In situ structures of the ciliary tip show the multi-step mechanism of axoneme assembly

Motile cilia are highly complex, ordered organelles which extend from the cell surface to generate fluid flow. During motile cilia assembly, components rapidly self-organize at the ciliary tip. Here, over two hundred different components bind microtubules to form an intricate, repeating structure called the axoneme. Despite in-depth structural characterization of axonemal complexes, we have limited understanding of how these structures are generated. Here, we directly visualize the steps of axonemal assembly in situ by performing cryo-electron tomography of Chlamydomonas reinhardtii cilia tips. Using subtomogram averaging and classification, we identified conserved microtubule luminal proteins that establish periodicity and show that controlled microtubule lattice perforation can facilitate incorporation of late-binding components. By uncovering unexpected assembly intermediates, we explain how structural integrity and the emergence of motility are coordinated within the axoneme. Overall, we demonstrate that self-assembly proceeds through regulated, sequential construction steps and provide a molecular framework for understanding axoneme biogenesis.