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

Abstract

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 Ca²⁺ 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 <i>cyb5d1</i> mutant increases <i>cis</i>-flagellar Ca²⁺ 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 Ca²⁺ spikes across both <i>cis</i>- and <i>trans</i>-flagella of <i>cyb5d1</i> and increased flagellar beating coordination. In addition, loss of CYB5D1 raised the intraflagellar Ca²⁺ pool. These results indicate that CYB5D1 links redox sensing to Ca²⁺ signaling in ciliary coordination and reveal how the two flagella of the same cell achieve synchronized beating through redox-gated Ca²⁺ dynamics. Furthermore, CYB5D1 loss impairs gliding motility by dysregulating Ca²⁺ spiking specifically in the leading flagellum, extending the redox-Ca²⁺ regulatory axis to surface-associated flagellar behaviors. Given the evolutionary conservation of both CYB5D1 and the redox-Ca²⁺ signaling axis, this mechanism likely regulates ciliary function across eukaryotes, with implications for understanding ciliopathies and respiratory diseases.