Dimensions of a Living Cochlear Hair Bundle

Abstract

The hair bundle is the mechanosensory organelle of hair cells that detects mechanical stimuli caused by sounds, head motions, and fluid flows. Each hair bundle is an assembly of cellular-protrusions called stereocilia, which differ in height to form a staircase. Stereocilia have different heights, widths, and separations in different species, sensory organs, positions within an organ, hair-cell types, and even within a single hair bundle. The dimensions of the stereociliary assembly dictate how the hair bundle responds to stimuli. These hair-bundle properties have been measured previously only to a limited degree. In particular, mammalian data are either incomplete, lack control for age or position within an organ, or have artifacts owing to fixation or dehydration. Here, we provide a complete set of measurements for postnatal day (P) 11 C57BL/6J mouse apical inner hair cells (IHCs) obtained from living tissue, tissue mildly-fixed for fluorescent imaging, or tissue strongly fixed and dehydrated for scanning electronic microscopy (SEM). We found that hair bundles mildly-fixed for fluorescence had the same dimensions as living hair bundles, whereas SEM-prepared hair bundles shrank uniformly in stereociliary heights, widths, and separations. By determining the shrinkage factors, we imputed live dimensions from SEM that were too small to observe optically. Accordingly, we created the first complete blueprint of a living IHC hair bundle. We show that SEM-prepared measurements strongly affect calculations of a bundle’s mechanical properties – overestimating stereociliary deflection stiffness and underestimating the fluid coupling between stereocilia. The methods of measurement, the data, and the consequences we describe illustrate the high levels of accuracy and precision required to understand hair-bundle mechanotransduction.

Control of stereocilia length during development of hair bundles

“…We determined dimensions of stereocilia F-actin cores, labeled with fluorescent phalloidin, which were imaged in apical IHCs of C57BL/6 mice at precise times during early postnatal development. Our measurements should reflect native lengths and widths; dimensions of mildly-fixed, phalloidin-labeled stereocilia are not significantly different from dimensions of live stereocilia labeled with membrane dyes [43]. We improved resolution over conventional confocal microscopy by using lattice structured illumination microscopy (lattice SIM) [44–46], which has a point-spread function (PSF) of ∼150 nm under our conditions [46]; typical confocal microscopy PSFs are ∼230 nm [47].…”

High-resolution immunofluorescence imaging of mouse cochlear hair bundles

“… After imaging, adjust the high and low intensity levels using an image processing software (e.g., Imaris or Fiji) to obtain low background and clear signal. Either select a z-slice containing the staining of interest, or select a range of z-slices to generate a maximum intensity projection, or generate a 3D-reconstruction of the image (for an example of 3D-reconstructed bundles, see Miller et al., 2021 ) to localize the protein of interest.

Figure 6 Immunofluorescence imaging of the bundles (A and B) (A) A confocal microscope (or better) is used to localize (B) fluorescently labeled proteins of interest located within the stereocilia in samples mounted below a coverslip.

…”

“…Either select a z-slice containing the staining of interest, or select a range of z-slices to generate a maximum intensity projection, or generate a 3D-reconstruction of the image (for an example of 3D-reconstructed bundles, see Miller et al., 2021 ) to localize the protein of interest.…”

“… Note: At the apical surface of each inner and outer hair cell is a staircase assembly of stereocilia (actin-filled cellular protrusions), which make up the hair bundle ( Tilney and Saunders, 1983 ). Cochlear stereocilia are typically a few microns tall, and less than half a micron wide ( Miller et al., 2021 ). Each shorter stereocilium is connected to a taller stereocilium through a thin extracellular filament, the tip link ( Figure 1 H) ( Pickles et al., 1984 ).…”

High-resolution imaging of the mouse-hair-cell hair bundle by scanning electron microscopy

“…Upon completion of this protocol, you should obtain crisp images of the hair bundle ( Figures 6 A–6C). Tip links are fragile structures but can easily be preserved and observed with this protocol ( Figures 6 D and 6E) ( Miller et al., 2021 ; Trouillet et al., 2021 ).

Figure 6 Examples of good SEM imaging of the cochlear hair bundles and tip links Areas corresponding to zoomed-in pictures presented in the next panel are indicated with a red border square or rectangle.

…”