Multi-scale mechanisms of microtubule-based transport within cilia and flagella

The goal of this proposal is to obtain mechanistic insight the microtubule-based transport system that enables cilia/flagella to assemble and perform essential functions in cell motility, signaling, and sensing. Strikingly, this process of intraflagellar transport (IFT) involves multi-megadalton polymers, termed IFT 'trains', which move under the power of oppositely-directed microtubule motors dynein-2 and kinesin Kif3. To address outstanding motor mechanisms of IFT, I propose a multi-disciplinary approach spanning three scales. At the scale of individual motors, we will determine structures of the dynein-2 and Kif3 complexes to elucidate how their subunits come together and enable regulated motor activity. At the scale of multi-motor assemblies, we will reconstitute IFT trains with purified proteins to dissect how they polymerize and coordinate bidirectional motility. At the scale of whole cilia, we will determine how dynein-2 powers IFT turnaround at the ciliary tip and define spatial regulators of dynein-2 in mammalian cells. To achieve these goals, we will use reconstitution and cryo-EM to determine structures, fluorescence microscopy to visualize dynamics, and genome editing to interrogate cellular factors. Together, these studies will illuminate the multi-scale mechanisms that cells use to build and signal through cilia/flagella, which I will integrate into a molecular movie.