SummaryCytoplasmic dynein-1 binds dynactin and cargo adaptor proteins to form a transport machine capable of long-distance processive movement along microtubules. However, it is unclear why dynein-1 moves poorly on its own or how it is activated by dynactin. Here, we present a cryoelectron microscopy structure of the complete 1.4-megadalton human dynein-1 complex in an inhibited state known as the phi-particle. We reveal the 3D structure of the cargo binding dynein tail and show how self-dimerization of the motor domains locks them in a conformation with low microtubule affinity. Disrupting motor dimerization with structure-based mutagenesis drives dynein-1 into an open form with higher affinity for both microtubules and dynactin. We find the open form is also inhibited for movement and that dynactin relieves this by reorienting the motor domains to interact correctly with microtubules. Our model explains how dynactin binding to the dynein-1 tail directly stimulates its motor activity.

Anterograde intraflagellar transport (IFT) trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFT-A and IFT-B proteins that link structural and signaling cargos to microtubule motors for import into cilia. It remains unknown how the IFT-A/-B proteins are arranged into complexes and how these complexes polymerize into functional trains. Here we use in situ cryo-electron tomography of Chlamydomonas reinhardtii cilia and AlphaFold2 protein structure predictions to generate a molecular model of the entire anterograde train. We show how the conformations of both IFT-A and IFT-B are dependent on lateral interactions with neighboring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. Following three-dimensional classification, we reveal how IFT-B extends two flexible tethers to maintain a connection with IFT-A that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that govern cilia assembly.

Dynactin is a 1.1 MDa complex that activates the molecular motor dynein for ultra‐processive transport along microtubules. In order to do this, it forms a tripartite complex with dynein and a coiled‐coil adaptor. Dynactin consists of an actin‐related filament whose length is defined by its flexible shoulder domain. Despite previous cryo‐EM structures, the molecular architecture of the shoulder and pointed end of the filament is still poorly understood due to the lack of high‐resolution information in these regions. Here we combine multiple cryo‐EM datasets and define precise masking strategies for particle signal subtraction and 3D classification. This overcomes domain flexibility and results in high‐resolution maps into which we can build the shoulder and pointed end. The unique architecture of the shoulder securely houses the p150 subunit and positions the four identical p50 subunits in different conformations to bind dynactin’s filament. The pointed end map allows us to build the first structure of p62 and reveals the molecular basis for cargo adaptor binding to different sites at the pointed end.

The ability of cytoskeletal motors to move unidirectionally along filamentous tracks is central to their role in cargo transport, motility and cell division. While kinesin and myosin motor families have members that move in opposite directions 1 , 2 , all dyneins studied to date exclusively move towards the microtubule (MT) minus-end 3 . In order to understand the mechanism of dynein’s directionality, we sought to engineer a plus-end-directed dynein guided by cryo-electron microscopy and molecular dynamics simulations. As shown by single-molecule assays, elongation or shortening of the coiled-coil stalk that connects the motor to the MT controls helical directionality of S. cerevisiae dynein around MTs. By changing the length and angle of the stalk, we successfully reversed the motility towards the MT plus-end. These modifications act by altering the direction dynein’s linker swings relative to the MT, not by reversing the asymmetric unbinding of the motor from MT. Because the length and angle of dynein’s stalk are fully conserved among species, our findings provide an explanation for why all dyneins move towards the MT minus-end.