Processive movement of single 22S dynein molecules occurs only at low ATP concentrations

Abstract

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...

Does axonemal dynein push, pull, or oscillate?

“…Therefore, the tip displacement that is caused by the head rotation is very close to 8 nm. Suggestively, this is the same as the length of a tubulin dimer, and the measured step size for isolated dynein [Hirakawa et al, 2000]. Multiplying the tip displacement by the stiffness of the B-link gives a force of 1.1 pN in the Vi condition and 3.8 pN in the Apo condition.…”

“…Multiplying the tip displacement by the stiffness of the B-link gives a force of 1.1 pN in the Vi condition and 3.8 pN in the Apo condition. The force that can be generated in the Apo condition could account for most of the Ϸ 5 pN dynein force that has been reported by direct measurement [Shingyoji et al, 1998;Sakakibara et al, 1999;Hirakawa et al, 2000].…”

“…The elasticity of the B-link is sufficient to support this mechanism up to a limit of about 3.8 pN, at which point rotation of the B-link would produce zero displacement (the motor is stalled). Measurements of the dynein force by laser trap [Shingyoji et al, 1998;Sakakibara et al, 1999;Hirakawa et al, 2000] and estimates from whole flagella [Schmitz et al, 2000] suggest that the maximum force per dynein is approximately 5 pN per head. Therefore, most of the force could be generated by lateral displacement of the B-links.…”

Myosin Va and microtubule-based motors are required for fast axonal retrograde transport of tetanus toxin in motor neurons

“…Third, a kinesin inhibitor also affected transport, although less dramatically. Moreover, cytoplasmic dynein supports long-range intracellular movements of cargo in vivo but does not appear to be a processive motor (Hirakawa et al, 2000;King and Schroer, 2000). It could therefore require other MT and actin-based motors to achieve maximal speed and processivity.…”

Effects of external strain on the regulation of microtubule sliding induced by outer arm dynein of sea urchin sperm flagella

“…This suggests that an ensemble of outer arm dynein molecules can not only be switched between on/off or stop/go, but can modify collective sliding velocity in a reversible manner in response to a mechanical signal in the presence of a high concentration (1 mmol l −1 ) of ATP. It is not yet clear whether the curvature of microtubules is important, or if some kind of external strain is important for the induction of velocity change, but considering the previous reports that decrease in sliding velocity of a single axonemal and cytoplasmic dynein molecule can be induced by backward strain imposed by an optical trap (Hirakawa et al, 2000;Rai et al, 2013), backward strain resulting from the elasticity of bent microtubules may be important for induction of decrease in sliding velocity in the present study.…”