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bioRxiv · 10.1101/2025.08.03.668368

Physiological Reconstitution of Microtubule Doublets

Li, M.·Chen, G.·Chen, Z.·Guo, Z.·Wang, Z.·Chai, Y.·Li, W.·Ou, G.
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Abstract

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 crucial factor that promotes B-tubule assembly. Molecular dynamics simulations revealed that FAP53 alleviates steric hindrance from the -tubulin C-terminal tail and stabilizes B-tubule docking at the A-tubule surface. In vitro reconstitution demonstrated that recombinant FAP53 is sufficient to drive MTD formation under physiological tubulin conditions. In cultured HeLa cells, co-expression of CFAP53 and CFAP20--an inner junction protein--induced ectopic MTD-like structures in the cytoplasm. Furthermore, a structurally homologous protein in C. elegans, WFAP-53, localized to sensory cilia; however, when overexpressed, it triggered ectopic MTD formation in neuronal dendrites and concomitantly led to sensory cilia disassembly. These findings uncover a conserved mechanism of B-tubule initiation and underscore the necessity for spatially restricted expression of MTD assembly factors during ciliogenesis.

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Li, M., Chen, G., Chen, Z., Guo, Z., Wang, Z., Chai, Y., Li, W., Ou, G.. 2025-08-04. Physiological Reconstitution of Microtubule Doublets. https://doi.org/10.1101/2025.08.03.668368

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