title: SHG-specificity of cellular Rootletin filaments enables naïve imaging with universal conservation.
abstract: Despite growing demand for truly naïve imaging, label-free observation of cilium-related structure remains challenging, and validation of the pertinent molecules is correspondingly difficult. In this study, in retinas and cultured cells, we distinctively visualized Rootletin filaments in rootlets in the second harmonic generation (SHG) channel, integrated in custom coherent nonlinear optical microscopy (CNOM) with a simple, compact, and ultra-broadband supercontinuum light source. This SHG signal was primarily detected on rootlets of connecting cilia in the retinal photoreceptor and was validated by colocalization with anti-Rootletin staining. Transfection of cells with Rootletin fragments revealed that the SHG signal can be ascribed to filaments assembled from the R234 domain, but not to cross-striations assembled from the R123 domain. Consistent with this, Rootletin-depleted cells lacked SHG signal expected as centrosome linker. As a proof of concept, we confirmed that similar fibrous SHG was observed even in unicellular ciliates. These findings have potential for broad applications in clinical diagnosis and biophysical experiments with various organisms.
authors.affiliation: Department of Applied Physics, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan.
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Akiyama
authors.affiliation: Division of Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya, Aichi 464-8681, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Inoko
authors.affiliation: Department of Ophthalmology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Kaji
authors.affiliation: Ultrastructural Research Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.|Department of Cell Biology, Tokushima University Graduate School of Medical Science, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
authors.forename: Shigenobu
authors.initials: S
authors.lastname: Yonemura
authors.affiliation: Ultrastructural Research Team, RIKEN Center for Life Science Technologies, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
authors.forename: Kisa
authors.initials: K
authors.lastname: Kakiguchi
authors.affiliation: Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Segawa
authors.affiliation: Department of Applied Physics, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan.
authors.forename: Kei
authors.initials: K
authors.lastname: Ishitsuka
authors.affiliation: Faculty of Life &Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572 Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Yoshida
authors.affiliation: Faculty of Life &Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572 Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Numata
authors.affiliation: Institut de Recherche XLIM, UMR CNRS No. 7252, 123 avenue Albert Thomas, 87060 Limoges CEDEX, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Leproux
authors.affiliation: Institut de Recherche XLIM, UMR CNRS No. 7252, 123 avenue Albert Thomas, 87060 Limoges CEDEX, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Couderc
authors.affiliation: Department of Ophthalmology, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Oshika
authors.affiliation: Department of Applied Physics, Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan.|Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan.|Tsukuba Research Center for Interdisciplinary Materials Science (TIMS), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8571, Japan.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Kano
mesh_terms: D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D002923:Cilia; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006801:Humans; D051381:Rats; D012160:Retina / Q000378:metabolism / Q000648:ultrastructure*; D000073798:Second Harmonic Generation Microscopy / Q000379:methods*
chemical_list: C000628797:Crocc protein, rat; D003598:Cytoskeletal Proteins