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bioRxiv · 10.1101/2025.03.12.642377

Cell-free Reconstitution Reveals Synergistic Stabilization of Microtubule Doublets by PACRG and FAP20

Khuntsariya, D.·Batman, U.·Krishnan, A.·Rozbesky, D.·Lemaitre, F.·Janke, C.·Hamel, V.·Guichard, P.·Braun, M.·Lansky, Z.
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Abstract

Ciliary microtubule doublets, consisting of an incomplete B-tubule on the surface of a complete A-microtubule, form the fundamental structural framework of motile and sensory cilia/flagella. Ciliary proteins PACRG and FAP20, which are essential for flagellar stability and function, localize to the junction between the A-and the B-tubule. However, their precise role in microtubule doublet assembly and dynamics remains unknown. Here, using a cell-free assay, TIRF-microscopy, and cryo-electron tomography, we demonstrate that in combination PACRG and FAP20 stabilize B-tubule dynamics and microtubule doublet architecture. We show that together these proteins localize to the B-tubules in distinct, high-density patches, which locally stabilize B-tubule by decreasing its depolymerization velocity and increasing its rescue frequency. Cryo-tomography of in vitro reconstructed microtubule doublets in presence of PACRG and FAP20 reveals reduced B-tubule curvature fluctuations, promoting a more rigid and aligned conformation. Altogether, our findings suggest that PACRG and FAP20 function synergistically to reinforce microtubule doublet stability, ensuring ciliary integrity and function.

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Khuntsariya, D., Batman, U., Krishnan, A., Rozbesky, D., Lemaitre, F., Janke, C., Hamel, V., Guichard, P., Braun, M., Lansky, Z.. 2025-03-12. Cell-free Reconstitution Reveals Synergistic Stabilization of Microtubule Doublets by PACRG and FAP20. https://doi.org/10.1101/2025.03.12.642377

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