Nano-dissection of inner-arm dynein-c by high-speed AFM

Daisuke Yamamoto1, Atsushi Miyagi1, Kazuhiro Oiwa2, Toshio Ando1, and Hitoshi Sakakibara2

1) Department of Physics, Kanazawa University, Kanazawa, Japan 2) Kobe Advanced ICT Research Center, National Institute of Information and Communications Technology, Kobe, Japan

The high-speed AFM is a new imaging tool which allows for dynamic visualization of bio-molecules. We applied it to inner-arm dynein-c, as a "nano-dissection tool". When we obseve a dynein-c molecule with high-speed AFM for more than 10 sec, structures of dynein-c are often destroyed by numerous probing. Using high-speed AFM, we can quickly observe the destruction process and thereby examine the architectures of domains of dynein-c. Previous EM studies revealed that the dynein-c tail and the head are connected with a flexible linker and the linker binds to the head. However, it is still unknown which side of the head ring the linker binds to. AFM images of dynein-c placed on a mica surface were observed mostly as "left view"; the head faces the mica surface and the stalk protrudes from the head to the northwest direction while the tail protrudes towards to the South. During imaging of left view of dynein-c, the head was occasionally peeled off from the mica surface while the tail remained on the surface. After that, a hook-shaped structure with a low height appeared at a place where the head had lain. One end of the hook was connected to the proximal tail end while the other end was connected to the peeled head. The hook is, therefore, the linker itself. On the right view orientated head, a ridge was observed lying and seemed to continuously link to the proximal tail end. Thus, we conclude that the linker lies on the right-half upper side of the head surface supposing that we look at dynein in the right view. During imaging of dynein-c in the left view, we observed an additional change in the structure. On the head surface of the left view, a ridge, which was more prominent than that of the right view, was observed. The ridge was detached from the head and the detached fragment was connected to the head through a thin strand. Even after the detachment, the shape of remaining head and the tail seemed unchanged. The ridge on the upper side of the dynein-c head in the left viewis was most likely the C-terminal segment of dynein-c.

e-mail address of presenting author: sakaki@nict.go.jp