Abstract
Cilia and flagella are important organelles that perform both motile and sensory functions. For more than half a century, electron microscopy has provided crucial insights into the fundamental architecture and function of these organelles, such as the characteristic [9+2] microtubule arrangement of the axoneme or the dynein-driven microtubule sliding as the basis of motility. However, we are just starting to explore the molecular organization and mechanisms that drive and regulate axonemal bending. Recently, electron tomography (ET) of rapidly frozen, that is, life-like preserved specimen, has emerged as a cutting-edge technique that provides three-dimensional (3D) views of cellular structures. Cryo-ET and subtomogram averaging has provided high-resolution 3D images of intact flagella and axonemes, allowing us to discover new structures and gain a better understanding of their molecular organization. This chapter provides an overview of the principles of cryo-preservation, ET, and tomographic averaging, and it highlights both strengths and limitations of combining these methods to study axonemal organization. The chapter gives a comprehensive overview of the major technical steps involved in cryo-ET and 3D averaging, and explains successful strategies to generate structural data of the axoneme with 3 to 4nm resolution. Basic equipment requirements, available software packages and how to use them, as well as common problems, artifacts and future challenges are discussed. The chapter is addressed to both scientists who already use or consider using cryo-tomography of cilia and flagella, as well as researchers who would like to learn more about the process and how to "read" these new 3D images.