Xu, X.; Yu, Y.; Zheng, T.; Clark, F.; Ross, J.; Tavares, A. L. P.; Wei, S.; Sun, J.

Motile cilia generate directional fluid flow critical for development and tissue homeostasis, and their dysfunction underlies a range of human diseases. Prior proteomic studies have focused on isolated axonemes, leaving the regulatory components located in other cellular compartments of multiciliated cells (MCCs) largely unidentified. To bridge this gap, we performed whole-cell proteomic profiling of multicilin-induced Xenopus animal cap-derived mucociliary organoids, which are highly enriched in MCCs. Leveraging On-Filter In-Cell processing and mass spectrometry, we identified over 9,000 proteins, including 832 upregulated in MCCs. These encompassed both established ciliary components and novel candidates localized to distinct ciliary structures. Functional validation confirmed the involvement of previously uncharacterized proteins in cilium formation and MCC differentiation. This study presents a high-resolution, whole-cell proteome of vertebrate MCCs, providing critical insights into the molecular networks that govern ciliogenesis and offering a valuable resource for understanding the mechanisms of cilia-related disorders.