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Ckb and Ybx2 interact with Ribc2 and are necessary for the ciliary beating of multi-cilia

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Abstract

Background

Motile cilia in a vertebrate are important to sustaining activities of life. Fluid flow on the apical surface of several tissues, including bronchial epithelium, ependymal epithelium, and fallopian tubules is generated by the ciliary beating of motile cilia. Multi-ciliated cells in ependymal tissue are responsible for the circulation of cerebrospinal fluid (CSF), which is essential for the development and homeostasis of the central nervous system, and airway tissues are protected from external contaminants by cilia-driven mucosal flow over the top of the airway epithelium.

Objective

A previous study reported that reduction of Ribc2 protein leads to disruption of ciliary beating in multi-ciliated cells. However, knowledge regarding the molecular function of Ribc2 is limited, thus currently available information is also limited. Therefore, we evaluated the importance of proteins involved in the interaction with Ribc2 in the process of ciliary beating.

Methods

Immunoprecipitation and mass spectrometry analysis was performed for the discovery of proteins involved in the interaction with Ribc2. Expression of the target gene was inhibited by injection of antisense morpholinos and measurement of the fluid flow on the embryonic epidermis of Xenopus was performed using fluorescent beads for examination of the ciliary beating of multi cilia. In addition, the flag-tagged protein was expressed by injection of mRNA and the changes in protein localization in the cilia were measured by immunostaining and western blot analysis for analysis of the molecular interaction between Ribc2 and Ribc2 binding proteins in multi-cilia.

Results

The IP/MS analysis identified Ckb and Ybx2 as Ribc2 binding proteins and our results showed that localization of both Ckb and Ybx2 occurs at the axoneme of multi-cilia on the embryonic epithelium of Xenopus laevis. In addition, our findings confirmed that knock-down of Ckb or Ybx2 resulted in abnormal ciliary beating and reduction of cilia-driven fluid flow on multi-cilia of Xenopus laevis. In addition, significantly decreased localization of Ckb or Ybx2 in the ciliary axoneme was observed in Ribc2-depleted multi-cilia.

Conclusion

Ckb and Ybx2 are involved in the interaction with Ribc2 and are necessary for the ciliary beating of multi-cilia.

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Availability of data and materials

The data and materials in the current study are available on request.

Abbreviations

Ribc2:

RIB43A domain with coiled-coils 2

Ckb:

Creatine kinase, brain

Ybx2:

Y-box-binding protein 2

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Funding

This work was supported by the Korea National Research Foundation. (2021R1A2B5B02002285, 2022R1A4A1021318).

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Author notes

  1. Keun Yeong Kwon, Hyeongsun Jeong, and Dong Gil Jang have contributed equally to this study.

Authors and Affiliations

  1. Department of Biological Sciences, College of Information-Bio Convergence Engineering, Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea

    Keun Yeong Kwon, Dong Gil Jang & Tae Joo Park

  2. Department of Biological Medical Engineering, College of Information-Bio Convergence Engineering, Ulsan National Institute of Science and Technology, Ulsan, 44919, Republic of Korea

    Hyeongsun Jeong & Taejoon Kwon

  3. Center for Genomic Integrity, Institute for Basic Science, Ulsan, 44919, Republic of Korea

    Taejoon Kwon & Tae Joo Park

Authors
  1. Keun Yeong Kwon
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  2. Hyeongsun Jeong
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  4. Taejoon Kwon
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Contributions

All authors contributed to the conception and design of the study. Material preparation, data collection, and analysis were performed by KYK and DGJ. The analysis of IP/MS data was performed by HJ and TK. The draft of the manuscript was written by KYK and TJP. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Taejoon Kwon or Tae Joo Park.

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Informed consent was obtained from all individual participants included in the study.

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Movie 1: Movie of fluid flow shown by fluorescent beads over the control embryonic skin Supplementary file1 (AVI 21008 KB)

Supplementary Movie 2: Movie of fluid flow shown by fluorescent beads over the Ribc2 morphant embryonic skin Supplementary file2 (AVI 22119 KB)

Supplementary Movie 3: Movie of fluid flow shown by fluorescent beads over the Ckb morphant embryonic skin Supplementary file3 (AVI 21875 KB)

Supplementary Movie 4: Movie of fluid flow shown by fluorescent beads over the Ybx2 morphant embryonic skin Supplementary file4 (AVI 24344 KB)

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Kwon, K.Y., Jeong, H., Jang, D.G. et al. Ckb and Ybx2 interact with Ribc2 and are necessary for the ciliary beating of multi-cilia. Genes Genom 45, 157–167 (2023). https://doi.org/10.1007/s13258-022-01350-w

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  • DOI: https://doi.org/10.1007/s13258-022-01350-w

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