Myosin XVa localizes to the tips of inner ear sensory cell stereocilia and is essential for staircase formation of the hair bundle

Abstract

Mutations of the gene encoding unconventional myosin XVa are associated with sensorineural deafness in humans (DFNB3) and shaker (Myo15sh2) mice. In deaf Myo15sh2/sh2 mice, stereocilia are short, nearly equal in length, and lack myosin XVa immunoreactivity. We previously reported that myosin XVa mRNA and protein are expressed in cochlear hair cells. We now show that in the mouse, rat, and guinea pig, endogenous myosin XVa localizes to the tips of the stereocilia of the cochlear and vestibular hair cells. Myosin XVa localization overlaps with the barbed ends of actin filaments and extends to the apical plasma membrane of the stereocilia. Gene gun-mediated transfection of mouse inner ear sensory epithelia explants shows selective accumulation of myosin XVa-GFP at the tips of stereocilia, confirming the localization of native myosin XVa. Expression in COS7 cells also reveals targeting of myosin XVa-GFP to the dynamic actin region at the tips of filopodia. In a wild-type mouse, during auditory and vestibular hair cell development, myosin XVa appears at the tips of stereocilia at the time when the hair bundle begins to develop its characteristic staircase pattern. We propose that myosin XVa is essential for the graded elongation of stereocilia during their functional maturation.

Fig. 1.

Evaluation of the specificity of antimyosin XVa antibodies in COS7 cells transiently transfected with [-N]Myo15a-GFP. (A) Domain structure of the chimeric protein encoded by [-N]Myo15a-GFP construct. Red bars depict the location of myosin XVa epitopes to which TF1 and PB48 antisera were raised. Shown are COS7 cells transfected with [-N]Myo15a-GFP (green; B) and stained with TF1 antibody (red; C). (D) Colocalization of the GFP signal of myosin XVa-GFP with TF1 antibody staining. (B–D Insets) Enlarged from the areas marked by the squares. Arrows point to myosin XVa-GFP at the tips of filopodia. (Bar = 10 μm.)

Fig. 2.

Localization of myosin XVa protein in OC hair cell stereocilia visualized by using TF1 antiserum (green) in rat, guinea pig, and mouse. The actin core of each stereocilium was counterstained with rhodamine–phalloidin (red). (A) Localization of myosin XVa at the tips of stereocilia of three rows of OHCs and one row of IHCs of P5 mouse. (B) Myosin XVa at the tips of OHC stereocilia of adult guinea pig OC. (C) IHC stereocilia of adult rat OC were splayed by pressing on the coverslip to better visualize the myosin XVa signal at each stereocilium tip. Arrow indicates stereocilium enlarged in D–F. (D) Myosin XVa staining is concentrated at the tip (green). No detectable staining of myosin XVa is evident along the length of a stereocilium. (E) Red channel of the same image. We were unable to detect F-actin at the tip. (F) Double staining reveals partial overlap of myosin XVa signal (green, D) with F-actin core of stereocilia (red, E). (G–I) Myosin XVa immunoreactivity (green) in the OC of E18.5 mouse at basal (G), middle (H), and apical (I) turns. Maturation of hair cells progresses from base to apex of the cochlea with the development of IHCs slightly preceding OHCs (28). Myosin XVa staining (green) is prominent at the tips of stereocilia of IHC and OHC in the basal turn, faint in the middle turn, and not detectable in apical turn. Arrows point to the immature stereocilia, which lack myosin XVa signal at the tips. (Bars = 5 μm.)

Fig. 4.

Myosin XVa is absent from stereocilia of P21 Myo15^sh2/sh2 mice. Hair cell stereocilia of a P21 Myo15^sh2/sh2 mouse utricle (A) and OC (B) lack myosin XVa immunoreactivity at the tips (TF1, green; rhodamine–phalloidin, red). Note that stereocilia have a similar length in the different rows within a bundle but are much shorter than corresponding mature hair bundles of a control C57BL/6 P19 mouse utricle (C) and OC (D) hair cells, which have myosin XVa at the stereocilia tips. (Bars = 5 μm.)

Fig. 3.

Localization of myosin XVa (green) at the tips of stereocilia of mouse vestibular hair cells. (A) Myosin XVa, revealed by TF1 antibody, at the tips of stereocilia of E14.5 mouse hair cells of the ampulla, when the gradation in height of stereocilia is just beginning to appear. (B) Myosin XVa at the tips of stereocilia of hair cells of adult mouse ampulla stained with TF1 antiserum. (C) PB48 antibody also revealed myosin XVa at the tips of saccular hair cell stereocilia of adult mouse. (Bars = 5 μm.)

Fig. 5.

Gene gun-mediated transfection of mouse vestibular and cochlear sensory epithelia explants with [-N]Myo15a-GFP (green). The actin core of stereocilia was counterstained with rhodamine–phalloidin (red). (A) Saccular hair cell 24 h after transfection. (B) Myosin XVa-GFP (green) at the tips of stereocilia 18 h after transfection of IHC from P5 OC explants. (C) The image of utricular explants illustrating that stereocilia bundles of three hair cells 48 h after transfection have slightly different amounts of myosin XVa-GFP at the tips of stereocilia. (D) Distension of the stereocilia tips of a vestibular hair cell due to an excessive accumulation of myosin XVa-GFP 96 h after transfection (see also Fig. 8). (E) Myosin XVa-GFP surrounds the top of the actin filaments of stereocilia but does not penetrate inside the actin core. Unstained spots corresponding to the actin core are visible in the center of patches of green fluorescence. (F) Rhodamine–phalloidin staining of the same stereocilia bundle. (Bars = 2 μm.)