The N-terminal, WD40, and C-terminal domains of WDR-31 control ciliary localization and cooperate with ELMOD/RP2 gaps to maintain membrane compartmentalization.

Background/aim: WD40 repeats are found in many ciliary proteins. Although the WD40 repeat-containing WD repeat-containing protein 31 (WDR-31) is known to regulate ciliary protein trafficking and morphology, the specific contributions of its N-terminal, WD40, and C-terminal domains to protein localization and ciliary gate integrity remain unclear. Aim of this study is to dissect the functional roles of WDR-31 domains by investigating their contributions to ciliary localization, ciliary protein trafficking, and gate integrity. Materials and methods: Using CRISPR/Cas9 technology, we generated Results: When the N-terminal domain was deleted, WDR-31 became less mobile at the basal body and mislocalized to the transition zone, suggesting a role in protein turnover. Furthermore, the WD40 domain is required for basal body confinement, whereas the C-terminus prevents WDR-31 from spreading into the distal axoneme. Any single domain deletion resulted in TRAM-1 leakage into the cilium in a sensitized mutant background. Remarkably, WDR-31 localization depends on IFT, the BBSome, and transition zone components; its mobility is significantly reduced in intraflagellar transport-defective mutants, supporting a model in which its steady-state distribution at the ciliary base appears to depend on intact intraflagellar transport machinery and may involve dynamic regulation of protein localization, retention, or turnover rather than being passively restricted. Conclusion: Our results indicate that the N-terminal, WD40, and C-terminal domains of WDR-31 are necessary for its dynamic positioning at the ciliary base. Together, these domains in WDR-31 are critical for maintaining TRAM-1 exclusion and periciliary membrane compartment/ciliary membrane compartmentalization.