Removal of the Ciliary Gate Allows Axoneme Extension in the Absence of Retrograde IFT

De-Castro, A. R. G.; De-Castro, M. J. G.; Haasnoot, G. H.; Gassmann, R.; Peterman, E. M. C.; Dantas, T. J.; Abreu, C. M. C.

doi:10.1101/2025.10.21.683645

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bioRxiv · 10.1101/2025.10.21.683645

Removal of the Ciliary Gate Allows Axoneme Extension in the Absence of Retrograde IFT

De-Castro, A. R. G.·De-Castro, M. J. G.·Haasnoot, G. H.·Gassmann, R.·Peterman, E. M. C.·Dantas, T. J.·Abreu, C. M. C.

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Abstract

The transition zone (TZ) is a selective barrier that maintains ciliary compartmentalization by controlling protein entry and exit. Cilia assembly requires the crossing of this barrier by intraflagellar transport (IFT) trains, scaffolded by IFT-A and IFT-B complexes, which move cargo bidirectionally using kinesin-2 and dynein-2 motors. In Caenorhabditis elegans, IFT-A loss abolishes retrograde transport, resulting in truncated cilia packed with IFT material. Here, we show that blocking TZ assembly prevents dynein-2 and IFT-B accumulation inside IFT-A-deficient cilia and partially rescues axoneme length. Single-particle imaging reveals that this rescue occurs without recovery of retrograde IFT. Instead, IFT particles exit cilia by passively diffusing through the disrupted TZ. Moreover, IFT-A/TZ double mutants shed ciliary extracellular vesicles (EVs) abnormally enriched in IFT components, providing a second clearance route. We conclude that TZ removal alters ciliary responses to retrograde transport defects, promoting diffusion and EV release to clear IFT machinery and facilitate axoneme extension. Highlights- TZ loss provides alternative routes for clearing IFT machinery stalled in IFT-A mutant cilia - Axoneme extension is possible without retrograde IFT when the TZ barrier is removed - Disrupted TZ enables exit of IFT particles by passive diffusion in retrograde IFT-deficient cilia - Excess IFT machinery is discarded in ciliary EVs when retrograde IFT and gating are compromised

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De-Castro, A. R. G., De-Castro, M. J. G., Haasnoot, G. H., Gassmann, R., Peterman, E. M. C., Dantas, T. J., Abreu, C. M. C.. 2025-10-23. Removal of the Ciliary Gate Allows Axoneme Extension in the Absence of Retrograde IFT. https://doi.org/10.1101/2025.10.21.683645

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