Single Protofilament is Enough to Support Unidirectional Walking of Cytoplasmic Dynein

Cytoplasmic dynein moves on microtubules toward its minus end with 8nm stepping. Dynein motor domain has a ring structure which consists of six AAA modules, and the diameter of the ring is 14 nm. This size is so large as compared with the 8 nm step size, and then question arises whether dynein can walk on a single protofirament of microtubule or dynein uses multiple protofilaments. To address this point, we examined dynein motility using zinc-induced tubulin sheet (Zn-sheet) which has an arrangement of adjacent protofilaments with anti-parallel and opposite orientations. From the structural analysis, the dynein binding sites are revealed to be exposed only at either edge of a Zn-sheet. Previously, we have shown that the Zn-sheet move on the glass surface coated with the dynein or kinesin molecules. Unlike microtubules, Zn-sheets followed the winding path and the tracks are often circular. In this study, we aimed to observe the processive movement of single dynein molecules on the edge of a Zn-sheet by TIRF microscopy. As a result of deliberate preparations, we have successfully observed that single dynein molecules walk on Zn-sheets. The velocity of the movement on Zn-sheets was almost the same as that on microtubules. These results demonstrate that the single protofilament is enough to support dynein motility, and suggest that dynein uses only one protofilament of microtubules as well as one protofilament at the edge of Zn-sheets. Considering the large size of the dynein head, it is hard for the two heads of the dynein molecules to move on a single protofilament by the hand-over-hand mechanism advocated for the two heads of kinesin, and it is necessary to investigate the coordination of the two head of dynein.