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An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.

Publisher Summary Tetrahymena is easy to grow and as ciliary dynein is easy to isolate it is an excellent source of large quantities of dynein for biochemical and in vitro motility studies. This chapter summarizes the methods employed for the isolation and characterization of Tetrahymena ciliary outer arm dynein. This dynein is isolated as a 22S particle composed of three different heavy chains, called α , β , and γ . The V1 photolysis reaction cleaves each heavy chain once, producing six distinct photolysis products. The specific activity of the 22S dynein is approximately 0.9 micromoles phosphate released per minute per milligram of protein. The axonemal dyneins are the molecular motors that drive the active sliding between adjacent outer doublet microtubules. Localized constraints on the active sliding give rise to the propagated bending of cilia and eukaryotic flagella. Under the appropriate conditions, the photolysis of the dynein heavy chain is specific and thus diagnostic for dynein. It is a convenient method used for generating peptide maps of each heavy chain and a useful way to evaluate whether an isolated dynein retains its MgATPase activity.