β-actin and γ-actin are each dispensable for auditory hair cell development but required for Stereocilia maintenance

Abstract

Hair cell stereocilia structure depends on actin filaments composed of cytoplasmic β-actin and γ-actin isoforms. Mutations in either gene can lead to progressive hearing loss in humans. Since β-actin and γ-actin isoforms are 99% identical at the protein level, it is unclear whether each isoform has distinct cellular roles. Here, we compared the functions of β-actin and γ-actin in stereocilia formation and maintenance by generating mice conditionally knocked out for Actb or Actg1 in hair cells. We found that, although cytoplasmic actin is necessary, neither β-actin nor γ-actin is required for normal stereocilia development or auditory function in young animals. However, aging mice with β-actin- or γ-actin-deficient hair cells develop different patterns of progressive hearing loss and distinct pathogenic changes in stereocilia morphology, despite colocalization of the actin isoforms. These results demonstrate overlapping developmental roles but unique post-developmental functions for β-actin and γ-actin in maintaining hair cell stereocilia.

Figure 1. β-actin is not required for stereocilia formation.

(A–F) Hair cells from control or Actb-flox Foxg1-cre mice. β-Actin staining of stereocilia in control pup overlays with phalloidin staining (A–B), but is absent from all cell types in Actb-flox Foxg1-cre mice (C) while phalloidin staining of stereocilia appears normal (D). At P21, phalloidin stained stereocilia are similar in control (E) and Actb-flox Foxg1-cre (F) hair cells. (G–L) Analysis of Actb-flox Atoh1-cre hair cells. In control hair cells (G–H), anti-β-actin stained stereocilia overlay with phalloidin staining. In Actb-flox Foxg1-cre hair cells β-actin is stained in support cells but is mostly absent from hair cell stereocilia (I), while phalloidin staining reveals normal stereocilia (J). (K–L) SEM analysis shows that control (K) and Actb-flox Atoh1-cre (L) stereocilia have similar architecture. In merged images, phalloidin staining is red and β-actin is green. Bar in A–J is 5 µm; K–L is 1 µm.

Figure 2. β-actin and γ-actin colocalize in hair cells.

(A–H) Comparison of γ-actin localization using unlabeled anti-γ-actin antibody with a fluorescent secondary antibody (A–D) or using the same anti-γ-actin antibody coupled directly to a fluorescent dye (E–H). Detection with a secondary antibody results in a peripheral localization pattern when viewed laterally (A–B) or end-on (C–D). Dye-conjugated anti-γ-actin antibody labels stereocilia uniformly and completely overlays with phalloidin in lateral (E–F) or end-on (G–H) views. In merged images, γ-actin is green and phalloidin is red. (I–Q) Localization of β-actin and γ-actin using primary antibodies conjugated to two different fluorescent dyes. γ-Actin and β-actin colocalize in stereocilia (I–K), cuticular plate (L–N) and lateral wall (O–Q). In merged images, β-actin is green and γ-actin is red. Arrowheads indicate the structure of interest and the asterisk in (N) marks the top of a support cell that borders outer hair cells. Bars in F and Q, 5 µm; H, 2 µm.

Figure 3. Actb Actg1 double knockout cells do not develop stereocilia.

(A, B) Phalloidin staining of organ of Corti from control (A) or Actb-flox Actg1-flox Atoh1-cre (double knockout) (B) P5 pups shows normal stereocilia in control but generally absent stereocilia in the double knockout. (C–H) double label with dye-conjugated antibodies to β-actin or γ-actin show that remaining stereocilia contain one of the actin isoforms. In merged images in (G–H) β-actin is green and γ-actin is red. Bars, 5 µm.

Figure 4. Mice with hair cells deficient for either β-actin or γ-actin develop distinct forms of progressive hearing loss.

Auditory brainstem response (ABR) thresholds at 6, 18 and 24 weeks of age. (A) Actg1-flox Atoh1-cre mice have near normal ABR thresholds for sound tones between 4 and 22 kHz at 6 weeks of age, but elevated thresholds at all frequencies at 18 and 24 weeks of age. (B) Actb-flox-Atoh1-cre mice have progressive loss of hearing starting with higher frequency sounds.

Figure 5. β-actin and γ-actin deficient stereocilia develop distinct pathology.

Analysis of stereocilia morphology by scanning electron microscopy at 18 weeks-of-age (A–L) and 24 weeks-of-age (M–P). (A–D) 18 Week-old control stereocilia. (E–H) 18 Week-old γ-actin deficient stereocilia from all cochlear locations are degraded with significant numbers of individual stereocilia missing (Q). (I–L) β-Actin deficient stereocilia at 18 weeks of age from the apical (J) and middle turns (I,K) are similar to control, while stereocilia from the basal turn are abnormally short (L). Bars, 2 µm. (M–O) Comparison of stereocilia pathology at 24 weeks-of-age. γ-Actin deficient bundles are missing individual stereocilia while β-actin deficient stereocilia are uniformly shortened. Bar, 1 µm. (P) β-Actin deficient stereocilia from the middle turn have developed pathology ranging from mild to severe shortening of most members of whole rows in the bundle. Bars, 2 µm. (Q) Number of stereocilia in 18 week-old hair cell bundles, only Actg1-flox Atoh1-cre stereocilia numbers are statistically different. 40 OHC bundles from 4 ears of each genotype were analyzed. (R–T) Analysis of the first row of stereocilia of bundles from 24 week-old mice. The number (R) and average length (S) of first row stereocilia were determined. The percent of these stereocilia that were of normal length (within 2 standard deviations of the control average) was calculated (T). In (R–T), each dot plotted represents one cell (more than 35 cells from 3 mice of each genotype) and the bar is the mean. All groups are statistically different from all other groups.

Figure 6. Onset and progression of hearing loss depends on γ-actin concentration.

(A) γ-actin protein levels in cochlea from Actg1^+/+, Actg1^+/−, Actg1^O/O, Actg1^O/− and Actg1^−/− mice were quantified using immunoblot analysis. (B) 22 kHz auditory brainstem response (ABR) thresholds were determined at the indicated ages, demonstrating that the onset and rate of hearing loss progression depend on the concentration of γ-actin. Actg1^−/− data is replotted from . (C) ABR thresholds from 28 week-old Actg1^+/+, Actg1^O/O and Actg1^O/− mice. (D) Scanning electron microscopy of outer hair cells from Actg1^O/− mice, demonstrating that mice with hypomorphic γ-actin expression develop a phenotype similar to that of hair cells devoid of γ-actin. Bars, 2 um.

Figure 7. Cdh23^Ahl+ does not rescue progressive hearing loss in mice with γ-actin–deficient hair cells.

(A) Actg1-flox Atoh1-cre Cdh^Ahl+/ahl mice have elevated ABR thresholds at 18 and 24 weeks of age. (B–G) SEM analysis of stereocilia morphology at 18 weeks of age. OHC hair bundles from control Actg1-flox Cdh^Ahl+/ahl mice (B–D) have normal stereocilia morphology while OHC hair bundles from Actg1-flox Atoh1-cre Cdh^Ahl+/ahl mice have a degraded appearance with missing individual stereocilia. Bar is 1 µm.