The Architecture of Outer Dynein Arms in Situ
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.
- SourceAvailable from: Gaia Pigino
- "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). "
ABSTRACT: Although eukaryotic flagella and cilia all share the basic 9+2 microtubule-organization of their internal axonemes, and are capable of generating bending-motion, the waveforms, amplitudes, and velocities of the bending-motions are quite diverse. To explore the structural basis of this functional diversity of flagella and cilia, we here compare the axonemal structure of three different organisms with widely divergent bending-motions by electron cryo-tomography. We reconstruct the 3D structure of the axoneme of Tetrahymena cilia, and compare it with the axoneme of the flagellum of sea urchin sperm, as well as with the axoneme of Chlamydomonas flagella, which we analyzed previously. This comparative structural analysis defines the diversity of molecular architectures in these organisms, and forms the basis for future correlation with their different bending-motions.Journal of Structural Biology 03/2012; 178(2):199-206. DOI:10.1016/j.jsb.2012.02.012 · 3.23 Impact Factor
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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). "
ABSTRACT: Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components.The Journal of Cell Biology 11/2011; 195(4):673-87. DOI:10.1083/jcb.201106125 · 9.69 Impact Factor