Structural analysis of dynein arms from Chlamydomonas flagella revealed by electron cryo-tomography

Takashi Ishikawa1, Khanh Huy Bui1, Tandis Movassagh1, Hitoshi Sakakibara2, Kazuhiro Oiwa2

1Department of Biology, ETH Zurich, Switzerland 2Kobe Advanced ICT Research Center, NICT, Japan

Inner and outer dynein arms are the force generators for axonemal motion. In flagella of Chlamydomonas, outer dynein arms form arrays with 24 nm periodicity on microtubule doublets in situ, although they are isolated in vitro as bouquet-like complexes with separated heads of multiple heavy-chains. Inner dynein arms follow 96 nm repeat together with radial spokes. Both dynein arms contain homologous, but not identical dynein heavy chains (each has ~4500 amino acids and is composed of a AAA-ring, N-terminal tail and a coiled-coil stalk) as well as many light and intermediate chains. We are analyzing the detailed 3D-structure of dynein arms of Chlamydomonas flagella in situ using the techniques of electron cryo-tomography and single particle averaging. Previously we proved that the three AAA-rings of heavy chains from the outer dynein arm, similar to three stacked plates, are connected in a striking manner on microtubule doublets (Ishikawa et al., (2007) J. Mol. Biol. 368, 1249). 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. We are now visualizing the interface between outer dynein arms and microtubule doublets, especially docking complexes. The docking complex, which is indispensable for the ATP-insensitive binding of outer dynein arms on the microtubule, arrays in elongated manner, parallel to a protofilament of the microtubule.

e-mail address of presenting author: takashi.ishikawa@mol.biol.ethz.ch

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