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: Masahide Kikkawa[show abstract] [hide abstract]
ABSTRACT: Dyneins and kinesins move in opposite directions on microtubules. The question of how the same-track microtubules are able to support movement in two directions remains unanswered due to the absence of details on dynein-microtubule interactions. To address this issue, we studied dynein-microtubule interactions using the tip of the microtubule-binding stalk, the dynein stalk head (DSH), which directly interacts with microtubules upon receiving conformational change from the ATPase domain. Biochemical and cryo-electron microscopic studies revealed that DSH bound to tubulin dimers with a periodicity of 80 A, corresponding to the step size of dyneins. The DSH molecule was observed as a globular corn grain-like shape that bound the same region as kinesin. Biochemical crosslinking experiments and image analyses of the DSH-kinesin head-microtubule complex revealed competition between DSH and the kinesin head for microtubule binding. Our results demonstrate that dynein and kinesin share an overlapping microtubule-binding site, and imply that binding at this site has an essential role for these motor proteins.The EMBO Journal 08/2004; 23(13):2459-67. · 9.82 Impact Factor
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ABSTRACT: Extraction of doublet microtubules from the sperm flagella of the sea urchin Strongylocentrotus purpuratus with sarkosyl (0.5%)-urea (2.5 M) yields a highly pure preparation of "tektin" filaments that we have previously shown to resemble intermediate filament proteins. They form filaments 2-3 nm in diameter as seen by negative stain electron microscopy and are composed of approximately equal amounts of three polypeptide bands with apparent molecular weights of 47,000, 51,000, and 55,000, as determined by SDS PAGE. We prepared antibodies to this set of proteins to localize them in the doublet microtubules of S. purpuratus and other species. Tektins and tubulin were antigenically distinct when tested by immunoblotting with affinity-purified antitektin and antitubulin antibodies. Fixed sperm or axonemes from several different species of sea urchin showed immunofluorescent staining with antitektin antibodies. We also used antibodies coupled to gold spheres to localize the proteins by electron microscopy. Whereas a monoclonal antitubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol. 93:576-582) decorates intact microtubules along their lengths, antitektins labeled only the ends of intact microtubules and sarkosyl-insoluble ribbons. However, if microtubules and ribbons attached to electron microscope grids were first extracted with sarkosyl-urea, the tektin filaments that remain were decorated by antitektin antibodies throughout their length. These results suggest that tektins form integral filaments of flagellar microtubule walls, whose antigenic sites are normally masked, perhaps by the presence of tubulin around them.The Journal of Cell Biology 02/1985; 100(1):126-35. · 10.82 Impact Factor
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ABSTRACT: Software for the processing of electron micrographs in structural biology suffers from incompatibility between different packages, poor definition and choice of conventions, and a lack of coherence in software development. The solution lies in adopting a common philosophy of interaction and conventions between the packages. To understand the choices required to have such common interfaces, I am developing a package called "Bsoft." Its foundations lie in the variety of different image file formats used in electron microscopy-a continually frustrating experience to the user and programmer alike. In Bsoft, this problem is greatly diminished by support for many different formats (including MRC, SPIDER, IMAGIC, SUPRIM, and PIF) and by separating algorithmic issues from image format-specific issues. In addition, I implemented a generalized functionality for reading the tag-base STAR (self-defining text archiving and retrieval) parameter file format as a mechanism to exchanging parameters between different packages. Bsoft is written in highly portable code (tested on several Unix systems and under VMS) and offers a continually growing range of image processing functionality, such as Fourier transformation, cross-correlation, and interpolation. Finally, prerequisites for software collaboration are explored, which include agreements on information exchange and conventions, and tests to evaluate compatibility between packages.Journal of Structural Biology 02/2001; 133(2-3):156-69. · 3.36 Impact Factor