IN-VITRO MOTILITY FROM RECOMBINANT DYNEIN HEAVY-CHAIN

The dyneins are a class of motor protein involved in ciliary and flage llar motility, organelle transport, and chromosome segregation, Becaus e of their large size and subunit complexity, relatively little is kno wn about their mechanisms of force production and regulation. We repor t here on the expression and analysis of the entire rat cytoplasmic dy nein heavy chain (M(r) 532,000). Full-length tDNAs were constructed fr om a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His(6)-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection, The recombinant protein was mostly s oluble and showed good microtubule binding. It exhibited a broad sedim entation profile, indicative of the formation of dimers as well as hig her order multimers. Good microtubule gliding motility activity was ob served in assays of heavy chain expressed in either insect or COS-7 ce lls, Average microtubule gliding velocities of 1.2-1.8 mu m/sec were o bserved, comparable with the rates determined for calf brain cytoplasm ic dynein. These results represent the first indication that recombina nt heavy chain alone is capable of force production, and should lead t o rapid progress In defining the dynein motor domain.