FAP53 facilitates formation of microtubule doublets.

The formation of microtubule doublets (MTDs) is a foundational step in cilia biogenesis, yet how B-tubule nucleation is initiated at the molecular level remains elusive. Here, we identify FAP53 as a factor that facilitates B-tubule assembly in a scaffold-dependent manner. In vitro reconstitution demonstrated that recombinant FAP53 enhances MTD formation in the presence of preassembled microtubule scaffolds, but is insufficient to drive de novo assembly from free tubulin alone. In cultured HeLa cells, coexpression of CFAP53 and CFAP20-an inner junction protein-induced ectopic MTD-like structures in the cytoplasm. Furthermore, we identified a structurally homologous protein in Caenorhabditis elegans, WFAP-53, which localized to sensory cilia. Loss of wfap-53 does not abolish MTD formation in vivo, but its overexpression triggered ectopic MTD formation in neuronal dendrites and concomitantly led to sensory cilia disassembly. Molecular dynamics simulations suggested that FAP53 could stabilize B-tubule docking at the A-tubule surface. These findings uncover a conserved mechanism of B-tubule initiation and underscore the necessity for spatially restricted expression of MTD assembly factors during ciliogenesis.