The radial spoke (RS) is a T-shaped macromolecular complex composed of more than twenty subunits. It transmits mechanochemical signals between central pair (CP) and axonemal dynein arms to coordinate ciliary motility. Large numbers of mutations in RS head-neck protein genes have been linked to primary ciliary dyskinesia (PCD) and asthenospermia. The structural information of the mammalian RS remains very limited, and a high-resolution structure of the RS head-neck need to be determined.
Ependymal cilia generate a unidirectional cerebrospinal fluid (CSF) flow through their rhythmic beating. Dyskinesia of ependymal cilia leads to the obstruction of CSF flow, which is closely related to hydrocephalus and idiopathic scoliosis. The in situ cryo-electron tomography (cryo-ET) structure of ependymal cilia remains unclear.
In a study published in Nature Communications, a team led by Prof. CONG Yao and Prof. ZHU Xueliang from the Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences, collaborating with Prof. YAN Xiumin from Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, reported the first near-atomic resolution cryo-EM structure of mammalian RS head-neck complex, and determined the first in situ cryo-ET axonemal structure of ependymal cilia.
The researchers in vitro assembled and determined the first high-resolution cryo-EM structure of the mammalian RS head-neck complex in both monomer and dimer forms. The structural analysis revealed the precise head-neck interaction networks, and the intrinsic motions of the dimer. They also proposed a step-by-step assembly mechanism of the RS head-neck, which provides insights into the potential etiology of RS head-neck gene mutations linked to PCD and asthenospermia.
In addition, the researchers utilized a 'primary culture–induced differentiation' method to derive multiciliated mouse ependymal epithelial cells. By combining cryo-ET with sub-tomogram averaging, they resolved the first cryo-ET axoneme structure of mammalian ependymal cilia, and demonstrated that the structure of the reconstituted RS head-neck dimer resembles the in situ cryo-ET structure of RS1/RS2 in ependymal cilia very well. They found the lack of IDA-b/c/e and the absence of Tektin filaments within the A-tubule of doublet microtubules in ependymal cilia. This tissue-specific feature may represent an evolutionary choice driven by the functional requirements on ependymal cilia.
By integrating structures from cryo-EM and cryo-ET, and incorporating AlphaFold2 predicted structures, the researchers proposed a structural model for mammalian RS3, and built more complete multi-scale models for the RS1/RS2, IDAs, and N-DRC of mouse ependymal cilia. They identified an extra subunit, AK8, situated in the stalk of RS1. Through multi-scale structural integration, they proposed that the intrinsic motion of the RS coordinates the RS-CP interaction to mediate the asymmetric beating of cilia.
In summary, this study sheds new lights on the RS head-neck assembly mechanism, how the RS-CP interaction mediates asymmetric ciliary beating, the etiology of PCD and asthenospermia, as well as the tissue-specific reduced features of ependymal cilia, and highlights an evolutionary choice driven by nature.
Contact: cong@sibcb.ac.cn / xlzhu@sibcb.ac.cn / yanx@shsmu.edu.cn
Reference: https://www.nature.com/articles/s41467-023-44577-1
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