論文

• Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis.
  Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Yoshitsuna Itagaki, Yuki Suzuki, Shige H Yoshimura
  PLoS biology, 16, 5, e2004786, 2018年05月, ［国際誌］
  英語, 研究論文（学術雑誌）, 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.
• In vivo dynamics of the cortical actin network revealed by fast-scanning atomic force microscopy.
  Yanshu Zhang, Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Masahiro Kumeta, Shige H Yoshimura
  Microscopy (Oxford, England), 66, 4, 272, 282, 2017年08月01日, ［国際誌］
  英語, 研究論文（学術雑誌）, 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).
  Eiji Usukura, Akihiro Narita, Akira Yagi, Nobuaki Sakai, Yoshitsugu Uekusa, Yuka Imaoka, Shuichi Ito, Jiro Usukura
  Scientific reports, 7, 1, 6462, 6462, 2017年07月25日, ［国際誌］
  英語, 研究論文（学術雑誌）, 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.
• Probing in vivo dynamics of mitochondria and cortical actin networks using high-speed atomic force/fluorescence microscopy.
  Aiko Yoshida, Nobuaki Sakai, Yoshitsugu Uekusa, Katashi Deguchi, Jamie L Gilmore, Masahiro Kumeta, Shuichi Ito, Kunio Takeyasu
  Genes to cells : devoted to molecular & cellular mechanisms, 20, 2, 85, 94, 2015年02月, ［国際誌］
  英語, 研究論文（学術雑誌）, 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 1.7×10(4) to 1.4×10(5) 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.
• Real-time AFMs combined with inverted optical microscopes for wet cell/tissue imaging
  Shuichi Ito, Nobuaki Sakai, Akira Yagi, Yoshitsugu Uekusa, Koichi Karaki, Yuki Suzuki, Kunio Takeyasu
  Atomic Force Microscopy in Nanobiology, 177, 189, 2014年04月
  論文集(書籍)内論文
• High-speed atomic force microscopy combined with inverted optical microscopy for studying cellular events.
  Yuki Suzuki, Nobuaki Sakai, Aiko Yoshida, Yoshitsugu Uekusa, Akira Yagi, Yuka Imaoka, Shuichi Ito, Koichi Karaki, Kunio Takeyasu
  Scientific reports, 3, 2131, 2131, 2013年, ［国際誌］
  英語, 研究論文（学術雑誌）, 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.
• High-resolution imaging of plasmid DNA in liquids in dynamic mode atomic force microscopy using a carbon nanofiber tip
  Masashi Kitazawa, Shuichi Ito, Akira Yagi, Nobuaki Sakai, Yoshitugu Uekusa, Ryo Ohta, Kazuhisa Inaba, Akari Hayashi, Yasuhiko Hayashi, Masaki Tanemura
  Japanese Journal of Applied Physics, 50, 8 PART 4, 2011年08月
  研究論文（国際会議プロシーディングス）, 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) × 9μm (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 0.2fps (5s/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. © 2011 The Japan Society of Applied Physics.