Lammers, L. G. | Search bioRxiv

bioRxiv2023

Conserved Roles for the Dynein Intermediate Chain and Ndel1 in Assembly and Activation of Dynein

Cytoplasmic dynein, the primary retrograde microtubule transport motor within cells, must be activated for processive motility through the regulated assembly of a dynein-dynactin-adapter (DDA) complex. The interaction between dynein and dynactin was initially ascribed to the N-terminus of the dynein intermediate chain (IC) and a coiled-coil of the dynactin subunit p150Glued. However, cryo-EM structures of DDA complexes have not resolve these regions of the IC and p150Glued, raising questions about the importance of this interaction. The IC N-terminus (ICN) also interacts with the dynein regulators Nde1/Ndel1, which compete with p150Glued for binding to ICN. Using a combination of approaches, we reveal that the ICN plays critical, evolutionarily conserved roles in DDA assembly by interacting with dynactin and Ndel1, the latter of which recruits the DDA assembly factor LIS1 to the dynein complex. In contrast to prior models, we find that LIS1 cannot simultaneously bind to Ndel1 and dynein, indicating that LIS1 must be handed off from Ndel1 to dynein in temporally discrete steps. Whereas exogenous Ndel1 or p150Glued disrupts DDA complex assembly in vitro, neither perturbs preassembled DDA complexes, indicating that the IC is stably bound to p150Glued within activated DDA complexes. Our study reveals previously unknown regulatory steps in the dynein activation pathway, and provides a more complete model for how the activities of LIS1/Ndel1 and dynactin/cargo-adapters are integrated to regulate dynein motor activity.

biochemistry↗

bioRxiv2022

Microtubule binding-induced allostery promotes LIS1 dissociation from dynein prior to cargo transport

The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (e.g., to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein-LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or -unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly bound to microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is an evolutionarily conserved phenomenon. Three cryo-EM structures of dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.

cell biology↗