title: Semi-in situ atomic force microscopy imaging of intracellular neurofilaments under physiological conditions through the 'sandwich' method.
abstract: Neurofilaments are intermediate filament proteins specific for neurons and characterized by formation of biochemically stable, obligate heteropolymers in vivo While purified or reassembled neurofilaments have been subjected to morphological analyses by electron microscopy and atomic force microscopy, there has been a need for direct imaging of cytoplasmic genuine intermediate filaments with minimal risk of artefactualization. In this study, we applied the modified 'cells on glass sandwich' method to exteriorize intracellular neurofilaments, reducing the risk of causing artefacts through sample preparation. SW13vim(-) cells were double transduced with neurofilament medium polypeptide (NF-M) and alpha-internexin (α-inx). Cultured cells were covered with a cationized coverslip after prestabilization with tannic acid to form a sandwich and then split into two. After confirming that neurofilaments could be deposited on ventral plasma membranes exposed via unroofing, we performed atomic force microscopy imaging semi-in situ in aqueous solution. The observed thin filaments, considered to retain native structures of the neurofilaments, exhibited an approximate periodicity of 50-60 nm along their length. Their structural property appeared to reflect the morphology formed by their constituents, i.e. NF-M and α-inx. The success of semi-in situ atomic force microscopy of exposed bona fide assembled neurofilaments through separating the sandwich suggests that it can be an effective and alternative method for investigating cytoplasmic intermediate filaments under physiological conditions by atomic force microscopy.
authors.affiliation: Department of Neuroanatomy and Cellular Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Japan Society for the Promotion of Science (DC1), Kojimachi Business Center Building, 5-3-1 Kojimahchi, Chiyoda-ku, Tokyo 102-0083, Japan.
authors.forename: Fumiya
authors.initials: F
authors.lastname: Sato
authors.affiliation: Super-resolution AFM R&D Division, Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Asakawa
authors.affiliation: Super-resolution AFM R&D Division, Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan Division of Electrical Engineering, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa 920-1192, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fukuma
authors.affiliation: Department of Neuroanatomy and Cellular Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan terada.nana@tmd.ac.jp.
authors.forename: Sumio
authors.initials: S
authors.lastname: Terada
mesh_terms: D000818:Animals; D003599:Cytoskeleton / Q000648:ultrastructure*; D006801:Humans; D007381:Intermediate Filament Proteins / Q000737:chemistry; D007382:Intermediate Filaments / Q000648:ultrastructure*; D018625:Microscopy, Atomic Force / Q000379:methods*; D016900:Neurofilament Proteins / Q000737:chemistry; D009474:Neurons / Q000648:ultrastructure*
chemical_list: D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; C046643:alpha-internexin; C051774:neurofilament protein H
keywords: atomic force microscopy; cytoskeleton; intermediate filament; neurofilament; surface structure; unroofing