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Fig. 2.
Distinct subcellular distributions of spectrin βV in the frog and mouse hair cells. (A–C) Frog (X. laevis). (A and B) In the vestibular hair cells (VHCs) of a frog utricular macula, spectrin βV (green) is invariably detected both in the cuticular plate (CP) and as an apical immunoreactive peripheral ring (arrowheads in A). The top views in B show spectrin βV labeling just above and under (asterisk) the cuticular plate. (C) At the CP apical surface, the spectrin βV immunostaining forms a well-organized punctate pattern, with seemingly uniformly spaced dots resembling lattice knots. (D–G) Mouse. (D and E) In the VHCs of a mouse crista ampullaris, spectrin βV is detected as a cytoplasmic punctate immunostaining both in type I and type II VHCs, which can be differentiated by the tubulin βIII immunolabeling of their afferent nerve fibers. Spectrin βV-immunoreactive puncta extend from the apical region near the cuticular plate down to the supranuclear region of the VHCs. (F and G) In the cochlea, spectrin βV is present both in inner hair cells (IHCs) and outer hair cells (OHCs). Note that the immunostaining of the IHC lateral wall is restricted to the neck region (arrowheads), whereas the protein is detected in (and restricted to) the entire lateral wall of OHCs, where it contributes to the cortical lattice (see diagrams) (F). (G) In the OHCs, the spectrin βV immunostaining matches the distribution of the lateral plasma membrane protein prestin (arrowheads). Bars, 5 µm. |
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Figure S4: Distribution of spectrin βV in the frog and mouse cochlear and vestibular hair cells
at embryonic and postnatal ages
(A) Whole-mount preparations of a frog (X. laevis) utricular macula (UM). In all frog hair cells,
spectrin βV is apically located just under the apical plasma membrane. Right panels: In addition to a
peripheral ring-shaped immunostaining at the level of the apical junctional complex (arrows), frog
hair cells displays a grid-like periodic pattern of spectrin βV immunoreactivity overlying the
cuticular plate (CP). As we go deeper into the cuticular plate, the spectrin βV immunostaining
becomes diffuse (arrowheads). The 2D spatial Fourier transformation of the distribution formed by
the immunoreactive spots displays an approximate hexagonal symmetry (yellow dashed lines).
Each
cell contains about to 139 ± 14 spots (mean ± s.e.m., n = 16 cells), with the mean spacing between
the spots being 0.53 ± 0.07 μm (mean ± s.e.m., measured on 64 rows manually selected on the same
16 cells).
(B) Left panel: Cryosections of a mouse vestibular sensory epithelium (ampullar crista) on
embryonic day 18 (E18). The myosin VIIa immunostaining indicates the position of the hair cells.
Spectrin βV is detected in a few hair cells at the periphery of the sensory organ (see magnified view of the boxed area). Upper right panel: Whole-mount preparations at developmental (E20) and
mature (postnatal day 10 (P10), P30, and P90) stages. The arrowhead at the midpoint of the sensory
epithelium stained for myosin VIIa indicates the eminentia cruciatum, a region devoid of sensory
cells. Unlike the spectrin αII immunostaining, the spectrin βV immunostaining of hair cells is
restricted to the periphery of the sensory epithelium at all stages analyzed. Lower right panel:
Isolated vestibular hair cells (VHCs) immunostained for spectrin βV (green). Actin filaments are
stained in red with TRITC-phalloidin and cell nuclei are stained in blue with DAPI. Unlike
spectrins αII and βII, which are mainly detected in the apical region of hair cells, specifically in the
CP, the spectrin βV staining displays a broad punctate distribution pattern, extending from the
pericuticular region down to the supranuclear region.
(C) Whole-mount preparations of the mouse cochlear sensory epithelium on E20 and P8. The
myosin VIIa immunostaining indicates the position of the hair cells. The spectrin βV
immunostaining is not detected before birth. Note the intense spectrin βV immunostaining of the
lateral wall in the outer hair cells (OHCs) on P8.
Bars = 5 μm in (A,C) and 25 μm in (B).
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