Time-resolved Cryo-EM visualizes snapshots of dynein's activation pathway.

Regulation of cytoplasmic dynein-1 (dynein) is critical for diverse functions of eukaryotic cells, including cell division and long-range intracellular transport. Both dynein and Lis1, an essential dynein regulator, are mutated in patients with neurodevelopmental diseases and are conserved from fungi to mammals. Dynein activity is controlled by an autoinhibited state called “Phi”, in which its two motor domains interact in a way that prevents motility. Lis1 is important for promoting the formation of active dynein complexes and it has been proposed to do so by stabilizing a dynein conformation that is not autoinhibited. Recently, we solved a structure of dynein bound to Lis1 in which two Lis1 dimers are inserted between two dynein motor domains; this “Chi” conformation appears to capture an early step in the dynein activation pathway. What other steps are involved in the full activation of dynein remains unknown. Here, we use time-resolved Cryo-EM to visualize as many of these steps as possible and determine the structural changes underlying dynein activation by Lis1. We do so by purposely introducing heterogeneity by adding ATP during our sample preparation, which allows dynein to go through its mechanochemical cycle. Using this approach, we capture eight distinct dynein and dynein-Lis1 structures from the same sample. We also show that the presence of these different conformations changes at different time points emphasizing the significance of the time-resolved Cryo-EM approach. Based on the novel dynein-Lis1 structures that we capture using this approach we propose a new model for how Lis1 relieves dynein autoinhibition and promotes conformations that are compatible with motility.