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.

Successful viral infection, replication, and dissemination depend on the ability of a virus to subvert host cell processes. The molecular interplay between viruses and their hosts is an ancient and ongoing evolutionary “arms race,” with each side constantly evolving new strategies to outwit the other. The existing literature regarding the impact of viral conflicts on mammalian evolution has predominantly concentrated on proteins that are directly involved in immune responses. Thus, in known cases of host-virus interactions, we see evolutionary signatures of conflict in host genes that encode viral antagonists. However, the principles of natural selection postulate that this paradigm is not exclusive to immune genes. Instead, we can infer that any protein interacting with viruses is subject to the selective pressures driving genetic adaptation. As such, we reasoned that genes involved in the active transport of macromolecules within the cell are one potential source of conflict. Due to the large size of eukaryotic cells, along with the high density of macromolecules, diffusion is limited. Cells transport intracellular cargo using dynein and kinesin motors, which move on microtubules in opposite directions to overcome this diffusion-limited environment. Viruses co-opt this cellular machinery for cellular entry, the transportation of viral components to replication sites, remodeling of cellular compartments, and viral egress. Conversely, the host adaptive and innate immune responses require movement of signaling components, transport of endocytic and exocytic vesicles, organelle recycling, and cellular remodeling, all of which require the microtubule-based trafficking machinery. Motivated by the competing interest of microtubule-based transport in aiding and inhibiting viral replication, we set out to identify conflicts over the intracellular trafficking machinery between viruses and their host cells. In this dissertation, I present collaborative work that identifies several intracellular trafficking genes exhibiting evolutionary signatures of conflict. Of these candidate genes, I characterize a novel role in the innate antiviral immune response for ninein-like (NINL), a dynein activating adaptor. Overall, this work provides a glimpse into the impact that viruses may have on the evolution of the intracellular transport machinery and demonstrates how leveraging evolutionary signatures of conflict can uncover novel host-pathogen interactions that could become therapeutic targets.

Leucine-Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinson’s Disease (PD). This neurodegenerative disease affects over 10 million people worldwide, and defects in LRRK2 function have also been linked to the sporadic form of the disease. LRRK2 is a large (286 kDa) multi-domain, predominantly cytosolic, protein and has been shown to have roles in membrane trafficking. While a subset of LRRK2 is localized to membranes in cells, LRRK2 has also been shown to colocalize with microtubules and form helical filaments that wrap around the microtubules. Intriguingly, many PD-linked mutations demonstrate an enhanced association with microtubules when expressed in cells. In vitro, microtubule-associated LRRK2 can act as a roadblock for the molecular motor proteins dynein and kinesin. However, many questions remain about the impact this microtubule-associated LRRK2 has on intracellular transport and how these LRRK2 filaments form. In this dissertation, I present interdisciplinary work investigating the mechanisms by which LRRK2 filament formation occurs. Here, we report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, LRRK2’s closest human homolog, which, while structurally similar to LRRK2, is not linked to PD and does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2’s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells without affecting LRRK2’s kinase activity. Overall, this work provides important insights into the basis of LRRK2 binding to microtubules and presents novel mutants that will enable future work to better probe the physiological roles of the LRRK2/microtubule interaction in the pathogenesis of PD. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.