Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca 2+ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca 2+ -binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized.Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca 2+ -binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF-hand Ca 2+ -binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross-linking experiment showed that calaxin binds to β-tubulin in situ. Overlay experiments further indicated that calaxin binds the β-dynein heavy chain of outer arm dynein in the presence of Ca 2+ .Conclusions. These results suggest that calaxin is a potential Ca 2+ -dependent modulator of outer arm dynein in metazoan cilia and flagella.

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We previously identified a 66 kDa axonemal protein (Ci-Axp66.0) in sperm of the ascidian Ciona intestinalis. Here we found that Ci-Axp66.0 shows sequence similarity to the DC2 subunit of the Chlamydomonas outer arm docking complex. Analysis of secondary structure of Ci-Axp66.0 suggested that the N-terminal two-thirds of the molecule is rich in coiled coil structure, as in Chlamydomonas DC2. Immunogold localization revealed that it is located in the vicinity of outer arm dynein. Ci-Axp66.0 was partly extracted from the axonemes by a high salt solution and co-purified with outer arm dynein. This co-purification was not affected by the absence of Mg(2+) in isolation buffer, indicating that Ci-Axp66.0 is associated with outer arm dynein. These results suggest that Ci-Axp66.0 is a component of the outer arm dynein docking complex in the axonemes of Ciona sperm.