title: Resolving bundled microtubules using anti-tubulin nanobodies.
abstract: Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20-40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50-70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.
authors.affiliation: 1] Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands [2] RG Neuroplasticity, Leibniz Institute for Neurobiology, Brenneckestr. 6, Magdeburg 39118, Germany.
authors.forename: Marina
authors.initials: M
authors.lastname: Mikhaylova
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Bas M C
authors.initials: BM
authors.lastname: Cloin
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors.forename: Kieran
authors.initials: K
authors.lastname: Finan
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Robert
authors.identifier: 0000000348063599
authors.initials: R
authors.lastname: van den Berg
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Jalmar
authors.identifier: 000000021637888X
authors.initials: J
authors.lastname: Teeuw
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Marta M
authors.initials: MM
authors.lastname: Kijanka
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Mikolaj
authors.initials: M
authors.lastname: Sokolowski
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Eugene A
authors.initials: EA
authors.lastname: Katrukha
authors.affiliation: Department of Neuro- and Sensory Physiology, University of Göttingen, Humboldtallee 23, Göttingen 37073, Germany.
authors.forename: Manuel
authors.initials: M
authors.lastname: Maidorn
authors.affiliation: Department of Neuro- and Sensory Physiology, University of Göttingen, Humboldtallee 23, Göttingen 37073, Germany.
authors.forename: Felipe
authors.initials: F
authors.lastname: Opazo
authors.affiliation: 1] Institut Curie, Research Center, 26, rue d'Ulm, Paris, F-75248, France [2] CNRS UMR144, 26, rue d'Ulm, Paris, France [3] Translational Research Department, Institut Curie, 26, rue d'Ulm, Paris F-75248, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Moutel
authors.affiliation: Inserm, U836, F-38000, Grenoble, France. Univ. Grenoble Alpes, Grenoble Institut des Neurosciences, Grenoble F-38000, France.
authors.forename: Marylin
authors.initials: M
authors.lastname: Vantard
authors.affiliation: 1] Institut Curie, Research Center, 26, rue d'Ulm, Paris, F-75248, France [2] CNRS UMR144, 26, rue d'Ulm, Paris, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Perez
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen en Henegouwen
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Casper C
authors.initials: CC
authors.lastname: Hoogenraad
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, UK.
authors.forename: Helge
authors.identifier: 0000000339484332
authors.initials: H
authors.lastname: Ewers
authors.affiliation: Division of Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, Utrecht 3584 CH, The Netherlands.
authors.forename: Lukas C
authors.initials: LC
authors.lastname: Kapitein
mesh_terms: D000818:Animals; D000906:Antibodies; D002460:Cell Line; D003198:Computer Simulation; D006801:Humans; D008853:Microscopy / Q000379:methods*; D008870:Microtubules / Q000648:ultrastructure*; D061905:Single-Domain Antibodies
chemical_list: D000906:Antibodies; D061905:Single-Domain Antibodies