UC San Diego

Cytoplasmic dynein-1 (dynein) is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions ranging from retrograde axonal transport to mitotic spindle assembly. For human dynein to be motile it must form an activated complex with two cofactors, dynactin and one of a set of coiled-coil containing activating adaptor proteins. In cells dynein requires additional cofactors, including the conserved dynein regulator Lis1. Lis1 is critical for all of dynein’s known functions and is unique among dynein’s regulators because it is the only one which binds directly to the motor domain. This makes it important to consider Lis1 regulation both of activated dynein complexes as well as regulation of the dynein motor itself. In this thesis I present work studying the mechanisms of dynein regulation by Lis1 using a combination of biochemistry, single molecule imaging, cell biology, and cryo-electron microscopy. In chapter 2 we investigated the role of Lis1 in the context of activated dynein complexes containing dynactin, an activating adaptor and one or two dynein dimers and found that Lis1 aids the formation of these complexes. Interestingly, Lis1 is not required for the motility of these complexes once they form. Lis1 aids dynein in overcoming its auto-inhibition and assume the correct configuration for proper addition into the complex before Lis1 dissociates as the complex moves along microtubules. In chapter 3 we solved a 3.1Å structure of a dynein-Lis1 complex, which we used to understand how Lis1 directly influences the mechanochemistry of the dynein motor. Lis1 has two opposing modes of regulating the dynein motor, either increasing or decreasing dynein’s microtubule binding affinity. We found novel contact sites important for both modes of regulation in vitro in single molecule assays and in vivo in Saccharomyces cerevisiae. Overall, this work reveals the complicated nature of dynein regulation by Lis1 and allows us to make a unified model, which is presented in chapter 4.