Dyneins are microtubule-based motor enzymes that convert chemical energy into mechanical work. The dynein motors occur either in the cytoplasm, where they mediate retrograde transport, or within the integral structure of cilia and flagella, where they generate the forces that drive these motile organelles. While progress has been made in understanding aspects of dynein's function, the complexity and size of this motor enzyme have made it difficult to elucidate its molecular mechanism. Electron tomography of rapidly frozen specimens has been shown to be an exciting new technique for imaging well-preserved biological structures in an unperturbed cellular environment. Axonemes are excellent specimens for cryo-electron tomography and for the study of dynein in situ, thanks to the small diameter and the highly ordered arrangement of the microtubules and associated protein complexes in this organelle. We are using the cutting-edge technology of cryo-electron tomography to study the three-dimensional ultrastructures of dynein and intact flagella both preserved in their native states. Our approaches use modern and innovative tools, including integrated genetic and structural approaches that allow us to overcome current limitations in imaging technology and to directly visualize gene products in cells. Our work is aimed at contributing fundamental knowledge to our understanding of the mechanisms underlying motor function and control on a molecular level and to our understanding of the functional organization of cells in general. A major benefit of the proposed experiments will be the development of new tools for 3D imaging and image processing, which in the future will provide insights into cellular events including those gone amiss as in major diseases. PROJECT NARRATIVE: In humans the normal function of several organs requires the activity of cilia. Defects in the motility and assembly of cilia and flagella have been linked to important human diseases, such as primary ciliary dyskinesia (PCD), polycystic kidney disease (PKD), chronic respiratory disease, male sterility, and human obesity disorders (reviewed by Snell et al., 2004). In addition dynein-driven transport along the microtubule cytoskeleton has major impact on cell behavior and organization, including cell division, signaling and cell shape;defects in this organization are often hallmarks of cancer. We expect that the proposed project will lay the foundation for our long-term goal to understand the mechanisms of ciliary-linked disorders in humans, a prerequisite to the development of therapeutic protocols capable of attenuating these disease processes.