Electron microscopy of flagella, primary cilia, and intraflagellar transport in flat-embedded cells.

Intraflagellar transport (IFT) is an evolutionarily highly conserved, microtubule-based, bidirectional transport system found in eukaryotic cilia/flagella and is indispensable for their assembly, maintenance, and sensory functions. Powered by two different motor complexes, linear arrays of protein particles, called IFT trains, are transported from the base to the tip of the cilium/flagellum and back, carrying axonemal precursors to the tip for assembly and turnover products back to the cell body for recycling. The dynamics of IFT can be visualized using various types of live-cell microscopy techniques, but for analyzing the ultrastructure of IFT trains, transmission electron microscopy is indispensable. The focus of this chapter is to describe the application of the flat embedding technique to Chlamydomonas reinhardtii and monolayers of mammalian culture cells. Such flat embeddings are well suited for the analysis of the ultrastructure of the IFT system by standard electron microscopy and electron tomography.