CFAP100 forms phase-separated condensates required for ciliary transition zone assembly.

Cilia are microtubule-based cellular protrusions critical for cell signaling and motility. The ciliary transition zone connects axonemal microtubules to the ciliary membrane and functions as a gatekeeper by regulating selective targeting and sorting of proteins. However, the molecular mechanisms underlying its assembly remain incompletely understood. Herein, we identify an important role for cilia and flagella associated protein 100 (CFAP100) in the transition zone assembly. Cfap100 knockout mice exhibit ciliary defects in multiple organs, including the brain, trachea, and kidney. Super-resolution imaging reveals that CFAP100 forms a ring-like structure at the distal end of centrioles. Molecular analyses demonstrate that CFAP100 stimulates the transition zone assembly and interacts with nephrocystin-3 (NPHP3), a component of the inversin compartment that localizes adjacent to the transition zone. Further investigation shows that CFAP100 contains an intrinsically disordered region and forms dynamic biomolecular condensates during ciliogenesis, which are sufficient to promote transition zone assembly. Moreover, the phase separation capacity of CFAP100 correlates with its ability to recruit NPHP3. Together, our findings reveal a mechanism through which CFAP100 phase separation promotes ciliary transition zone assembly, providing insights into the physicochemical principle governing ciliogenesis.