We have analyzed the movement of single 22S dynein molecules from Tetrahymena cilia by using a nanometer measuring system equipped with optical tweezers. Statistical analysis proved that a single molecule of 22S dynein can move processively and develop force at low concentrations of ATP (<20 M). The maximum force was Ϸ4.7 pN, and the force-velocity curve was convex down. During force development, dynein molecules showed stepwise displacement of Ϸ8 nm and frequently exhibited backward steps of Ϸ8 nm. At higher concentrations of ATP (м20 M) single molecules of 22S dynein were not observed to move processively. Twenty-two S dynein seems to switch over from a processive mode to a nonprocessive mode, sensing a subtle change of ATP concentrations. These observations indicate that the processivity, maximum force, and step size of dynein are similar to those of kinesin, but the ATP concentration-dependence, force-velocity relationship, and backward steps are clearly distinct from kinesin. Dynein is the first microtubule motor protein to be discovered and functions as a molecular engine for ciliary and flagellar movement (1). Dynein constitutes the outer and inner arms of axonemes, and multiple forms (monomer, dimer, and trimer) of dyneins have been isolated from axonemes, even in a single species. More than 20 years after the first discovery of axonemal dynein, cytoplasmic dynein was identified (2) and found to be involved in transport of organelles and vesicles (3) and in spindle assembly (4, 5) and chromosome segregation (6, 7). Both axonemal and cytoplasmic dynein consist of multiple subunits referred to as heavy chains (Ͼ500 kDa), intermediate chains (60-150 kDa), and light chains (Ͻ50 kDa). Each dynein heavy chain comprises a globular motor domain that hydrolyzes ATP and interacts with microtubules.Molecular genetics revealed that there are more than 10 different dynein heavy chain genes in eukaryotes that produce ciliated or flagellated cells (8,9). From the amino acid sequence similarity, dyneins are classified into three groups: namely, axonemal outer arms, axonemal inner arms, and cytoplasmic dynein (10). Each dynein heavy chain contains four conserved ATP binding sites, P1-P4. The P1 site is thought to be an ATP catalytic site, but the functions of the P2-P4 sites are unknown. Because of its large size and complexity, the structure and function of the dyneins are not well characterized yet, compared with myosin and the kinesins.Among the dyneins, Tetrahymena ciliary 22S dynein is relatively well characterized. 22S dynein is derived from the outer arms of ciliary axonemes. The molecular weight of 22S dynein is about two million, and it comprises three different heavy chains and has a three-headed ''flower bouquet '' structure (11, 12). The kinetics of ATP hydrolysis by 22S dynein is thought to be similar to that of actomyosin (13). In microtubule gliding assays, 22S dynein translocates microtubules at Ϸ8 m͞s directed toward the minus end of microtubules (14).Recently, very high resolution systems have been deve...
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