Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia. Significance Impairment of the coordinated beating of multiple cilia results in primary ciliary dyskinesia (PCD) phenotypes such as chronic respiratory problems. In this study, we find CYB5D1 is a radial spoke stalk protein that binds heme under oxidizing conditions. Mutation of CYB5D1 in zebrafish and green algae lead to uncoordinated ciliary beating. Importantly, lack of CrCYB5D1 changes the redox state of flagella. Our findings reveal the important role of redox in the coordinated beating of multiple cilia and suggest a possible PCD therapy by modulating the redox poise in cilia.

The flagellar (ciliary) membrane of Chlamydomonas reinhardtii is continuous with the plasma membrane but maintains a distinct protein composition through the ciliary gate mechanism; however, a systematic comparison of the membrane proteomes of these two compartments has not been reported. Here, we performed comparative proteomics on highly enriched plasma and flagellar membrane fractions and identified 2779 proteins, with hundreds differentially abundant. Plasma membrane-enriched proteins were mainly involved in proton transport and ATP synthesis, whereas flagellar membrane-enriched proteins were specialized for cilium movement, axoneme assembly, and intraflagellar transport. Fluorescence localization validated the proteomic data and revealed previously uncharacterized subcellular patterns. Notably, PLD1 showed dual localization at the plasma membrane and punctate cytoplasmic structures, and FAP215 (validated by subflagellar fractionation) displayed a broader flagellar signal than the central microtubule protein FAP178, consistent with its localization in the flagellar matrix or membrane. This work provides the first systematic comparison of plasma and flagellar membrane proteomes in Chlamydomonas, offering a valuable resource for understanding flagellar membrane specialization.

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.