High-Speed Atomic Force Microscopic Observations On Demembranated Chlamydomonas Axonemes And Dynein Arms

In Chlamydomonas axoneme, nine doublet microtubules surround a pair of singlet microtubules. On each of doublet microtubules, protein motor dyneins are precisely aligned in two rows, outer- and inner-arm dyneins. The dynein arms are regulated in a coordinated fashion to produce periodic flagellar beating. The coordination among the dynein arms under strict spatiotemporal regulation makes flagella beat in an organized manner. To understand the coordination mechanism, we have performed video imaging of demembranated flagellar axonemes and dynein arms under the aqueous environment using the high-speed atomic force microscope. By using this technique, unstained, unfixed axonemes in their natural physiological surroundings was imaged. Axonemes of the wild-type and the outer-dynein arm-less mutant oda1 were demembranated and attached on poly-L-lysine coated mica surface. To visualize the axonemes, we used a tapping-mode high speed-AFM. AFM images clearly highlighted many details of axonemes and difference between wild-type and oda1 axonemes. In the wild-type axoneme topography, the doublet microtubules were recognized but the furrows between the doublets were not clearly seen because of the presence of bulky blobs between the doublets. In contrast, the oda1-axoneme topography showed clearly the furrows and individual doublets. The difference between two topography was probably derived from the presence of outer dynein arms in wild-type axonemes. Phase imaging of wild-type axonemes revealed the detail of the bulky blobs since it provides information about surface stiffness variations. The structural repeat of 24nm was confirmed in the phase images. The high-speed AFM images thus defined a detailed map of axonemal components under the aqueous environment and will provide the dynamics of these components. This work was supported by Grant-in-Aids for Scientific Research (C), JSPS, 26440089 and 17K07376 to K.O., and the Takeda Science Foundation (K.O.).