Dynein, kinesin, and myosin are motor proteins that power the majority of movements of eukaryotic cells. Many human cardiac, kidney, auditory, reproductive, and nervous system diseases have been linked to mutations in these cytoskeletal motors, and small molecule drugs that modulate the activities of molecular motors are now being tested in clinical trials. Of the three types of cytoskeletal motor proteins, dynein is least well understood. The majority of the dyneins encoded by the human genome power the movement of sperm and the beating of cilia and flagella in epithelial cells. One goal of this grant is to understand the structures and biophysical mechanisms of axonemal dyneins, which overall are much less well understood than cytoplasmic dynein (which our lab has studied for the past 12 years). Our general approach will be first to produce recombinant human axonemal dyneins and then develop a suite of single molecule assays to measure how they produce and respond to force. These studies on the force-dependent behavior of dynein will provide insight into a long-standing question of how dynein-induced sliding of microtubules in axonemes is converted into the oscillatory bending of cilia/flagella. We also will determine the subunit compositions of different axonemal dyneins (e.g. outer and inner arm dynein) and, by cryo-EM, determine the structures of the intact holoenzymes both in solution and bound to microtubules. Using our recombinant protein system, we will study the effects of dynein mutations that give rise to human diseases called primary ciliary dyskinesias so as to understand the underlying molecular defects. A second major focus of this grant is to investigate how motors dock onto specific cargoes in cells. Humans contain approximately fifty different microtubule-based motors that attach onto hundreds of different cargos. However, the rules that govern these interactions remain poorly understood. We will focus on a few motor-adaptor-receptor interactions and study them in great detail by obtaining crystal structures of these complexes and developing in vitro and in vivo cargo transport assays. In addition to membrane cargo, we will also investigate how axonemal dyneins recognize specific docking sites on outer doublet microtubules. In summary, by the end of this grant period, we will generate new reagents (e.g. recombinant proteins) and develop motility assays for axonemal dyneins. With these tools, we will gain new insights into the structures and biophysical mechanisms of axonemal dyneins. We also will develop new assays and obtain high resolution structures of motor interacting with cargo receptors. These studies will reveal how motors bind to organelles and transport them to particular intracellular locations.