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Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man

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Abstract

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This “battery” is located “off-site” in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

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Abbreviations

Cx30:

Connexin30

Cx26:

Connexin26

GJ:

Gap junction

TJ:

Tight junction

TEM:

Transmission electron microscopy

EP:

Endocochlear potential

MIP:

Maximal intensity projection

SR-SIM:

Super-resolution structured illumination microscopy

IHC:

Immunohistochemistry

GJB2:

GJ protein gene beta 2

GJB6:

GJ protein gene beta 6

SV:

Stria vascularis

KCNJ10:

Gene encoding a member of the inward rectifier-type K+ channel family Kir4.1

NKCC1:

Gene encoding the furosemide-sensitive Na+/K+/2Cl- membrane co-transporter

KCNQ1/KCNE1:

Gene encoding the voltage-gated potassium channel Kv7.1 (KvLQT1)

3-D:

Three-dimensional

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Authors and Affiliations

  1. Department of Surgical Sciences, Head and Neck Surgery, Section of Otolaryngology, Department of Otolaryngology, Uppsala University Hospital, SE-751 85, Uppsala, Sweden

    Wei Liu, Fredrik Edin, Hao Li, Göran Laurell & Helge Rask-Andersen

  2. Science for Life laboratory, Department of Applied Physics, Royal Institute of Technology, Tomtebodavägen 23A, 17121, Solna, Sweden

    Hans Blom

  3. Department of Immunology, Genetics and Pathology, Clinical Immunology, Dag Hammarskjölds väg 20, 751 85, Uppsala, Sweden

    Peetra Magnusson

  4. Department of Otolaryngology, Medical University of Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria

    Annelies Schrott-Fischer & Rudolf Glueckert

  5. Department of Otolaryngology, University of Minnesota, 121, Lions Research Bldg., 2001 Sixth St. SE, Minneapolis, MN 55455, USA

    Peter A. Santi

Authors
  1. Wei Liu
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  2. Fredrik Edin
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  3. Hans Blom
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  4. Peetra Magnusson
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  5. Annelies Schrott-Fischer
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  6. Rudolf Glueckert
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  7. Peter A. Santi
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  8. Hao Li
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  9. Göran Laurell
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  10. Helge Rask-Andersen
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Corresponding authors

Correspondence to Rudolf Glueckert or Helge Rask-Andersen.

Additional information

This study was supported by ALF grants from Uppsala University Hospital and Uppsala University and by the Foundation of “Tysta Skolan,” the Swedish Deafness Foundation (HRF) and generous private donations from Börje Runögård and David Giertz, Sweden. This work was also funded in part by Medel (Fuerstenweg 77a, 6020 Innsbruck, Austria). Funding was also provided by NIDCD (PS - U24DC011968). Imaging was performed with equipment maintained by the Science for Life Lab BioVis Platform, Uppsala University. For skillful art work we thank Karin Lodin.

Electronic supplementary material

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ESM 1

Image and data precision were evaluated by means of an EMCCD camera and SIM focus from ZEN calibration on 40-nm beads. A lateral precision of approximately 80 nm and 250 nm axially was obtained. (PPTX 72.4 kb)

ESM 2

3-D video recording of the SR-SIMZ-stacks of Cx26 and Cx30 protein expression in the lateral cochlear wall demonstrating the separate expression. (WMV 7.80 mb)

ESM 3

Confocal IHC show basal cells identified by their expression of Claudin (green), a TJ protein expressed selectively in the basal cells and type I fibrocytes. These TJs help to maintain the particular electrochemical environment of the intrastrial space. Marginal and intermediate cells express Na+−K+−ATPase (red). (PPTX 2.01 mb)

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Liu, W., Edin, F., Blom, H. et al. Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man. Cell Tissue Res 365, 13–27 (2016). https://doi.org/10.1007/s00441-016-2359-0

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