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Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells

Abstract

The organ of Corti is an auditory organ located in the cochlea, comprising hair cells (HCs) and other supporting cells. Cellular shape changes of HCs are important for the development of auditory epithelia and hearing function. It was previously observed that HCs and inner sulcus cells (ISCs) demonstrate cellular shape changes similar to the apical constriction of the neural epithelia. Apical constriction is induced via actomyosin cable contraction in the apical junctional complex and necessary for the physiological function of the epithelium. Actomyosin cable contraction is mainly regulated by myosin regulatory light chain (MRLC) phosphorylation by myosin light chain kinase (MLCK). However, MRLC and MLCK isoforms expressed in HCs and ISCs are unknown. Hence, we investigated the expression patterns and roles of MRLCs and MLCKs in HCs. Droplet digital PCR revealed that HCs expressed MYL12A/B and MYL9, which are non-muscle MRLC and smooth muscle MLCK (smMLCK), respectively. Immunofluorescence staining throughout the organ of Corti demonstrated that only MYL12 was expressed in the apical portion of HCs, whereas MYL12 and MYL9 were expressed on ISCs. In addition, purified MYL12B was phosphorylated by smMLCK in vitro, and the harvested HCs contained phosphorylated MYL12. Furthermore, accompanied by the expansion of the cell area of outer HCs, MYL12 phosphorylation was reduced by ML-7, which is an inhibitor of smMLCK. In conclusion, MYL12 phosphorylation by smMLCK contributed to the apical constriction-like cellular shape change of HCs possibly relating to the development of auditory epithelia and hearing function.

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Availability of Data and Material

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgments

The authors would like to thank Enago (www.enago.jp) for the English language review.

Funding

This study was supported by the GSK Japan Research Grant 2019.

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Authors

Contributions

R.O. and T.S. designed the study. R.O. performed all the experiments in this study and wrote the manuscript. O.T. and S.T. provided the advice for protein purification. H.K., K.M., T.K., Y.O., and T.I. provided critical advice and reviewed the article. K.O. and H.I. supervised the project.

Corresponding author

Correspondence to Osamu Tsukamoto.

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Ethics Approval

All animal procedures and the protocol for these experiments, including genomic recombination, were reviewed and approved by the Institute of Experimental Animal Sciences of the Faculty of Medicine at Osaka University (No. 26-080-038).

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The authors declare that they have no conflicts of interest.

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The present study does not include human subjects. Therefore, consent to participate was waived.

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The present study does not include human subjects. Therefore, consent for publication was waived.

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Oya, R., Tsukamoto, O., Sato, T. et al. Phosphorylation of MYL12 by Myosin Light Chain Kinase Regulates Cellular Shape Changes in Cochlear Hair Cells. JARO 22, 425–441 (2021). https://doi.org/10.1007/s10162-021-00796-1

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Keywords

  • cochlea
  • hair cells
  • apical junctional complex
  • non-muscle myosin II
  • myosin light chain kinase