An amino acid-resolution interactome for motile cilia identifies the structure and function of ciliopathy protein complexes

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain a myriad of different proteins that assemble into an array of distinct machines, and understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry in Tetrahymena thermophila. From over 19,000 cross-links, we identified over 4,700 unique amino acid interactions among over 1,100 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the intraflagellar transport system, axonemal dynein arms, radial spokes, the 96-nm ruler, and microtubule inner proteins. Guided by this dataset, we used vertebrate multiciliated cells to reveal functional interactions among several poorly defined human ciliopathy proteins. This dataset provides a resource for studying the biology of an ancient organelle and the molecular etiology of human genetic disease.