RESUMEN
Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.
Asunto(s)
Proteínas de Unión al Calcio , Espermatozoides , Proteínas de Pez Cebra , Pez Cebra , Animales , Masculino , Animales Modificados Genéticamente , Calcio/metabolismo , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Cilios/metabolismo , Dineínas/metabolismo , Dineínas/genética , Flagelos/metabolismo , Flagelos/fisiología , Motilidad Espermática/genética , Motilidad Espermática/fisiología , Espermatozoides/metabolismo , Espermatozoides/fisiología , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas del Citoesqueleto/metabolismoRESUMEN
Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of armc4 caused complete loss of OAD, whereas mutation of calaxin caused only partial loss of OAD. Detailed structural analysis revealed that calaxin-/- OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates.
Asunto(s)
Cilios , Proteínas del Citoesqueleto , Dineínas , Microtúbulos , Pez Cebra , Animales , Masculino , Axonema/metabolismo , Cilios/genética , Cilios/metabolismo , Dineínas/metabolismo , Microtúbulos/metabolismo , Mutación , Pez Cebra/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Espermatozoides/metabolismo , Microscopía Fluorescente , Microscopía por Crioelectrón , Modelos Moleculares , Estabilidad ProteicaRESUMEN
[This corrects the article DOI: 10.1038/s42003-019-0462-y.].
RESUMEN
Calaxin is a Ca2+-binding dynein-associated protein that regulates flagellar and ciliary movement. In ascidians, calaxin plays essential roles in chemotaxis of sperm. However, nothing has been known for the function of calaxin in vertebrates. Here we show that the mice with a null mutation in Efcab1, which encodes calaxin, display typical phenotypes of primary ciliary dyskinesia, including hydrocephalus, situs inversus, and abnormal motility of trachea cilia and sperm flagella. Strikingly, both males and females are viable and fertile, indicating that calaxin is not essential for fertilization in mice. The 9 + 2 axonemal structures of epithelial multicilia and sperm flagella are normal, but the formation of 9 + 0 nodal cilia is significantly disrupted. Knockout of calaxin in zebrafish also causes situs inversus due to the irregular ciliary beating of Kupffer's vesicle cilia, although the 9 + 2 axonemal structure appears to remain normal.
