Dynein project

Axonemal dyneins in Chlamydomonas flagella

Flagella and cilia share a “9+2” structure, in which two central singlet microtubules are surrounded by nine outer doublet microtubules. Outer and inner dynein arms slide on each outer doublet microtubule, hydrolyzing ATP (Fig. 1a). Dynein arms consist of one to three dynein heavy chains and many light chains (Fig. 1b).

Fig. 1 Flagella of Chlamydomonas. (a) The horizontal section shows a “9+2” structure. Dynein arms slide on microtubules hydrolyzing ATP to generate bending motion of flagella. (b) One outer microtubule and bound dynein arms are enlarged from (a) and shown as a side view. Dynein arms, consisting of one to three dynein heavy chains, and many light chains are highly complicated and heterogeneous, but their structure is approachable by electron tomography.

Fig. 2 Cryo-electron micrograph of Chlamydomonas flagellum.

We will study axonemal dyneins in Chlamydomonas flagella, addressing mainly two questions. One is the architecture of the whole flagellum. This information is necessary to reveal the mechanism of converting linear sliding movements of dynein molecules on microtubules into the bending motion of flagella. Recently, the research of Chlamydomonas flagella has been substantially increased by the discovery of the intraflagellar machinery that transports elements to the right location in flagella. Three-dimensional information of the whole flagella will also give an insight into the mechanism of flagellar assembly/disassembly.

The second question is on the structure of the dynein molecule itself. Dynein belongs to AAA protein superfamily and has a much more complicated structure than other motor proteins such as myosin and kinesin. The dynein heavy chain has six different AAA domains as well as a stalk (~15 nm, microtubule binding) and a stem (cargo binding) in a single polypeptide chain. It is pointed out that only the first and the forth AAA domains in dynein can bind ATP, and only the first one can hydrolyze it. Thus, the function of each AAA domain in dynein must be investigated, especially in complex with a microtubule.

For addressing both questions, we will utilize electron tomography to reconstruct the whole structure of Chlamydomonas flagella (Fig.2). By using electron tomography, our first aim is mapping locations of dynein arms on microtubules, and determining the angles of individual dynein heavy chains at each step of bending motion. The information about the locations and angles will help us understand how outer doublet microtubules slide from each other to generate the bending motion. When we arrive at medium resolution (~25 Å), such subdomains as the six AAA domains, the stem and the stalk will be distinguishable so that we can estimate how the dynein molecule generates force on the microtubule. If higher resolution (~15 Å) becomes available by image classification and averaging, more detailed structures of these subdomains and their changes accompanied by the ATP hydrolysis cycle will be revealed. Various dynein mutants that cause malfunctions on bending motion have been reported.