AM-AFM measurements of interfacial phenomena on various surfaces in water.

Interface structure between tetraglyme and graphite.

Clarification of the details of the interface structure between liquids and solids is crucial for understanding the fundamental processes of physical functions. Herein, we investigate the structure of the interface between tetraglyme and graphite and propose a model for the interface structure based on the observation of frequency-modulation atomic force microscopy in liquids. The ordering and distorted adsorption of tetraglyme on graphite were observed. It is found that tetraglyme stably adsorbs on graphite. Density functional theory calculations supported the adsorption structure. In the liquid phase, there is a layered structure of the molecular distribution with an average distance of 0.60 nm between layers.

Molecular-scale quantitative charge density measurement of biological molecule by frequency modulation atomic force microscopy in aqueous solutions.

Surface charge distributions on biological molecules in aqueous solutions are essential for the interactions between biomolecules, such as DNA condensation, antibody-antigen interactions, and enzyme reactions. There has been a significant demand for a molecular-scale charge density measurement technique for better understanding such interactions. In this paper, we present the local electric double layer (EDL) force measurements on DNA molecules in aqueous solutions using frequency modulation atomic force microscopy (FM-AFM) with a three-dimensional force mapping technique. The EDL forces measured in a 100 mM KCl solution well agreed with the theoretical EDL forces calculated using reasonable parameters, suggesting that FM-AFM can be used for molecular-scale quantitative charge density measurements on biological molecules especially in a highly concentrated electrolyte.

Spatiotemporal resolution in high-speed atomic force microscopy for studying biological macromolecules in action.

High-speed atomic force microscopy (HS-AFM) is a unique approach that allows direct real-time visualization of biological macromolecules in action under near-physiological conditions, without any chemical labeling. Typically, the temporal resolution is sub-100 ms, and the spatial resolution is 2-3 nm in the lateral direction and ∼0.1 nm in the vertical direction. A wide range of biomolecular systems and their dynamic processes have been studied by HS-AFM, providing deep mechanistic insights into how biomolecules function. However, the level of mechanistic detail gleaned from an HS-AFM experiment critically depends on the spatiotemporal resolution of the system. In this review article, we explain the principle of HS-AFM and describe how the resolution is determined. We also discuss recent attempts to improve the resolution of HS-AFM to further extend the observable range of biological phenomena.

Removing the parachuting artifact using two-way scanning data in high-speed atomic force microscopy.

The high-speed atomic force microscopy (HS-AFM) is a unique and prominent method to observe structural dynamics of biomolecules at single molecule level at near-physiological condition. To achieve high temporal resolution, the probe tip scans the stage at high speed which can cause the so-called parachuting artifact in the HS-AFM images. Here, we develop a computational method to detect and remove the parachuting artifact in HS-AFM images using the two-way scanning data. To merge the two-way scanning images, we employed a method to infer the piezo hysteresis effect and to align the forward- and backward-scanning images. We then tested our method for HS-AFM videos of actin filaments, molecular chaperone, and duplex DNA. Together, our method can remove the parachuting artifact from the raw HS-AFM video containing two-way scanning data and make the processed video free from the parachuting artifact. The method is general and fast so that it can easily be applied to any HS-AFM videos with two-way scanning data.

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.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

Imaging single CaMKII holoenzymes at work by high-speed atomic force microscopy.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in synaptic plasticity. It is a dodecameric serine/threonine kinase that has been highly conserved across metazoans for over a million years. Despite the extensive knowledge of the mechanisms underlying CaMKII activation, its behavior at the molecular level has remained unobserved. In this study, we used high-speed atomic force microscopy to visualize the activity-dependent structural dynamics of rat/hydra/C. elegans CaMKII with nanometer resolution. Our imaging results revealed that the dynamic behavior is dependent on CaM binding and subsequent pT286 phosphorylation. Among the species studies, only rat CaMKIIα with pT286/pT305/pT306 exhibited kinase domain oligomerization. Furthermore, we revealed that the sensitivity of CaMKII to PP2A in the three species differs, with rat, C. elegans, and hydra being less dephosphorylated in that order. The evolutionarily acquired features of mammalian CaMKIIα-specific structural arrangement and phosphatase tolerance may differentiate neuronal function between mammals and other species.

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.

Nanomechanics of self-assembled surfactants revealed by frequency-modulation atomic force microscopy.

Surfactants play a critical role in bottom-up nanotechnologies due to their peculiar nature of controlling the interfacial energy. Since their operational mechanism originates from the molecular-scale formation and disruption processes of molecular assemblies (i.e., micelles), conventional static-mode atomic force microscopy has made a significant contribution to unravel the detailed molecular pictures. Recently, we have successfully developed a local solvation measurement technique based on three-dimensional frequency-modulation atomic force microscopy, whose spatial resolution is not limited by jump-to-contact. Here, using this novel technique, we investigate molecular nanomechanics in the formation and disruption processes of micelles formed on a hydrophobic surface. Furthermore, an experiment employing a hetero-nanostructure reveals that the nanomechanics depends on the form of the molecular assembly. Namely, the hemifusion and disruption processes are peculiar to the micellar surface and cause a higher energy dissipation than the monolayer surface. The technique established in this study will be used as a generic technology for further development of bottom-up nanotechnologies.

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.

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3.

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific single-stranded DNA (ssDNA) cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target double-stranded DNA (dsDNA) substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

An ultra-wide scanner for large-area high-speed atomic force microscopy with megapixel resolution.

High-speed atomic force microscopy (HS-AFM) is a powerful tool for visualizing the dynamics of individual biomolecules. However, in single-molecule HS-AFM imaging applications, x,y-scanner ranges are typically restricted to a few hundred nanometers, preventing overview observation of larger molecular assemblies, such as 2-dimensional protein crystal growth or fibrillar aggregation. Previous advances in scanner design using mechanical amplification of the piezo-driven x,y-positioning system have extended the size of HS-AFM image frames to several tens of micrometer, but these large scanners may suffer from mechanical instabilities at high scan speeds and only record images with limited pixel numbers and comparatively low lateral resolutions (> 20-100 nm/pixel), complicating single-molecule analysis. Thus, AFM systems able to image large sample areas at high speeds and with nanometer resolution have still been missing. Here, we describe a HS-AFM sample-scanner system able to record large topographic images (≤ 36 × 36 µm2) containing up to 16 megapixels, providing molecular resolution throughout the image frame. Despite its large size, the flexure-based scanner features a high resonance frequency (> 2 kHz) and delivers stable operation even at high scans speeds of up to 7.2 mm/s, minimizing the time required for recording megapixel scans. We furthermore demonstrate that operating this high-speed scanner in time-lapse mode can simultaneously identify areas of spontaneous 2-dimensional Annexin A5 crystal growth, resolve the angular orientation of large crystalline domains, and even detect rare crystal lattice defects, all without changing scan frame size or resolution. Dynamic processes first identified from overview scans can then be further imaged at increased frame rates in reduced scan areas after switching to conventional HS-AFM scanning. The added ability to collect large-area, high-resolution images of complex samples within biological-relevant time frames extends the capabilities of HS-AFM from single-molecule imaging to the study of large dynamic molecular arrays. Moreover, large-area HS-AFM scanning can generate detailed structural data sets from a single scan, aiding the quantitative analysis of structurally heterogenous samples, including cellular surfaces.