Lis1 slows force-induced detachment of cytoplasmic dynein from microtubules.

Lis1 is a key cofactor for the assembly of active cytoplasmic dynein complexes that transport cargo along microtubules. Lis1 binds to the AAA+ ring and stalk of dynein and slows dynein motility, but the underlying mechanism has remained unclear. Using single-molecule imaging and optical trapping assays, we investigated how Lis1 binding affects the motility and force generation of yeast dynein in vitro. We showed that Lis1 slows motility by binding to the AAA+ ring of dynein, not by serving as a roadblock or tethering dynein to microtubules. Lis1 binding also does not affect force generation, but it induces prolonged stalls and reduces the asymmetry in the force-induced detachment of dynein from microtubules. The mutagenesis of the Lis1-binding sites on the dynein stalk partially recovers this asymmetry but does not restore dynein velocity. These results suggest that Lis1-stalk interaction slows the detachment of dynein from microtubules by interfering with the stalk sliding mechanism.

Studying Dynein Mechanochemistry with an Optical Trap.

Molecular motors generate force and mechanical work to perform some of the most energy-demanding cellular processes, such as whole cell motility and cell division. These motors experience resistance from the viscoelastic environment of the surrounding cytoplasm, and opposing forces that can originate from other motors bound to cytoskeleton. Optical trapping is the most widely used method to measure the force-generating and force-response characteristics of motor proteins. Here we present the methodologies of three different optical trapping assays we use to measure how forces originating from external factors affect the microtubule-detachment rate and velocity of dynein. We also briefly discuss the remaining challenges and future directions of optical trapping studies of dyneins and other microtubule-based motors.

Structure and Mechanics of Dynein Motors.

Dyneins make up a family of AAA+ motors that move toward the minus end of microtubules. Cytoplasmic dynein is responsible for transporting intracellular cargos in interphase cells and mediating spindle assembly and chromosome positioning during cell division. Other dynein isoforms transport cargos in cilia and power ciliary beating. Dyneins were the least studied of the cytoskeletal motors due to challenges in the reconstitution of active dynein complexes in vitro and the scarcity of high-resolution methods for in-depth structural and biophysical characterization of these motors. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. This review synthesizes the results of structural and biophysical studies for each class of dynein motors. We highlight several outstanding questions about the regulation of bidirectional transport along microtubules and the mechanisms that sustain self-coordinated oscillations within motile cilia.

Lis1 activates dynein motility by modulating its pairing with dynactin.

Lissencephaly-1 (Lis1) is a key cofactor for dynein-mediated intracellular transport towards the minus-ends of microtubules. 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 favours 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 autoinhibited state, which provides a mechanistic explanation for why Lis1 is required for efficient transport of many dynein-associated cargos in cells.