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, 106 (24), 9703-8

Gamma-actin Is Required for Cytoskeletal Maintenance but Not Development

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Gamma-actin Is Required for Cytoskeletal Maintenance but Not Development

Inna A Belyantseva et al. Proc Natl Acad Sci U S A.

Abstract

Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
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 γcyto-actin, βcyto-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).
Fig. 2.
Fig. 2.
Differential localization of βcyto- and γcyto-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 βcyto-actin (D) and γcyto-actin (E) along the length of adult wild-type (wt) OHC and IHC stereocilia. (F) Absence of γcyto-actin (green) in 6-week-old Actg1−/− OC. (G–L) At E16.5, βcyto-actin immunoreactivity follows rhodamine-phalloidin labeling in wt hair cells (G–I), whereas γcyto-actin is detected in supporting cells but not in hair cells (J–L). (M–P) At E18.5, βcyto-actin is present in all stereocilia of hair cells throughout the cochlea (M, N), whereas γcyto-actin begins to appear only in stereocilia of more developed basal turn of the cochlea (O, P). (Q–T) βcyto-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidin staining, whereas γcyto-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.
Fig. 3.
Fig. 3.
γcyto-Actin concentrates at the sites of stereocilia core disruptions. (A–C) γcyto-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) γcyto-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 γcyto-actin along adult guinea pig IHC stereocilia not exposed to damaging noise. (H) Redistribution of γcyto-actin in noise-damaged guinea pig IHC stereocilia. γcyto-Actin absent from the tips and evenly distributed along stereocilia which appear unaffected (inset: second and fifth stereocilium from the left). (I–K) γcyto-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) βcyto-Actin concentrates in the F-actin gap of Actg1−/− IHC stereocilium. βcyto-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.
Fig. 4.
Fig. 4.
Morphology of stereocilia bundles in adult wild-type (Actg+/+) and γcyto-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, 5th–95th 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.

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