Abstract
Cytoplasmic dynein is an ATP-driven motor that transports intracellular cargos along microtubules. Dynein adopts an inactive conformation when not attached to a cargo, and motility is activated when dynein assembles with dynactin and a cargo adaptor. It was unclear how active dynein-dynactin complexes step along microtubules and transport cargos under tension. Using single-molecule imaging, we showed that dynein-dynactin advances by taking 8 to 32-nm steps toward the microtubule minus end with frequent sideways and backward steps. Multiple dyneins collectively bear a large amount of tension because the backward stepping rate of dynein is insensitive to load. Recruitment of two dyneins to dynactin increases the force generation and the likelihood of winning against kinesin in a tug-of-war but does not directly affect velocity. Instead, velocity is determined by cargo adaptors and tail-tail interactions between two closely packed dyneins. Our results show that cargo adaptors modulate dynein motility and force generation for a wide range of cellular functions.