The Architecture of Outer Dynein Arms in Situ

Department of Biology, ETH Zürich (Swiss Federal Institute of Technology, Zurich), HPK F7 ETH Hönggerberg, CH8093 Zürich, Switzerland.
Journal of Molecular Biology (Impact Factor: 4.33). 05/2007; 368(5):1249-58. DOI: 10.1016/j.jmb.2007.02.072
Source: PubMed

ABSTRACT Outer dynein arms, the force generators for axonemal motion, form arrays on microtubule doublets in situ, although they are bouquet-like complexes with separated heads of multiple heavy chains when isolated in vitro. To understand how the three heavy chains are folded in the array, we reconstructed the detailed 3D structure of outer dynein arms of Chlamydomonas flagella in situ by electron cryo-tomography and single-particle averaging. The outer dynein arm binds to the A-microtubule through three interfaces on two adjacent protofilaments, two of which probably represent the docking complex. The three AAA rings of heavy chains, seen as stacked plates, are connected in a striking manner on microtubule doublets. The tail of the alpha-heavy chain, identified by analyzing the oda11 mutant, which lacks alpha-heavy chain, extends from the AAA ring tilted toward the tip of the axoneme and towards the inside of the axoneme at 50 degrees , suggesting a three-dimensional power stroke. The neighboring outer dynein arms are connected through two filamentous structures: one at the exterior of the axoneme and the other through the alpha-tail. Although the beta-tail seems to merge with the alpha-tail at the internal side of the axoneme, the gamma-tail is likely to extend at the exterior of the axoneme and join the AAA ring. This suggests that the fold and function of gamma-heavy chain are different from those of alpha and beta-chains.

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    • "This is true especially for analyses performed at room temperature, either after chemical fixation (see, e.g., Mayorovits et al., 2008; Acehan et al., 2009; Liu et al., 2006) or freeze substitution (see, e.g., Noske et al., 2008; Marsh et al., 2007; Marsh, 2005; Richter et al., 2008), as well as for the small number of high-tech laboratories able to perform tomography of frozen hydrated samples (see, e.g., Beck et al., 2007; Morris and Jensen, 2008; He et al., 2009; Izard et al., 2008). Together with the use of standard ET, i.e., the collection of the tilt series and the 3D reconstruction of the tomogram , is also progressively increasing the number of ultra-structural studies in which a post-processing of the tomograms is performed, consisting of 3D alignment and averaging of equivalent structures contained in the map (see, e.g., Nicastro et al., 2006; Ishikawa et al., 2007; Liu et al., 2008; Bui et al., 2008). "
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    • "Surface rendering was done by Chimera (Pettersen et al., 2004). Segmentation of components (ODA, IDA and RS) was carried out based on our analysis of mutants which lack these components (Ishikawa et al., 2007; Bui et al., 2008; Pigino et al., 2011). DRC was located based on Heuser et al., 2009, except one part which we proved to belong to dynein, not DRC (in Section 3). "
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    • "The grids with frozen-hydrated samples were transferred to a cryoholder (626; Gatan) cooled by liquid nitrogen. Images were collected as described previously (Ishikawa et al., 2007; Bui et al., 2008, 2009; Movassagh et al., 2010) using a transmission electron microscope (Tecnai F20; FEI) equipped with a field emission gun, an energy filter (GIF Tridiem; Gatan), and a 2,048 × 2,048 charge-coupled device camera (UltraScan 1000; Gatan) at the accelerating voltage of 200 kV, a magnification of 19,303×, and an under focus of 3–5 µm. Tomographic image series from 60 to 60°, with 2° tilt increments, were acquired using Explore3D software (FEI). "
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