Differential localization of β_cyto- and γ_cyto-actin in the mouse organ of Corti (OC). (A) The OC has three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs). Each hair cell is surrounded by non-sensory supporting cells. (B) Scanning electron microscopy images of OHC and IHC stereocilia bundles. (C) Stereocilium core consists of tightly packed unidirectional actin filaments (F-actin). In (D–T), rhodamine-phalloidin highlights F-actin (red), and actin stained by antibodies (green). Isoform-specific antibodies detect β_cyto-actin (D) and γ_cyto-actin (E) along the length of adult wild-type (wt) OHC and IHC stereocilia. (F) Absence of γ_cyto-actin (green) in 6-week-old Actg1^−/− OC. (G–L) At E16.5, β_cyto-actin immunoreactivity follows rhodamine-phalloidin labeling in wt hair cells (G–I), whereas γ_cyto-actin is detected in supporting cells but not in hair cells (J–L). (M–P) At E18.5, β_cyto-actin is present in all stereocilia of hair cells throughout the cochlea (M, N), whereas γ_cyto-actin begins to appear only in stereocilia of more developed basal turn of the cochlea (O, P). (Q–T) β_cyto-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidin staining, whereas γ_cyto-actin (S, T) is concentrated toward the periphery of the IHC stereocilia F-actin core in adult wt mice. Scale bars (B, Q–T), 2 μm; scale bars (D–P), 5 μm.

Morphology of stereocilia bundles in adult wild-type (Actg^+/+) and γ_cyto-actin deficient (Actg1^−/−) mice. (A–D) Scanning electron micrographs of stereocilia from (A, B) 6-week-old Actg1^+/+ and (C, D) 6-week-old Actg1^−/− mice. (E–H) Scanning electron microscopy images of OHC stereocilia from 16-week-old Actg1^+/+ (E, F) and 16-week-old Actg1^−/− mice (G, H). There is a loss of individual stereocilia from all three rows of OHC hair bundle from Actg1^−/− mice. Images are from the middle turn of the cochlea. (I) Box and whisker plot (whiskers, maximum and minimum; box, 5^th–95^th percentile; line, mean) of the number of individual stereocilia in individual OHC bundles from Actg1^+/+ or Actg1^−/− mice at 6 and 16 weeks of age, *P < 0.005. (J–K) enlargements of image in (H) with arrows indicating missing and shortened stereocilia. Scale bars (A, C, E, and G), 5 μm; scale bars (B, D, F, H, J, and K), 1 μm.

Characterization of live-born homozygous mutant Actg1^−/− mice. (A) Body mass growth curve of Actg1^+/+ (wild-type, closed squares), Actg1^+/− (heterozygous, open circles) and Actg1^−/− (homozygous mutant, open triangles) mice from P28 until P300 (n = 12 Actg1^+/+, 18 Actg1^+/−, 11 Actg1^−/−, mean ± SEM). (B) Kaplan-Meier survival curve of Actg1^+/− and Actg1^−/− mice from P0 to P350, (n = 31 for each genotype). (C) Representative immunoblots of SDS extracts from Actg1^+/+, Actg1^+/− and Actg1^−/− cochlear extracts probed with antibodies specific for γ_cyto-actin, β_cyto-actin, pan-actin, or tubulin antibody. Protein levels were quantified and are expressed relative to the wild-type level (mean ± SEM). (D) Actg1^−/− mice develop progressive hearing loss. Auditory brainstem response (ABR) thresholds were determined for Actg1^+/+ and Actg1^−/− mice at 6, 16, and 24 weeks of age using stimulus frequencies from 4 to 22 kHz, presented at half-octave steps (n > 5, mean ± SEM).

γ_cyto-Actin concentrates at the sites of stereocilia core disruptions. (A–C) γ_cyto-Actin antibody highlights gaps (segments of F-actin depolymerization; arrows) in wild-type (wt) mouse vestibular hair cell (VHC) stereocilia. In all panels, rhodamine-phalloidin highlights F-actin in red, and labeling with antibodies is in green. (D) γ_cyto-Actin at the base and within the F-actin gaps of longest stereocilia in wt mouse VHC (arrows). (E) DNase I stains globular actin within F-actin gaps of VHC stereocilia (arrows). (F) Espin concentrates in gaps of wt VHC stereocilia. (G) Uniform distribution of γ_cyto-actin along adult guinea pig IHC stereocilia not exposed to damaging noise. (H) Redistribution of γ_cyto-actin in noise-damaged guinea pig IHC stereocilia. γ_cyto-Actin absent from the tips and evenly distributed along stereocilia which appear unaffected (inset: second and fifth stereocilium from the left). (I–K) γ_cyto-Actin concentrates at sites of F-actin damage (arrows) and at tips of shortened stereocilia (asterisks, inset in H) in a noise-damaged bundle from (H). (L) The F-actin gaps in IHC stereocilia from Actg1^−/− mouse (arrows). (M) β_cyto-Actin concentrates in the F-actin gap of Actg1^−/− IHC stereocilium. β_cyto-Actin staining along stereocilia is barely visible because of intense gap staining. (N–P) Espin concentrates in gaps of Actg1^−/− VHC stereocilia. Scale bars, 2 μm.