Living cells are complex factories that simultaneously perform thousands of different processes using a variety of molecular machines. These molecular machines must operate in a coordinated way, but how they are assembled and positioned in the cell, and coordinated in space and time to form a functioning organism is one of the profound mysteries of biology. The long-term goal of my laboratory is to gain a molecular understanding of the functional organization of cells by studying the three-dimensional structure of macromolecules and organelles in situ, i.e. in their native environment. Our tool is electron microscope tomography of rapidly frozen (and thus well-preserved) cellular structures. We have focused on the eukaryotic flagellum and its major motor protein, dynein, as a model for exploring and dissecting a well-defined genetic and cellular system (and its sub-components). Our studies are beginning to provide a comprehensive understanding of the molecular structure, function and regulation underlying motor function and flagellar beating in health and disease. Defects in the motility and assembly of cilia and flagella are linked to a variety of human genetic diseases (e.g. polycystic kidney disease, Bardet-Biedl Syndrome, and primary ciliary dyskinesia) so these studies will provide insights into the mechanisms and structural manifestations of ciliary-linked disorders in humans. An important side-benefit of our research will be the development of new tools for 3D imaging, image processing and integrative genetic-structural studies. These advances will allow investigators to perform experiments not previously feasible, which will provide greater insights into cellular events in general.