Yuta Watanabe

Recombinant human cytoplasmic dynein heavy chain 1 and 2: Observation of dynein-2 motor activity in vitro

DYNC2H1 (Homo sapiens) binds to DYNC2H1 (Homo sapiens) by electron microscopy (View interaction)

A Single Protofilament Is Sufficient to Support Unidirectional Walking of Dynein and Kinesin

Cytoplasmic dynein and kinesin are two-headed microtubule motor proteins that move in opposite directions on microtubules. It is known that kinesin steps by a ‘hand-over-hand’ mechanism, but it is unclear by which mechanism dynein steps. Because dynein has a completely different structure from that of kinesin and its head is massive, it is suspected that dynein uses multiple protofilaments of microtubules for walking. One way to test this is to ask whether dynein can step along a single protofilament. Here, we examined dynein and kinesin motility on zinc-induced tubulin sheets (zinc-sheets) which have only one protofilament available as a track for motor proteins. Single molecules of both dynein and kinesin moved at similar velocities on zinc-sheets compared to microtubules, clearly demonstrating that dynein and kinesin can walk on a single protofilament and multiple rows of parallel protofilaments are not essential for their motility. Considering the size and the motile properties of dynein, we suggest that dynein may step by an inchworm mechanism rather than a hand-over-hand mechanism.

Simple Method of Synthesizing Nickel?Nitrilotriacetic Acid Gold Nanoparticles with a Narrow Size Distribution for Protein Labeling

We developed a simple method to synthesize nickel–nitrilotriacetic acid gold nanoparticles (Ni–NTA Au NPs) with a narrow size distribution for site-specific labeling in protein complexes. Au NPs were synthesized by the reduction of HAuCl4 using trisodium citrate and tannin acid. Then, the nanoparticle surfaces were modified with NTA and subsequent complexation with Ni2+. The mean diameter of the synthesized Ni–NTA Au NPs was 4.3 nm, and the coefficient of variation was 9%. The specific binding of the Ni–NTA Au NPs to polyhistidine-tagged (His-tagged) proteins was determined by transmission electron microscopy using kinesin and the p62 subunit of dynactin. Consequently, our method is useful for analyzing the substructures of protein complexes.

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.

Structural Analysis of Cytoplasmic Dynein Tail Domain

Cytoplasmic dynein is microtubule-based motor protein utilizing the energy from ATP hydrolysis and plays fundamental roles in various cellular activities including mitosis, vesicle transport and cell migration. Cytoplasmic dynein is a huge protein complex consisting of the head domain and the tail domain. The head domain consists of six AAA modules and a stalk. So far, many studies have investigated about the head domain, because it contains ATP hydrolysis and motor activities. On the other hand, the tail domain consists of N-terminal third of heavy chains (HC), intermediate chains (IC), light intermediate chains (LIC), and light chains (LC). Although the tail domain is implicated in binding of cargos and some of dynein binding proteins which control motor activities of dynein, for example dynactin, the detailed molecular architecture of the tail domain remains to be unveiled. In this study, we observed cytoplasmic dynein molecules with or without polyhistidine tag (His-tag) by transmission electron microscopy (TEM), performed single particle image analysis and obtained the averaged image of the tail domain. Using nickel nitrilotriacetic acid (Ni-NTA) conjugated gold nanoparticles to label His-tag of recombinant proteins, we identified the positions of N-terminus and C-terminus of IC and determined its orientation in the tail domain. Furthermore, we identified the position of C-terminus of LIC relative to IC. Based on our observations, we propose a new model of the architecture of the dynein tail domain which differs from currently advocating model.