Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules (MTs). It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 promotes the formation of an active complex with dynactin. Lis1 also favors the recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. We propose that Lis1 binding releases dynein from its auto-inhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargoes in cells.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minusends of microtubules (MTs). It remains unclear whether Lis1 serves as an inhibitor or an activator of mammalian dynein motility. Here we use single-molecule imaging and optical trapping to show that Lis1 does not directly alter the stepping and force production of individual dynein motors assembled with dynactin and a cargo adaptor. Instead, Lis1 binding releases dynein from its auto-inhibited state and thereby promotes the formation of an active complex with dynactin. Lis1 also favors recruitment of two dyneins to dynactin, resulting in increased velocity, higher force production and more effective competition against kinesin in a tug-of-war. Lis1 dissociates from motile complexes, indicating that its primary role is to orchestrate the assembly of the transport machinery. These results provide a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargoes in cells.Cytoplasmic dynein (dynein hereafter) is an AAA+ motor responsible for nearly all motility and force generation towards the microtubule minus-end 1-3 . Dynein is involved in a wide variety of cellular functions, such as positioning of intracellular organelles, breakdown of the nuclear envelope and assembly of the mitotic spindle during mitosis 4-6 . The partial loss of dynein function has been implicated in a range of neurogenerative and neurodevelopmental conditions, including spinal muscular atrophy, amyotrophic lateral sclerosis, Alzheimer's disease and schizophrenia 7-9 .The dynein complex (1.4 MDa) is a homodimer of two heavy chains, which recruit smaller associated polypeptides 10 . The C-terminal motor domain of the heavy chain is a catalytic ring of six AAA modules (AAA1-6). ATPase activity at AAA1 powers dynein motility 11, 12 , while AAA3 regulates MT attachment and force generation cycles [13][14][15] . In contrast to kinesin, whose MT interface is located on the surface of the ATPase core, dynein's MTBD is separated from the catalytic domain by a coiled-coil stalk 16 . Nucleotidedependent conformational changes of the linker drive the motility towards the MT minus-end 17, 18 . The tail domain dimerizes the heavy chains 19-21 , and binds a light intermediate chain (LIC) and an intermediate chain (IC) complexed with three light chains (LCs: LC7, LC8 and Tctex) 1, 22, 23 .When dynein is not bound to its cargo, it forms two distinct conformations, the phi-particle and open conformation, both of which move poorly along MTs 24, 25 . In the phi conformation, two motor domains self-dimerize through interactions between their linker, AAA+ ring and stalk regions and weakly interact with MTs. In the open conformation, the motor domains are more flexible and point towards each other, which is unfavorable for processive motility 24,26 . Dynein interacts with its activating cofactor dynactin and binds to its cellular cargos in the open conformation. Transitions between the phi and open conformation are propos...