Cilia and eukaryotic flagella are microtubule-based organelles that are crucial for cell motility and signaling. SAXO proteins (denoted for ‘stabilizers of axonemal microtubules’) are found exclusively in flagellated or ciliated organisms, but their physiological functions remain unclear. We investigated four SAXO proteins (SAXO1–SAXO4) in Chlamydomonas reinhardtii, identified via bioinformatics. All localize to cilia but differ in axonemal binding and spatial distribution. Single SAXO knockouts had no effect, whereas double mutants (saxo1/2, saxo1/3 and saxo2/3) showed shorter cilia. This phenotype intensified in the saxo1/2/3 triple mutant but not further in the quadruple mutant. Ciliary beating remained normal in saxo1/2/3 mutants, even under mechanical stress, indicating that SAXO1–SAXO3 are not essential for ciliary rigidity. Biochemical and proteomic analyses revealed no significant changes in the ciliary proteome or in tubulin acetylation, tyrosination and glutamylation within cilia. However, dikaryon assays with labeled tubulin showed that there was increased axonemal tubulin turnover in saxo1/2/3 mutant. Our findings underscore a crucial role of SAXO proteins in stabilizing axonemal microtubules by reducing tubulin turnover, thereby regulating ciliary length and assembly, and provide new insights into their function in cilia.

Keywords:
Chlamydomonas, Cilia, Eukaryotic flagella, Axonemal microtubules, SAXO, Ciliogenesis, Microtubule dynamics
Subjects:
Cell migration, Cilia and flagella, Cytoskeleton

Author contributions

Conceptualization: J.P., Y.Z., X.L., Z.W.; Data curation: J.P., Y.Z., X.L., Z.W.; Formal analysis: J.P., Y.Z., X.L., Z.W.; Funding acquisition: J.P.; Investigation: J.P., Y.Z., X.L., Z.W.; Methodology: Y.Z., X.L., Z.W.; Project administration: J.P.; Resources: J.P.; Supervision: J.P.; Validation: Y.Z., X.L., Z.W.; Visualization: Y.Z., X.L., Z.W.; Writing – original draft: J.P., Y.Z., X.L.; Writing – review & editing: J.P., Y.Z., X.L.

Funding

This work was supported by the National Natural Science Foundation of China (32370813, 32461160292, 31991191).

Data and resource availability

All relevant data can be found within the article and its supplementary information.

First Person

This article has an associated First Person interview with the co-first authors of the paper.

Special Issue

This article is part of the Special Issue ‘Cilia and Flagella: from Basic Biology to Disease’, guest edited by Pleasantine Mill and Lotte Pedersen. See related articles at https://journals.biologists.com/jcs/issue/138/20.

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