title: Direct observation of the actin filament by tip-scan atomic force microscopy.
abstract: Actin filaments, the actin-myosin complex and the actin-tropomyosin complex were observed by a tip-scan atomic force microscope (AFM), which was recently developed by Olympus as the AFM part of a correlative microscope. This newly developed AFM uses cantilevers of similar size as stage-scan AFMs to improve substantially the spatial and temporal resolution. Such an approach has previously never been possible by a tip-scan system, in which a cantilever moves in the x, y and z directions. We evaluated the performance of this developed tip-scan AFM by observing the molecular structure of actin filaments and the actin-tropomyosin complex. In the image of the actin filament, the molecular interval of the actin subunits (∼5.5 nm) was clearly observed as stripes. From the shape of the stripes, the polarity of the actin filament was directly determined and the results were consistent with the polarity determined by myosin binding. In the image of the actin-tropomyosin complex, each tropomyosin molecule (∼2 nm in diameter) on the actin filament was directly observed without averaging images of different molecules. Each tropomyosin molecule on the actin filament has never been directly observed by AFM or electron microscopy. Thus, our developed tip-scan AFM offers significant potential in observing purified proteins and cellular structures at nanometer resolution. Current results represent an important step in the development of a new correlative microscope to observe nm-order structures at an acceptable frame rate (∼10 s/frame) by AFM at the position indicated by the fluorescent dye observed under a light microscope.
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan JST PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012, Japan narita.akihiro@f.mbox.nagoya-u.ac.jp.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Narita
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Usukura
authors.affiliation: R&D Group, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Yagi
authors.affiliation: R&D Group, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan.
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Tateyama
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Akizuki
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Mahito
authors.initials: M
authors.lastname: Kikumoto
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Tomoharu
authors.initials: T
authors.lastname: Matsumoto
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Maéda
authors.affiliation: R&D Group, Olympus Corporation, 2-3 Kuboyama-cho, Hachioji, Tokyo 192-8512, Japan.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Ito
authors.affiliation: Structural Biology Research Center, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Usukura
mesh_terms: D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D000818:Animals; D018625:Microscopy, Atomic Force / Q000379:methods*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*
chemical_list: D014335:Tropomyosin; D009218:Myosins
keywords: actin filament; atomic force microscopy; correlative microscopy; cytoskeleton; tip-scan AFM; tropomyosin