title: Impact-Free Measurement of Microtubule Rotations on Kinesin and Cytoplasmic-Dynein Coated Surfaces.
abstract: Knowledge about the three-dimensional stepping of motor proteins on the surface of microtubules (MTs) as well as the torsional components in their power strokes can be inferred from longitudinal MT rotations in gliding motility assays. In previous studies, optical detection of these rotations relied on the tracking of rather large optical probes present on the outer MT surface. However, these probes may act as obstacles for motor stepping and may prevent the unhindered rotation of the gliding MTs. To overcome these limitations, we devised a novel, impact-free method to detect MT rotations based on fluorescent speckles within the MT structure in combination with fluorescence-interference contrast microscopy. We (i) confirmed the rotational pitches of MTs gliding on surfaces coated by kinesin-1 and kinesin-8 motors, (ii) demonstrated the superiority of our method over previous approaches on kinesin-8 coated surfaces at low ATP concentration, and (iii) identified MT rotations driven by mammalian cytoplasmic dynein, indicating that during collective motion cytoplasmic dynein side-steps with a bias in one direction. Our novel method is easy to implement on any state-of-the-art fluorescence microscope and allows for high-throughput experiments.
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany.
authors.forename: Aniruddha
authors.initials: A
authors.lastname: Mitra
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: Ruhnow
authors.affiliation: Max Plank Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Bert
authors.initials: B
authors.lastname: Nitzsche
authors.affiliation: B CUBE-Center for Molecular Bioengineering, Technische Universität Dresden, Dresden, Germany; Max Plank Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Diez
mesh_terms: D000818:Animals; D056445:Cytoplasmic Dyneins / Q000737:chemistry / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000378:metabolism; D016547:Kinesins / Q000737:chemistry / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism*; D012399:Rotation; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine
chemical_list: D029721:Drosophila Proteins; D029701:Saccharomyces cerevisiae Proteins; D056445:Cytoplasmic Dyneins; D016547:Kinesins