Heme-binding protein CYB5D1 couples intraflagellar redox to calcium signaling for coordinated flagellar beating

Coordinated ciliary/flagellar beating requires precise spatiotemporal regulation of molecular motors such as dyneins, yet the molecular mechanisms governing ciliary synchrony remain poorly understood. Here, we demonstrate that a heme-binding axonemal protein CYB5D1 functions as a redox-sensitive switch that controls flagellar beating coordination by regulating Ca ²⁺ dynamics. Both the D58G point mutation, which abolishes heme-binding activity, and the complete loss of CYB5D1 lead to a reduction in the flagellar redox potential. More importantly, the hyperreductive intraflagellar redox shift in the cyb5d1 mutant increases cis -flagellar Ca ²⁺ spike frequency and amplitude, similar to reductive treatment of wild-type flagella, resulting in the loss of flagellar beating coordination. Interestingly, oxidative treatments induced synchronized Ca ²⁺ spikes across both cis - and trans -flagella of cyb5d1 and increased flagellar beating coordination. In addition, loss of CYB5D1 raised the intraflagellar Ca ²⁺ pool. These results indicate that CYB5D1 links redox sensing to Ca ²⁺ signaling in ciliary coordination and reveal how the two flagella of the same cell achieve synchronized beating through redox-gated Ca ²⁺ dynamics. Furthermore, CYB5D1 loss impairs gliding motility by dysregulating Ca ²⁺ spiking specifically in the leading flagellum, extending the redox-Ca ²⁺ regulatory axis to surface-associated flagellar behaviors. Given the evolutionary conservation of both CYB5D1 and the redox-Ca ²⁺ signaling axis, this mechanism likely regulates ciliary function across eukaryotes, with implications for understanding ciliopathies and respiratory diseases.