Conserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein

Abstract

Processive transport by the microtubule motor cytoplasmic dynein requires the regulated assembly of a dynein-dynactin-adapter complex. Interactions between dynein and dynactin were initially ascribed to the dynein intermediate chain N-terminus and the dynactin subunit p150Glued. However, recent cryo-EM structures have not resolved this interaction, questioning its importance. The intermediate chain also interacts with Nde1/Ndel1, which compete with p150Glued for binding. We reveal that the intermediate chain N-terminus is a critical evolutionarily conserved hub that interacts with dynactin and Ndel1, the latter of which recruits LIS1 to drive complex assembly. In additon to revealing that the intermediate chain N-terminus is likely bound to p150Glued in active transport complexes, our data support a model whereby Ndel1-LIS1 must dissociate prior to LIS1 being handed off to dynein in temporally discrete steps. Our work reveals previously unknown steps in the dynein activation pathway, and provide insight into the integrated activities of LIS1/Ndel1 and dynactin/cargo-adapters.

Molecular mechanism of dynein-dynactin complex assembly by LIS1

“…Only the edges of the sites are partially blocked by the ICD and CC1A domains. This accessibility explains many pull-down studies showing an interaction between DIC-N and p150 ( 32 – 37 , 39 ). Curiously, other studies using gel filtration ( 5 , 6 ), sucrose gradients ( 40 ), or single-molecule assays ( 4 ) reported limited interaction of dynein and dynactin unless an activating adaptor is present.…”

“…The interaction of DIC-N with p150 appears similar to its interaction with another dynein regulator, Ndel1 ( 39 ). This explains the proposed competition between Ndel1 and p150 for dynein binding ( 37 , 39 , 82 , 83 ) and suggests that Ndel1 will need to be displaced before DIC-N can initiate DDA assembly ( 39 , 82 , 83 ).…”

“…This may reflect the tendency of these methods to detect higher-affinity interactions than pull-downs, suggesting that although dynein and dynactin can interact through DIC-N:p150, the formation of DDA further stabilizes this interaction. It was previously not clear why DDA complex formation requires DIC-N binding to CC1B ( 39 ). Here, we observe that LIS1 requires the presence of DIC-N to bind p150 (fig.…”

“…The dynein-dynactin machinery contains two long flexible regions, the p150 arm and the DIC N termini, both of which are important for complex assembly and function ( 32 , 33 , 39 , 60 – 62 ) (Fig. 4A).…”

“…Dynein’s DHC tails bind the filament grooves ( 6 ), and its DIC N terminus (DIC-N) interacts with p150 ( 32 – 37 ). The DIC-N:p150 interaction is critical for dynein function in cells ( 38 ) and formation of DDA complexes in vitro ( 39 ). However, as several studies suggest that dynein and dynactin interact only weakly in the absence of an adaptor ( 4 – 6 , 40 ), it is unclear when DIC-N binds p150 during DDA complex formation and why the interaction is critical for dynein’s activity.…”