The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia

Eukaryotic cells use cytoskeletal motor proteins to transport many different intracellular cargos. Numerous kinesins and myosins have evolved to cope with the various transport needs that have arisen during eukaryotic evolution. Surprisingly, a single cytoplasmic dynein (a minus end-directed microtubule motor) carries out similarly diverse transport activities as the many different types of kinesin. How is dynein coupled to its wide range of cargos and how is it spatially and temporally regulated? The answer could lie in the several multifunctional adaptors, including dynactin, lissencephaly 1, nuclear distribution protein E (NUDE) and NUDE-like, Bicaudal D, Rod-ZW10-Zwilch and Spindly, that regulate dynein function and localization.

Vesicles, organelles and other intracellular cargo are transported by kinesin and dynein motors, which move in opposite directions along microtubules. This bidirectional cargo movement is frequently described as a 'tug of war' between oppositely directed molecular motors attached to the same cargo. However, although many experimental and modelling studies support the tug-of-war paradigm, numerous knockout and inhibition studies in various systems have found that inhibiting one motor leads to diminished motility in both directions, which is a 'paradox of co-dependence' that challenges the paradigm. In an effort to resolve this paradox, three classes of bidirectional transport models--microtubule tethering, mechanical activation and steric disinhibition--are proposed, and a general mathematical modelling framework for bidirectional cargo transport is put forward to guide future experiments.

Summary Cytoplasmic 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.