Nobuaki Sakai

Fast-scanning atomic force microscopy reveals the ATP/ADP-dependent conformational changes of GroEL

In order to fold non‐native proteins, chaperonin GroEL undergoes numerous conformational changes and GroES binding in the ATP‐dependent reaction cycle. We constructed the real‐time three‐dimensional‐observation system at high resolution using a newly developed fast‐scanning atomic force microscope. Using this system, we visualized the GroES binding to and dissociation from individual GroEL with a lifetime of 6 s (k=0.17 s−1). We also caught ATP/ADP‐induced open–closed conformational changes of individual GroEL in the absence of qGroES and substrate proteins. Namely, the ATP/ADP‐bound GroEL can change its conformation ‘from closed to open’ without additional ATP hydrolysis. Furthermore, the lifetime of open conformation in the presence of ADP (∼1.0 s) was apparently lower than those of ATP and ATP‐analogs (2–3 s), meaning that ADP‐bound open‐form is structurally less stable than ATP‐bound open‐form. These results indicate that GroEL has at least two distinct open‐conformations in the presence of nucleotide; ATP‐bound prehydrolysis open‐form and ADP‐bound open‐form, and the ATP hydrolysis in open‐form destabilizes its open‐conformation and induces the ‘from open to closed’ conformational change of GroEL.

High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events

A hybrid atomic force microscopy (AFM)-optical fluorescence microscopy is a powerful tool for investigating cellular morphologies and events. However, the slow data acquisition rates of the conventional AFM unit of the hybrid system limit the visualization of structural changes during cellular events. Therefore, high-speed AFM units equipped with an optical/fluorescence detection device have been a long-standing wish. Here we describe the implementation of high-speed AFM coupled with an optical fluorescence microscope. This was accomplished by developing a tip-scanning system, instead of a sample-scanning system, which operates on an inverted optical microscope. This novel device enabled the acquisition of high-speed AFM images of morphological changes in individual cells. Using this instrument, we conducted structural studies of living HeLa and 3T3 fibroblast cell surfaces. The improved time resolution allowed us to image dynamic cellular events.

Probing in vivo dynamics of mitochondria and cortical actin networks using high-speed atomic force/fluorescence microscopy.

The dynamics of the cell membrane and submembrane structures are closely linked, facilitating various cellular activities. Although cell surface research and cortical actin studies have shown independent mechanisms for the cell membrane and the actin network, it has been difficult to obtain a comprehensive understanding of the dynamics of these structures in live cells. Here, we used a combined atomic force/optical microscope system to analyze membrane‐based cellular events at nanometer‐scale resolution in live cells. Imaging the COS‐7 cell surface showed detailed structural properties of membrane invagination events corresponding to endocytosis and exocytosis. In addition, the movement of mitochondria and the spatiotemporal dynamics of the cortical F‐actin network were directly visualized in vivo. Cortical actin microdomains with sizes ranging from 1.7 × 104 to 1.4 × 105 nm2 were dynamically rearranged by newly appearing actin filaments, which sometimes accompanied membrane invaginations, suggesting that these events are integrated with the dynamic regulation of submembrane organizations maintained by actin turnovers. These results provide novel insights into the structural aspects of the entire cell membrane machinery which can be visualized with high temporal and spatial resolution.

Fabrication and Optical Characterization of Five-Layer Asymmetric Coupled Quantum Well (FACQW)

The five-layer asymmetric coupled quantum well (FACQW) is a new potential-tailored quantum well (QW) that is promising for ultrafast and ultralow-voltage optical modulators and switches. We succeeded in fabricating GaAs/AlGaAs FACQW with monolayer accuracy by the molecular beam epitaxy (MBE) method by monitoring reflection high-energy electron diffraction (RHEED) specular beam intensity oscillation. Photoabsorption current measurements of the FACQW sample showed good agreement with theoretical results, and a potential for much lower voltage operation. In addition, we studied the growth sequences of GaAs/AlGaAs QWs in the migration-enhanced epitaxy (MEE) method in order to fabricate the FACQW with steeper and flatter heterointerfaces. The sequence of supplying materials for Al0.3Ga0.7As growth, on which there is no report, was modified and optimized, and the QWs of higher quality were obtained at a growth temperature of 490°C using the optimized sequence. The results of photoluminescence measurements show that the MEE method modified as mentioned above is a promising growth technique for the fabrication of FACQWs of higher quality.

High-Resolution Imaging of Plasmid DNA in Liquids in Dynamic Mode Atomic Force Microscopy Using a Carbon Nanofiber Tip

To understand the motion of DNA and DNA complexes, the real-time visualization of living DNA in liquids is quite important. Here, we report the high-resolution imaging of plasmid DNA in water using a rapid-scan atomic force microscopy (AFM) system equipped with a carbon nanofiber (CNF) probe. To achieve a rapid high-resolution scan, small SiN cantilevers with dimensions of 2 (width) × 0.1 (thickness) × 9 µm (length) and a bent end (tip view structure) were employed as base cantilevers onto which single CNFs were grown. The resonant frequencies of the cantilever were 1.5 MHz in air and 500 kHz in water, and the spring constant was calculated to be 0.1 N/m. Single CNFs, typically 88 nm in length, were formed on an array of the cantilevers in a batch process by the ion-irradiation method. An AFM image of a plasmid DNA taken in water at 0.2 fps (5 s/image) using a batch-fabricated CNF-tipped cantilever clearly showed the helix turns of the double strand DNA. The average helical pitch measured 3.4 nm (σ: 0.5 nm), which was in good agreement with that determined by the X-ray diffraction method, 3.4 nm. Thus, it is presumed that the combined use of the rapid-scan AFM system with the ion-induced CNF probe is promising for the dynamic analysis of biomolecules.

In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy

Together with lamellipodia and stress fibers, a dynamic network of actin filaments in the cell cortex plays a major role in the maintenance of cell morphology and motility. In contrast to lamellipodia, which have been well studied in various motile cells, the dynamics of actin filaments in the cell cortex have not yet been clarified due to a lack of proper imaging techniques. Here, we utilized high-speed atomic force microscopy for live-cell imaging and analyzed cortical actin dynamics in living cells. We successfully measured the polymerization rate and the frequency of filament synthesis in living COS-7 cells, and examined the associated effects of various inhibitors and actin-binding proteins. Actin filaments are synthesized beneath the plasma membrane and eventually descend into the cytoplasm. The inhibitors, cytochalasin B inhibited the polymerization, while jasplakinolide, inhibited the turnover of actin filaments as well as descension of the newly synthesized filaments, suggesting that actin polymerization near the membrane drives turnover of the cortical actin meshwork. We also determined how actin turnover is maintained and regulated by the free G-actin pool and G-actin binding proteins such as profilin and thymosin β4, and found that only a small amount of free G-actin was present in the cortex. Finally, we analyzed several different cell types, and found that the mesh size and the orientation of actin filaments were highly divergent, indicating the involvement of various actin-binding proteins in the maintenance and regulation of cortical actin architecture in each cell type.

A Cryosectioning Technique for the Observation of Intracellular Structures and Immunocytochemistry of Tissues in Atomic Force Microscopy (AFM)

The use of cryosectioning facilitates the morphological analysis and immunocytochemistry of cells in tissues in atomic force microscopy (AFM). The cantilever can access all parts of a tissue sample in cryosections after the embedding medium (sucrose) has been replaced with phosphate-buffered saline (PBS), and this approach has enabled the production of a type of high-resolution image. The images resembled those obtained from freeze-etching replica electron microscopy (EM) rather than from thin-section EM. The AFM images showed disks stacked and enveloped by the cell membrane in rod photoreceptor outer segments (ROS) at EM resolution. In addition, ciliary necklaces on the surface of connecting cilium, three-dimensional architecture of synaptic ribbons, and the surface of the post-synaptic membrane facing the active site were revealed, which were not apparent using thin-section EM. AFM could depict the molecular binding of anti-opsin antibodies conjugated to a secondary fluorescent antibody bound to the disk membrane. The specific localization of the anti-opsin binding sites was verified through correlation with immunofluorescence signals in AFM combined with confocal fluorescence microscope. To prove reproducibility in other tissues besides retina, cryosectioning-AFM was also applied to elucidate molecular organization of sarcomere in a rabbit psoas muscle.

Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis

Clathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here, we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy (HS-AFM) combined with a confocal laser scanning unit. The plasma membrane starts to invaginate approximately 30 s after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which, within 30 s, rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open–close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of CME.

Divergence of structural strategies for homophilic E-cadherin binding among bilaterians.

ABSTRACT Homophilic binding of E-cadherins through their ectodomains is fundamental to epithelial cell–cell adhesion. Despite this, E-cadherin ectodomains have evolved differently in the vertebrate and insect lineages. Of the five rod-like, tandemly aligned extracellular cadherin domains of vertebrate E-cadherin, the tip extracellular cadherin domain plays a pivotal role in binding interactions. Comparatively, the six consecutive N-terminal extracellular cadherin domains of Drosophila E-cadherin, DE-cadherin (also known as Shotgun), can mediate adhesion; however, the underlying mechanism is unknown. Here, we report atomic force microscopy imaging of DE-cadherin extracellular cadherin domains. We identified a tightly folded globular structure formed by the four N-terminal-most extracellular cadherin domains stabilized by the subsequent two extracellular cadherin domains. Analysis of hybrid cadherins from different insects indicated that the E-cadherin globular portion is associated with determining homophilic binding specificity. The second to fourth extracellular cadherin domains were identified as the minimal portion capable of mediating exclusive homophilic binding specificity. Our findings suggest that the N-terminal-most four extracellular cadherin domains of insect E-cadherin are functionally comparable with the N-terminal-most single extracellular cadherin domain of vertebrate E-cadherin, but that their mechanisms might significantly differ. This work illuminates the divergence of structural strategies for E-cadherin homophilic binding among bilaterians. Summary: Atomic force microscopy imaging revealed that Drosophila E-cadherin has a tightly folded globular structure with multiple domains that contain determinants of homophilic binding.

Rapid Bactericidal Action of Propolis against Porphyromonas gingivalis

Propolis, a resinous substance produced by bees, is used as a folk medicine for treatment of periodontal diseases. However, its mode of the action and the compounds responsible for its activities remain obscure. In the present study, we comprehensively investigated the antibacterial activities of ethanol-extracted propolis (EEP) and EEP-derived compounds toward Porphyromonas gingivalis, a keystone pathogen for periodontal diseases. Broth microdilution and agar dilution assays were used to determine the minimum inhibitory concentrations of EEP against a range of oral bacterial species, of which P. gingivalis showed a higher level of sensitivity than oral commensals such as streptococci. Its antibacterial activity toward P. gingivalis was maintained even after extensive heat treatment, demonstrating a high level of thermostability. EEP also induced death of P. gingivalis cells by increasing membrane permeability within 30 min. Spatiotemporal analysis based on high-speed atomic force microscopy revealed that EEP immediately triggered development of aberrant membrane blebs, followed by bleb fusion events on the bacterial surface. Furthermore, we isolated artepillin C, baccharin, and ursolic acid from EEP as antibacterial compounds against P. gingivalis. Of those, artepillin C and baccharin showed bacteriostatic activities with membrane blebbing, while ursolic acid showed bactericidal activity with membrane rupture. In particular, ursolic acid demonstrated a greater ability to affect bacterial membrane potential with increased membrane permeability, probably because of its highly lipophilic nature as compared with other compounds. Taken together, these findings provide mechanistic insight into the antibacterial activities of EEP and its exquisite membrane-targeting antibacterial compounds and imply the applicability of narrow-spectrum therapeutics with EEP for treatment of periodontitis. In addition, the advanced technology utilized in the present study to visualize the nanometer-scale dynamics of microorganisms will contribute to expanding our understanding of the activities of antimicrobials and the mechanism of drug resistance in bacteria.