The Mechanism and Regulation of the Dynein Transport Machinery

University Of California Berkeley, Berkeley CA

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Abstract

Project Summary Cytoplasmic dynein is an AAA+ motor responsible for nearly all minus-end-directed motility and force generation functions along microtubules (MTs). Surprisingly, a single dynein-1 heavy chain gene is responsible for an enormous breadth of cellular activities in intracellular transport, MT organization, and mitosis, in comparison to more than 40 plus-end-directed kinesin motors performing complementary functions on MTs. 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 their in-depth structural and biophysical characterization. These challenges have been recently addressed, and there have been major advances in our understanding of the activation, mechanism, and regulation of dyneins. For example, we recently showed that the mammalian dynein complex is a robust motor that rapidly walks along MT tracks and produces forces comparable to kinesins. We also developed a robust mechanistic model for the activation of the dynein transport machinery by accessory proteins, Lis1 and Nde1. Our future goals are to dissect the mechanism of active dynein complexes and determine how dynein activation and motility are regulated across multiple scales using biochemical reconstitution, single-molecule imaging, and cryo-electron microscopy (cryo-EM). Specifically, we will use the recently developed MINFLUX method, single- molecule FRET (smFRET), and protein engineering approaches to directly monitor the conformational dynamics of the dynein motor domain during stepping. These experiments will fill critical gaps in our understanding of how dynein couples ATP hydrolysis to a mechanical step at unprecedented resolution. We will also investigate the mechanism of Lis1/Nde1-mediated activation of the dynein transport machinery using cryo-EM, smFRET, and mutagenesis studies. These studies will reveal how Nde1 recruits Lis1 to autoinhibited dynein and facilitates the opening of dynein for its assembly into functional transport complexes. A large body of evidence shows that dynein is recruited to intracellular cargos side-by-side with kinesins through cargo adaptors. These adaptors may also coordinate motor activity to avoid tug-of-war between the antagonistic motors and determine which direction the cargo moves on MTs. To investigate the mechanism of motor recruitment and coordination on a cargo, we will reconstitute the machinery that transports mitochondria (Miro/TRAK/dynein/dynactin/kinesin) and autophagosomes (HAP1/Huntingtin/dynein/dynactin/kinesin) from purified components. We will also investigate the assembly and regulation of dynein/dynactin by the NuMA adaptor for its mitotic functions. We will characterize the assembly and motility of these complexes in vitro, and use cryo-EM to gain structural insight into the coordination of motor activity on cargo adaptors. The success of our research program will reveal the fundamental mechanochemistry of dynein and how it achieves retrograde transport of intracellular cargos and performs specific functions in mitosis.