A cytoskeleton structure revealed by super-resolution fluorescence imaging in inner ear hair cells

Dear Editor,

F-actin is expressed in almost all cells, and plays a variety of important roles1,2. In the cuticular plate of hair cells, they are thought to be critical in mammalian hearing by holding the stereocilia rootlets in place and providing the rigidity and support necessary for auditory transduction3,4. Due to the high spatial resolution and molecular specificity, the F-actin structure in several types of cells has been recently revealed by super-resolution fluorescence imaging5,6. However, how F-actin performs their function in inner ear hair cells remains elusive, partly due to an incomplete understanding of their structure. Here we applied structured illumination microscopy (SIM) imaging to study the sub-diffraction-limit structures of F-actin in the cuticular plate of rodents in order to better understand their function in the development of hearing.

A dense F-actin meshwork has been reported to exist underneath the apical plasma membrane in the cuticular region of hair cells3,7,8. Using SIM imaging, we observed ring-like structures of F-actin with a small point cluster at the center, outlining the edges of both inner hair cell (IHC) and outer hair cell (OHC) stereocilia rootlets (Fig. 1a), consistent with the previous EM reconstructed results that F-actin forms two rows of ring-like structure wrapping around the stereocilia rootlets in OHCs7. Importantly, a previously unseen fan-shaped meshwork was observed in the OHCs (Fig. 1). The fan-shaped structure was observed in different regions of the cochlea (Fig. 1b), implying a common organization of F-actin in the cuticular plate. Similar to OHCs, ring-like structures of F-actin were observed outlining the edges of IHC stereocilia rootlets (Supplementary Fig. S1a). The small point cluster at the center of each F-actin ring is directly corresponding to individual rootlet (Supplementary Fig. S1b). To investigate how the ring and fan-shaped structure F-actin is organized in the cuticular plate, we visualized the structure of the other actin-associated proteins, such as α-actinin. α-actinin cross-links F-actin bundles and often plays a crucial role to organize F-actin structure. Earlier studies showed the expression of α-actinin in the cuticular plate9, and likely it plays a role in organizing the structure of F-actin in the cuticular plate. Here, we found that α-actinin was specifically expressed in the cuticular plate and did not observe the ring or fan-shape like structure of α-actinin with super-resolution imaging experiments (Supplementary Fig. S2).

Fig. 1: Structure of F-actin in the cuticular plate.

a Representative conventional epifluorescence vs. SIM fluorescence images of F-actin in the cuticular plates of OHCs from P12 mice (n = 5 mice). Magnifications of the boxed region and intensity profiles from the corresponding lines are shown. b Representative SIM images of F-actin in the cuticular plates from P12 mice at the apex (n = 3 mice), middle (n = 3 mice) and base (n = 3 mice) region of the cochlea. Intensity profiles along dashed lines are shown. x̅ shows the average distance between two adjacent peaks. c Representative serial optical sections (interval = 0.125 μm) of SIM images starting from the apical surface towards the deep region of the cuticular plate of OHC (P12, n = 5 mice) along the Z-axis. Intensity profiles along the dotted lines are shown. Scale bars: 1 μm in upper panel a, 500 nm in lower panel a, 1 μm in b, c

The regular structural pattern of F-actin prompted us to ask whether this structure remained in three-dimensional space of the cuticular plate. We performed three-dimensional SIM experiments. SIM images showed that the F-actin rings and fan meshwork were aligned at different depths that extended more than 750 nm into the cuticular plate of OHCs (Fig. 1c, Supplementary Fig. S3, Supplementary Movies S1-6). Such three-dimensional organized regular pattern of the F-actin cytoskeleton may strength its elasticity, increase the stability of cuticular plate and thus is suitable to hold stereocilia in place after deflections with sounds. F-actin rings were also observed in rat OHCs and IHCs (Supplementary Fig. S4), suggesting that this framework for the spatial patterning of the stereocilia rootlets in the cuticular plate may reflect a common phenomenon in mammalian HCs.

To test whether these structures existed in the early stages of hair cell development, we observed the F-actin structures at different postnatal developmental stages. F-actin concentrated in the cuticular plate of OHCs as early as 0 days after birth (P0), and interestingly no F-actin fan-shaped structure was detected (Supplementary Fig. S5a). Fan-shaped meshwork of F-actin were observed in the cuticular plate of OHCs as early as P7, and remained visible at P30 (Supplementary Fig. S5a-c, Supplementary Movies S1-6). Next, we found that F-actin rings were formed in early development and remained until the mature stage (Supplementary Movies S1-6). Notably, the average diameter of stereocilia rootlets is larger in P0 than that in mature hair cell (Supplementary Fig. S6). Earlier EM experiments elegantly reconstructed the ring-like structure of F-actin in the cuticular plate10, and here we extended the earlier results to observe the F-actin bundle which corresponds to each stereocilium and quantitated the properties of the F-actin bundle in the cuticular plate during development with SIM experiments (Supplementary Fig. S6). Our new results suggest that F-actin in rootlets may undergo a developmental structural organization and become more compact and thus support the stereocilia deflection during sound detection. SIM imaging demonstrated that F-actin rings and fan-shaped meshwork appeared in OHCs from the first week after birth and remained to the mature stage. Functionally, the structures of F-actin in the cuticular plate of the hearing impaired Atoh1-Brg1/ mice11 with OHC morphology disrupted (Supplementary Fig. S7a) showed that, as expected, their rings were severely disrupted in the OHCs, and characteristic fan-shaped meshwork of F-actin had entirely disappeared (Supplementary Fig. S7). Given our findings, it seems likely that the absence of the F-actin rings and the F-actin fan-shaped meshwork would lead to the disruption of the stereocilia rootlets, changing their spatial pattern in the cuticular plate and eventually leading to the disappearance of stereocilia in OHC (Supplementary Fig. S7b). These results support that the organization of the cuticular cytoskeleton is associated with the proper development of stereocilia and thus contributing to the hearing loss in these mice.

In summary, this study has characterized the F-actin nanoscale structures in the cuticular plate with super-resolution imaging method. Our results demonstrate that F-actin forms ring-like structures corresponding to each stereocilium and develops a previously unknown fan-shaped network. Such spatial organization of F-actin in cuticular plate may play a critical role in hearing function.


  1. 1.

    Bennett, V. & Baines, A. J. Spectrin and ankyrin-based pathways: Metazoan inventions for integrating cells into tissues. Physiol. Rev. 81, 1353–1392 (2001).

  2. 2.

    Baines, A. J. The spectrin-ankyrin-4.1-adducin membrane skeleton: adapting eukaryotic cells to the demands of animal life. Protoplasma 244, 99–131 (2010).

  3. 3.

    Tilney, L. G. & DeRosier, D. J. Actin filaments, stereocilia, and hair cells of the bird cochlea. IV. How the actin filaments become organized in developing stereocilia and in the cuticular plate. Dev. Biol. 116, 119–129 (1986).

  4. 4.

    Derosier, D. J. & Tilney, L. G. The structure of the cuticular plate, an in vivo actin gel. J. Cell Biol. 109, 2853–2867 (1989).

  5. 5.

    Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2013).

  6. 6.

    Zhong, G. et al. Developmental mechanism of the periodic membrane skeleton in axons. Elife 3, e04581(2014).

  7. 7.

    Furness, D. N., Mahendrasingam, S., Ohashi, M., Fettiplace, R. & Hackney, C. M. The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane. J. Neurosci. 28, 6342–6353 (2008).

  8. 8.

    Arima, T., Uemura, T. & Yamamoto, T. Three-dimensional visualizations of the inner ear hair cell of the guinea pig. A rapid-freeze, deep-etch study of filamentous and membranous organelles. Hear. Res. 25, 61–68 (1987).

  9. 9.

    Slepecky, N. & Chamberlain, S. C. Immunoelectron microscopic and immunofluorescent localization of cytoskeletal and muscle-like contractile proteins in inner ear sensory hair cells. Hear. Res. 20, 245–260 (1985).

  10. 10.

    Furness, D. N., Mahendrasingam, S., Ohashi, M., Fettiplace, R. & Hackney, C. M. The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high and low frequency cells and evidence for a connection to the lateral membrane. J. Neurosci. 28, 6342 (2008).

  11. 11.

    Jin, Y. et al. Deletion of Brg1 causes abnormal hair cell planer polarity, hair cell anchorage, and scar formation in mouse cochlea. Sci. Rep. 6, 27124 (2016).

Download references


We thank the Shanghai Municipal Government and ShanghaiTech University for financial support. We thank the Bioimaging Core Facilities of the iHuman Institute and the animal facility of Shanghai Biomodel Organism Science and Technology Development Co., Ltd for their support. We also thank Fei Hao and Haixiao Liu for helpful discussions. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Science (XDA16010302), the National Key Research and Development Program of China (2016YFC0905900, 2017YFA0103900, 2017YFC1001300, 2015CB965000), the 2015 Thousand Youth Talents Plan of China (Guisheng Zhong, Renjie Chai), and the National Natural Science Foundation of China (31200743, 31771130, 31671062, 81622013, 81470692, 31500852), the Natural Science Foundation from Jiangsu Province BK20150022, Boehringer Ingelheim Pharma GmbH, and the Yingdong Huo Education Foundation.

Author information

G.Z., R.C., and S.H. conceived and designed the experiments. J.Q. and Y.L. performed most of the experiments and data analyses. G.Z., J.Q., C.C., Y.L., X.C., W.Z., Y.S., and R.C. contributed to data analysis. G.Z., J.Q., S.H., and Y.L. discussed data analysis, interpretation and presentation. G.Z., J.Q., and Y.L. wrote the manuscript with contributions from all of the authors.

Correspondence to Shuijin He or Renjie Chai or Guisheng Zhong.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Supplementary Information

Supplementary Movie S1

Supplementary Movie S2

Supplementary Movie S3

Supplementary Movie S4

Supplementary Movie S5

Supplementary Movie S6

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark