NATURE STRUCTURAL & MOLECULAR BIOLOGY VOLUME 17 NUMBER 6 JUNE 2010 761 ARTICLES Eukaryotic flagella and motile cilia make a bending motion to drive cells or to generate extracellular flow by hydrolyzing ATP. Most of the flagella and motile cilia share a common cytoskeletal structure called an axoneme, in which nine microtubule doublets surround two singlet microtubules (central pair) (‘9 + 2’). Inner and outer dynein arms (IDAs and ODAs) are located between two adjacent doublets (A- and B-microtubules, Fig. 1a) with the periodicities of 96 nm and 24 nm (refs. 1–3), respectively, and they generate the sliding motion of the doublets (Fig. 1a). The mechanisms of force-generation by dynein motor proteins as well as how the sliding motions of dyneins are inte- grated into this highly orchestrated bending motion are still unclear. From studies on dynein-arm mutants of green algae Chlamydomonas reinhardtii, it is thought that the IDAs determine the waveform, whereas the ODAs amplify the bending motion 4 . An ODA consists of two or three heavy chains (α, β and γ in C. reinhardtii, Fig. 1b) and a number of intermediate and light chains (IC/LC), whereas the IDA contains a total of eight heavy chains (six monomeric chains (a, b, c, d, e and g) and one dimeric chain (f), Fig. 1b) as well as IC/LC (the three-dimensional architecture 5 is shown in Fig. 1b). Defects in human dyneins can cause diseases such as primary ciliary dyskinesia 6 . Each single heavy chain (4,500 residues) has an N-terminal tail (1,500 residues), six ATPase associated with diverse cellular activities (AAA) domains forming the ring 7 and a coiled-coil stalk (extending from the AAA ring, between the fourth and fifth AAA domains). The part connecting the tail and the AAA ring is called the linker (Fig. 1c). Because dynein heavy chains are ATP-driven molecular motors, their structures at various nucleotide-bound states are essential to explain the mechanism of flagellar/ciliary bending. Kinetics studies indicating that release of hydrolysis products is coupled with the power stroke of dyneins 8 suggest that the structures with and without ADP.Pi (phosphate) can be considered as pre– and post–power stroke states, respectively. Electron microscopy and two-dimensional (2D) image analysis of purified and negatively stained inner-arm dynein c showed a structural change in one dynein heavy chain induced by nucleotides in vitro. The change in the orientation of the linker due to the binding of ADP.vanadate (ADP.Vi), which should mimic the ADP.Pi state, induces a shift of the position where the N-terminal tail emerges (the neck) 9 . Electron tomography of flagella has revealed the three-dimensional (3D) conformation of ODAs and IDAs in situ in the absence of nucleotides 5,10–12 , but for an understanding of the molecular mechanisms of the sliding and bending motions, the nucleotide- induced structural changes of dynein arm complexes in situ still need to be elucidated. In this report, we reveal 3D conformational changes in the IDAs and ODAs of C. reinhardtii flagella in situ using a combination of electron cryotomography, single-particle image averaging and clas- sification in the presence and absence of nucleotides. We characterize the structural change of ODAs and IDAs induced by product release. Our image-classification results indicate that at least two different conformations of ODAs coexist in the presence of a saturating amount of nucleotides in situ and that these two conformations occur in clus- ters along microtubule doublets. We interpret our data in terms of a model for microtubule sliding and flagellar bending induced by ATP hydrolysis of ODAs and IDAs. RESULTS To understand the mechanism of ATP-driven force generation during the flagellar bending motion, we reconstructed 3D structures of flagella 1 Department of Biology, ETH Zurich, Zurich, Switzerland. 2 Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Iwaoka, Kobe, Japan. 3 Graduate School of Life Science, University of Hyogo, Harima Science Park City, Hyogo, Japan. 4 These authors contributed equally to this work. Correspondence should be addressed to T.I. (takashi.ishikawa@mol.biol.ethz.ch). Received 26 April 2009; accepted 24 March 2010; published online 9 May 2010; doi:10.1038/nsmb.1832 Nucleotide-induced global conformational changes of flagellar dynein arms revealed by in situ analysis Tandis Movassagh 1,4 , Khanh Huy Bui 1,4 , Hitoshi Sakakibara 2 , Kazuhiro Oiwa 2,3 & Takashi Ishikawa 1 Outer and inner dynein arms generate force for the flagellar/ciliary bending motion. Although nucleotide-induced structural change of dynein heavy chains (the ATP-driven motor) was proven in vitro, our lack of knowledge in situ has precluded an understanding of the bending mechanism. Here we reveal nucleotide-induced global structural changes of the outer and inner dynein arms of Chlamydomonas reinhardtii flagella in situ using electron cryotomography. The ATPase domains of the dynein heavy chains move toward the distal end, and the N-terminal tail bends sharply during product release. This motion could drive the adjacent microtubule to cause a sliding motion. In contrast to in vitro results, in the presence of nucleotides, outer dynein arms coexist as clusters of apo or nucleotide-bound forms in situ. This implies a cooperative switching, which may be related to the mechanism of bending. © 2010 Nature America, Inc. All rights reserved.