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. 2016 Mar 1;7:10833. doi: 10.1038/ncomms10833

Figure 6. MYO3A and MYO3B separately localize in bundles but replace each other in the ear.

Figure 6

(a) Immunolabelling of MYO3A (green) in rat auditory hair cells (P3); actin (magenta) labelled with phalloidin. (right) MYO3A label only. (b) MYO3A and ESPNL-GFP do not completely overlap in hair bundle of outer hair cell (see inset). Elongated ESPNL-GFP labelling in row 2. (c) SIM image of outer hair-cell stereocilia showing MYO3A cap and elongated ESPNL-GFP. (d) MYO3A labelling in utricle bundle. (left) MYO3A image with transect indicated. (right) Intensity profile along transect. (e) (left) MYO3B labelling is greatest in tallest stereocilia (asterisks). (right) Intensity profile along transect. (f) Representation of exons 1–7 (blue boxes) of WT mouse Myo3a locus. The targeting vector replaced exons 4 and 5 with a floxed neomycin-resistance gene cassette, introducing a premature stop codon in exon 6. (g) Exons 1–8 of the Myo3b locus; the targeting vector (middle) was designed to eliminate exons 3 and 4, replacing them with a neomycin-resistance gene cassette conjugated to LacZ; a premature stop codon was introduced in exon 5. (h) RT–PCR on inner-ear mRNA showing 578 bp amplicon from WT mouse and expected 205 bp truncated band in Myo3a−/−. (i) Mean ABR measurements (±s.d.) from Myo3a−/− (n=29) and Myo3a+/− (n=27) mice at 6 months. (j) RT–PCR on inner-ear mRNA showing 695 bp amplicon from WT mouse and expected 319 bp truncated band from Myo3b−/−. (k) ABR from Myo3b−/− (n=9) and Myo3b+/− (n=9) mice at 6 months. (l) WT extrastriolar bundle (phalloidin stained). (m) Myo3a−/− extrastriolar bundle. (n) Myo3b−/− extrastriolar bundle. (o) Heights of short and tall stereocilia from indicated genotypes. Mean±s.d.; n=148 (WT), 123 (Myo3a−/−) and 199 (Myo3b−/−). Student’s t-test indicated Myo3b−/− short stereocilia lengths are different from WT at P=10−21; tall lengths are different at P=0.007. (p) Distribution of short stereocilia lengths. (q) Distribution of tall stereocilia lengths. Scale bars, (a,b,d,e) 5 μm; (c) 300 nm; and (l–n) 2 μm.