A power stroke of dynein is thought to be responsible for the stepping of dimeric dynein. However, the actual size of the displacement driven by a power stroke has not been directly measured. Here, the displacements of single-headed cytoplasmic dynein were measured by optical tweezers. The mean displacement of dynein interacting with microtubule was ~8 nm at 100 µM ATP, and decreased sigmoidally with a decrease in the ATP concentration. The ATP dependence of the mean displacement was explained by a model that some dynein molecules bind to microtubule in pre-stroke conformation and generate 8-nm displacement, while others bind in the post-stroke one and detach without producing a power stroke. Biochemical assays showed that the binding affinity of the post-stroke dynein to a microtubule was ~5 times higher than that of pre-stroke dynein, and the dissociation rate was ~4 times lower. Taking account of these rates, we conclude that the displacement driven by a power stroke is 8.3 nm. A working model of dimeric dynein driven by the 8-nm power stroke was proposed.

RNA helicases are enzymes that generally unwind double-stranded RNA using ATP hydrolysis energy, mainly involved in RNA metabolism, transcription, translation, and mRNA splicing. While the helicase core is crucial for RNA unwinding activity, N- and C-terminal extensions of specific helicases may contain an intrinsically disordered region for electrostatic interaction, resulting in the formation of droplets in the cytoplasm. However, how the disordered region of the RNA helicase contributes to RNA unwinding and dissociation remains unclear. Here, we focused on Bombyx mori Vasa, which unwinds truncated target transposon RNAs from the piRNA-induced silencing complex piRISC. In this study, we used single-molecule techniques to visualise how Vasa dynamically interacts with piRISC and investigate how Vasa oligomerization is involved in the process of piRNA amplification, named the ping-pong pathway. We found that Vasa’s oligomerization is required during these processes in vitro and in vivo, and that Vasa triggers the dissociation of truncated RNA in heterogeneous pathways. Our single-molecule results suggest that oligomerized Vasa guides the timing of the process regulating overall dissociation efficiency.

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Cytoplasmic dynein-1 is a 1.4 MDa motor protein, which performs essential cargo transport to the minus end of microtubules. Long distance movement of single dynein-1 molecules is induced by binding to its 23-subunit cofactor dynactin and a cargo-adaptor protein. Here we present the cryo-EM structure of isolated human dynein-1 in its inhibited, phi-particle conformation. We achieved a resolution of 3.8Å for the motor domains and 8.4Å in the highly-flexible tail region. We reveal the architecture of the complete complex; identify all subunits, build helices in the tail and a full atomic model for the human dynein-1 motor domains. Mutagenesis to disrupt the interface between the two motor domains is not sufficient to activate dynein-1. Instead dynactin is required to orient the motors in a parallel fashion so that they can interact correctly with microtubules, thus explaining the structural mechanism of dynein-1 activation.