SAXO proteins mediate tubulin turnover in axonemal microtubules of Chlamydomonas

Cilia and eukaryotic flagella are microtubule-based organelles crucial for cell motility and signaling. SAXO proteins (stabilizers of axonemal microtubules) are found exclusively in flagellated or ciliated organisms, but their physiological functions remain unclear. We investigated four SAXO proteins (SAXO1-4) in Chlamydomonas, identified via bioinformatics. All localize to cilia but differ in axonemal binding and spatial distribution. Single SAXO knockouts had no effect, while 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 SAXO1-3 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 labelled tubulin show increased axonemal tubulin turnover in saxo1/2/3 mutant. Our findings underscore a critical 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.