Molecular and cellular approaches toward understanding dynein-driven motility

Active transport is integral to organelle localization and their distribution within the cell. Kinesins, myosins and dynein are the molecular motors that drive this long range transport on the actin and microtubule cytoskeleton. Although several families of kinesins and myosins have evolved, there is only one form of cytoplasmic dynein driving active retrograde transport in cells. While dynactin is an essential co-factor for most cellular functions of dynein, the mechanistic basis for this evolutionarily well conserved interaction remains unclear. Here, I use single molecule approaches with purified dynein to reconstitute processes in vitro, and implement an optogenetic tool in neurons to further dissect regulatory mechanisms of dynein-driven transport in cells. I demonstrate for the first time, at the single molecule level, that dynactin functions as a tether to enhance the initial recruitment of dynein onto microtubules but also acts as a brake to slow the motor. I then extend this work in neurons to understand regulation of the dynein motor at the cellular level. Neurons are particulary dependent on long-range transport as organelles and macromolecules must be efficiently moved over the extended length of the axon and further, have mechanisms in place for the compartment-specific regulation of trafficking in axons and dendrites. I use a light-inducible dimerization tool to recruit motor proteins or motor adaptors to organelles in real time to examine downstream effects of organelle motility and compartment-specific regulation of motors. I find that while dynein works efficiently in both axons and dendrites, kinesins are differentially regulated in a compartment-specific manner. I further demonstrate that dynein-driven motility in neurons is largely governed by microtubule orientation and requires microtubule dynamics for efficient navigation in axons and dendrites. Together, this work sheds light on the molecular and cellular mechanisms of dynein function both in vitro and in vivo using a combination of approaches. My findings converge to a model wherein dynactin enhances the recruitment of dynein onto microtubule plus ends, leading to efficient minus-end directed motility of dynein. This becomes especially critical in neuronal growth cones and dendrites owing to the large number of highly dynamic microtubules in these compartments