Journal Club: Myosin light chain and related kinase regulate the stiffness and oscillatory behaviour of stereocilia on hair cells.

on

Today's journal article

Oya R, Woo KM, Fabella B, Alonso RG, Bravo P, Hudspeth AJ. Influence of Myosin Regulatory Light Chain and Myosin Light Chain Kinase on the Physiological Function of Inner Ear Hair Cells. 

Why I picked this article

Sensory hair cells in the cochlea and vestibular system detect sound and head motion with hair bundles called stereocilia. Stereocilia are stiff structures on hair cells that, when bent, open mechanotransduction channels and allow signal transduction. For these bundles to work, the cell must set a proper “operating point” to set the right stiffness, resting open probability, and active motility need tight control.

Stereocilia are made up of a number of specialised proteins. Examples are "Non-muscle myosin II (NM2)" and its regulatory light chain, phosphorylated by yet another protein called the "myosin light chain kinase (MLCK)". Together, these proteins seem to regulate the shape changes of the stereocilia in auditory hair cells. This research aim to investigate the relationship between NM2 and MLCK with other proteins in how they regulate stiffness and sensitivity of stereocilia, using multiple species - frog and mice. 

Some of the research findings

Model and timing:
  • Frog: Adult American bullfrogs (Rana catesbeiana) 
    • saccule; 6–10 saccules per experiment were dissected.
    • mounted into a two-compartment experiment chamber to monitor stereocilia
    • Fibre was used to stimulate hair-bundle, and motion was recorded to estimate stiffness. 
  • Mammal: Female C57BL/6 N mice 7 to 8 weeks of age
Antibody detection of key proteins & manipulation:
  • Antibody labeling & microscopy: 
    • anti-MYH9 (ab238131; Abcam, Cambridge, UK; 1:50)
    • anti-MYH10 (ab230823; Abcam; 1:50)
    • anti-MYH14 (20,716–1-AP; Proteintech, Rosemont, IL, USA; 1:50)
    • anti-MYL12A (16,287–1-AP; Proteintech; 1:100)
    • anti-MYL12B (10,324–1-AP; Proteintech; 1:100)
    • anti-MYL9 (ab191393;Abcam; 1:50)
  • blebbistatin - 10 μM = NM2 inhibitor
  • NM2 inhibitors and MLCK inhibitors applied to bundles (frog); MLCK inhibitor delivered middle ear (mouse).
Part of Figure 1C. Schematics of the stereocilia, cuticular plate (where stereocilia are anchored) and where you find some key proteins of interest in this research. Oya2025

Finding: 
  • Proteins of interest - NM2A and NM2B were found on the apical surface of the hair cells, as well as under the cuticular plate. 
  • Apical surface: NM2A/B and MYL9 detected.
  • Hair bundles: NM2A and MYL12A present.
  • Stiffness: NM2 and MLCK inhibition reduced hair-bundle stiffness.
  • Active motility: MLCK inhibition suppressed spontaneous hair-bundle oscillations.
  • Operating point: MLCK inhibition increased the resting open probability of MET channels.
  • Hearing: MLCK inhibition elevated ABR thresholds in mice (poorer sensitivity).
The research together suggests that NM2-RLC-MLCK together sets the mechanics of the stereocilia and MET operating point. Disrupting phosphorylation of myosin softens bundles, shifts the resting status of the channel opening, and dampens active oscillations.

Haruna's takeaway

Wao! This is a very cool research drilling into the details of how exactly stereocilia are tuned and maintained for their stiffness, which is very critical for their sensitivity. It was kind of a timely research article to read, as I was just having a discussion with the team about what "cuticular plate" is and how stereocilia are situated/maintained on them. Microscopy images of different myosin and NM proteins are beautiful, and I personally get fascinated by those. I have not looked at frog hair cells myself, as I normally work with mammalian models, so the saccule of frogs looks very different to what I'm used to. I would love to see more detailed microscopy of various proteins in the mammalian cuticular plate, too. 

 ------- 

This is Haruna's 59/100 of the 100-day challenge to post a science blog article every day! I love inner ear biology & cochlear physiology.