␥- is required for cytoskeletal maintenance but not development

Inna A. Belyantsevaa,1, Benjamin J. Perrinb,1, Kevin J. Sonnemannb,1, Mei Zhuc, Ruben Stepanyand, JoAnn McGeee, Gregory I. Frolenkovd,f, Edward J. Walshe, Karen H. Fridericic, Thomas B. Friedmana, and James M. Ervastib,2

aLaboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850; bDepartment of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455; cMicrobiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824; dDepartment of Physiology, University of Kentucky, Lexington, KY 40536; fMolecular Biology and Genetics Section, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850; and eDevelopmental Auditory Physiology Laboratory, Boys Town National Research Hospital, Omaha, NE 68131

Edited by Carl Frieden, Washington University School of Medicine, St. Louis, MO, and approved April 24, 2009 (received for review January 8, 2009)

␤cyto-Actin and ␥cyto-actin are ubiquitous thought to be talline array of unidirectionally oriented actin filaments (Fig. 2C) essential building blocks of the in all non-muscle cells. (13–15). Despite this widely held supposition, we show that ␥cyto-actin null In the mammalian organ of Corti, the precise architecture of mice (Actg1؊/؊) are viable. However, they suffer increased mor- stereocilia is preserved for the life of the organism. Meanwhile, the tality and show progressive hearing loss during adulthood despite stereocilia actin core is reported to undergo renewal by continuous ␤ compensatory up-regulation of cyto-actin. The surprising viability actin polymerization at filament barbed ends and depolymerization ␤ ؊/؊ and normal hearing of young Actg1 mice means that cyto-actin at pointed ends, which is precisely coupled to maintain stereocilia can likely build all essential non-muscle actin-based cytoskeletal length (15, 16). The speed of stereocilia treadmilling is reported to structures including mechanosensory stereocilia of hair cells that be the same for all stereocilia of the same row and is proportional ␥ are necessary for hearing. Although cyto-actin–deficient stereo- to stereocilia length (17). Immuno-electron microscopy shows that cilia form normally, we found that they cannot maintain the in wild-type hair cells ␤cyto-actin is largely restricted to stereocilia, integrity of the stereocilia actin core. In the wild-type, ␥cyto-actin their rootlets, and the cuticular plate (2, 3, 18), whereas ␥cyto-actin localizes along the length of stereocilia but re-distributes to sites is reported to have more broad localization, including hair cell of F-actin core disruptions resulting from animal exposure to stereocilia and their rootlets, the cuticular plate in which stereocilia ؊/؊ damaging noise. In Actg1 stereocilia similar disruptions are are anchored, adherens junctions, and outer hair cell lateral walls ␥ observed even without noise exposure. We conclude that cyto- (2, 3, 18). Hair cells and their stereocilia are thus an attractive model actin is required for reinforcement and long-term stability of to study the structural consequences of perturbing actin isoform F-actin–based structures but is not an essential building block of composition. the developing cytoskeleton. ␤cyto- and ␥cyto-Actin are among the most abundant proteins in every mammalian cell, leading to the common assumption that ͉ ͉ actin cytoskeleton hearing both cytoplasmic are essential for function and viability. To test this supposition and to uncover the unique function of here are six encoding six vertebrate actins that are ␥cyto-actin, we generated a whole-body ␥cyto-actin knockout Tclassified according to where they are predominately ex- mouse (Actg1Ϫ/Ϫ). We show here that mice completely lacking ␣ ␣ ␣ ␥ Ϫ/Ϫ pressed. skeletal-Actin, smooth-actin, cardiac-actin, and smooth- ␥cyto-actin can survive to adulthood. Interestingly, Actg1 mice actin are primarily found in muscle cells, whereas cytoplasmic initially have normal hearing but develop progressive hearing ␤ ␥ cyto-actin and cyto-actin are ubiquitously and highly expressed loss during adulthood that is characterized by stereocilia actin in non-muscle cells, as reviewed elsewhere (1). Athough ␤cyto- core disruptions and stereocilia degradation. These findings led CELL BIOLOGY actin and ␥cyto-actin differ at only four biochemically similar us to conclude that ␥cyto-actin is not necessary for the formation residues in their N-termini, several lines of evidence of actin-based structures required for organogenesis and devel- suggest that each is functionally distinct. The amino acid ␤ ␥ opment, but is essential for maintenance of the hair cell actin sequences of cyto- and cyto-actin are each exactly conserved cytoskeleton. among avian and mammalian species. In addition, ␤cyto- and ␥ cyto-actin proteins are differentially localized (2–5) and post- Results translationally modified (6). Finally, although dominant mis- ␥ ␤ cyto-Actin Null Mice Are Viable. To determine whether there is a sense mutations in ACTB encoding cyto-actin are associated unique function of ␥ -actin that cannot be compensated by the with syndromic phenotypes including severe developmental mal- cyto other actin family members, we generated a ␥cyto-actin null formations and bilateral deafness (7), humans carrying a variety Ϫ Ϫ (Actg1 / ) mouse. Mice entirely devoid of ␥ -actin were of dominant missense mutations in ACTG1 develop postlingual cyto viable, but born at one-third of the expected Mendelian ratio, nonsyndromic progressive hearing loss (DFNA20, OMIM indicating that the absence of ␥ -actin caused some embryonic 604717) (8–11). cyto or perinatal lethality. Although the overall development of ␥cyto-Actin is widely expressed in the inner ear sensory epi- thelial cells on which mammalian hearing depends. These cells

are organized in rows along the cochlea length: one row of inner Author contributions: I.A.B., B.J.P., K.J.S., R.S., J.M., G.I.F., E.J.W., K.H.F., T.B.F., and J.M.E. hair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig. designed research; I.A.B., B.J.P., K.J.S., M.Z., R.S., J.M., and G.I.F. performed research; I.A.B., 2A). IHCs function as auditory receptors, converting sound B.J.P., K.J.S., M.Z., R.S., J.M., G.I.F., E.J.W., K.H.F., T.B.F., and J.M.E. analyzed data; and I.A.B., energy into neuronal signals, whereas OHCs enhance sensitivity B.J.P., K.J.S., G.I.F., K.H.F., T.B.F., and J.M.E. wrote the paper. to sound stimuli, as reviewed elsewhere (12). The apical surface The authors declare no conflict of interest. of a hair cell is topped with actin-rich microvilli-derived protru- This article is a PNAS Direct Submission. sions termed stereocilia, which deflect in response to sound 1I.A.B., B.J.P., and K.J.S. contributed equally to this work. stimuli, initiating mechanoelectrical transduction (Fig. 2B). 2To whom correspondence should be addressed. E-mail: [email protected]. ␤ ␥ cyto- and cyto-Actin are both thought to be essential compo- This article contains supporting information online at www.pnas.org/cgi/content/full/ nents of the stereocilia core (2–4), which consists of a paracrys- 0900221106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900221106 PNAS ͉ June 16, 2009 ͉ vol. 106 ͉ no. 24 ͉ 9703–9708 Downloaded by guest on September 25, 2021 40 ␥cyto-Actin Null Mice Show Progressive Loss of Hearing. We assessed A Ϫ/Ϫ 35 hearing in wild-type and Actg1 mice by measuring auditory 30 brainstem response (ABR) thresholds. ABR objectively mea- 25 sures synchronous electrical activity generated by the neurons in 20 the ascending auditory system and can be recorded from scalp 15 Actg1+/+ electrodes by averaging responses to short tone bursts (19, 20). Mass (grams) +/- Ϫ/Ϫ 10 Actg1 We found that Actg1 mice up to 6 weeks of age had Actg1-/- near-normal ABR thresholds (Fig. 1D). However, 16-week-old 0 Actg1Ϫ/Ϫ 0 50 100 150 200 250 300 mice demonstrated a marked hearing impairment at Age (days) each frequency tested, and by 24 weeks of age were profoundly deaf (Fig. 1D). This progressive hearing loss was not found in 100 ϩ/Ϫ B Actg1+/- Actg1 littermates, which exhibited wild-type ABR thresholds 80 up to 24 weeks of age (Fig. S2) despite expressing only 50% of wild-type levels of ␥cyto-actin (Fig. 1C). 60

40 Differential Localization of ␤cyto- and ␥cyto-Actin in Developing and -/- Survival (%) Actg1 Adult Mouse Hair Cells Revealed Delayed Appearance of ␥cyto-Actin in 20 Stereocilia. Consistent with previous reports in postnatal chicken 0 and mature guinea pig or rat, both ␤cyto- and ␥cyto-actin were 0 100 200 300 detected in stereocilia (Fig. 2 D and E) and the cuticular plate Age (days) of adult wild-type mouse hair cells. The three independently ␥ C 200 +/+ generated cyto-actin-specific antibodies used did not stain any γ -actin Actg1 Ϫ/Ϫ cyto Actg1+/- structures in Actg1 hair cells (Fig. 2F), demonstrating the 150 -/- β Actg1 ␥ cyto-actin specificity of these antisera for cyto-actin. We found that during 100 embryonic development of wild-type mice, ␤cyto-actin appeared total actin 50 in the body of hair cells and subsequently in stereocilia earlier Protein level

α- (% of wild-type) ␥ 0 than cyto-actin, which accumulated first in supporting cells and

+/+ +/- -/- only later appeared in hair cells (Fig. 2 G–P). We observed -actin -actin ␤cyto-actin in auditory hair cell stereocilia as soon as they appear Actg1 Actg1 Actg1 γ cyto β cyto total actin around E16.5 (Fig. 2 G–I) in the basal turn of the cochlea. The ␥ D 120 first appearance of cyto-actin within stereocilia was detected 100 after stereocilia emerged at approximately E18.5 (Fig. 2 O–P). Actg1+/+ 6 weeks These data are consistent with ␤ -actin primarily contributing 80 Actg1+/+ 16 weeks cyto +/+ 60 Actg1 24 weeks to the formation of the actin cytoskeleton of developing stere- -/- Actg1 6 weeks ocilia, whereas ␥cyto-actin may be important for cytoskeleton 40 Actg1-/- 16 weeks maintenance and/or reinforcement. Actg1-/- 24 weeks Level (dB SPL) 20 Although both actins are found in mouse stereocilia, we 0 observed differential localization within the stereocilia, again 46101625 ␤ ␥ Frequency (kHz) consistent with cyto- and cyto-actin having disparate functions. In the adult wild-type mouse stereocilia, ␤cyto-actin staining Fig. 1. Characterization of live-born homozygous mutant Actg1Ϫ/Ϫ mice. (A) overlapped completely with rhodamine-phalloidin staining, ϩ/ϩ ϩ/Ϫ Body mass growth curve of Actg1 (wild-type, closed squares), Actg1 whereas ␥cyto-actin was concentrated more toward the periphery (heterozygous, open circles) and Actg1Ϫ/Ϫ (homozygous mutant, open trian- of the stereocilia actin core, often only partially overlapping with ϩ ϩ ϩ Ϫ Ϫ Ϫ gles) mice from P28 until P300 (n ϭ 12 Actg1 / ,18Actg1 / ,11Actg1 / , rhodamine-phalloidin staining (Fig. 2 Q–T). mean Ϯ SEM). (B) Kaplan-Meier survival curve of Actg1ϩ/Ϫ and Actg1Ϫ/Ϫ mice from P0 to P350, (n ϭ 31 for each genotype). (C) Representative immunoblots Phalloidin-Negative Gaps in F-Actin Stereocilia Cores Contain Core of SDS extracts from Actg1ϩ/ϩ, Actg1ϩ/Ϫ and Actg1Ϫ/Ϫ cochlear extracts Components. In the course of characterizing the localization of probed with antibodies specific for ␥cyto-actin, ␤cyto-actin, pan-actin, or tubulin antibody. Protein levels were quantified and are expressed relative to the the cytoplasmic actins, we observed occasional gaps in phalloidin wild-type level (mean Ϯ SEM). (D) Actg1Ϫ/Ϫ mice develop progressive hearing staining of F-actin cores of vestibular hair cell stereocilia (Fig. 3 loss. Auditory brainstem response (ABR) thresholds were determined for A–C). The gaps were most frequently observed at the base and Actg1ϩ/ϩ and Actg1Ϫ/Ϫ mice at 6, 16, and 24 weeks of age using stimulus fre- along the length of stereocilia in the tallest row (Fig. 3D). Using quencies from 4 to 22 kHz, presented at half-octave steps (n Ͼ 5, mean Ϯ SEM). our ␥cyto-actin specific antibodies, which recognize both globular (G) and filamentous (F) forms of actin (see SI Text), we found that gaps were enriched in ␥cyto-actin. This actin population is surviving Actg1Ϫ/Ϫ mice appeared largely normal, their body likely to be predominantly monomeric, because phalloidin rec- ϩ ϩ ognizes only filamentous actin (Fig. 3 A–D). Usually gap staining weight was Ϸ20% lower than wild-type (Actg1 / ) and heterozy- ϩ Ϫ Ϫ Ϫ was much more intense relative to that along the length of gous (Actg1 / ) littermates (Fig. 1A). In addition, some Actg1 / stereocilium (Figs. 3 A–D and 3 F–M), which may be caused by B mice died prematurely of unknown cause(s) (Fig. 1 ). To enhanced antibody accessibility within the gaps. Alternatively, investigate whether the observed effects were caused by a intense gap staining could be partially explained by the redis- general depletion of cellular actin, we analyzed actin isoform tribution of ␥ -actin to F-actin gaps from a pool of available ϩ/Ϫ Ϫ/Ϫ cyto expression in wild-type, Actg1 and Actg1 tissues by West- non-filamentous actin within a stereocilium. A similar redistri- ern blot. We observed dose-dependent expression of bution to F-actin gaps was also seen for ␤ -actin (Fig. S3). It ␥ cyto cyto-actin and compensatory up-regulation of other actin family is likely that ␤cyto-actin is also recruited to the gaps from a pool members to maintain the total actin level in all tissues examined of non-filamentous actin, as staining intensity along a stereoci- (Fig. 1C and [supporting information (SI) Fig. S1]). Therefore, lium with a gap was not different from the intensity of staining the actin composition, but not the concentration, was altered in along a stereocilium without gaps (Fig. S3B). The same pattern Actg1Ϫ/Ϫ mice. of staining was also observed for DNase I (Fig. 3E), a marker for

9704 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900221106 Belyantseva et al. Downloaded by guest on September 25, 2021 A B C

D E F

G HI J KL

MNO P

Q R S T

Fig. 2. Differential localization of ␤cyto- and ␥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 support- ing 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 ␤cyto-actin (D) and ␥cyto-actin (E) along the length of adult wild-type (wt) OHC Ϫ/Ϫ and IHC stereocilia. (F) Absence of ␥cyto-actin (green) in 6-week-old Actg1 OC. (G–L) At E16.5, ␤cyto-actin immunoreactivity follows rhodamine-phalloidin labeling in wt hair cells (G–I), whereas ␥cyto-actin is detected in supporting cells ␥ CELL BIOLOGY but not in hair cells (J–L). (M–P) At E18.5, ␤cyto-actin is present in all stereocilia Fig. 3. cyto-Actin concentrates at the sites of stereocilia core disruptions. ␥ of hair cells throughout the cochlea (M, N), whereas ␥cyto-actin begins to (A–C) cyto-Actin antibody highlights gaps (segments of F-actin depolymeriza- appear only in stereocilia of more developed basal turn of the cochlea (O, P). tion; arrows) in wild-type (wt) mouse vestibular hair cell (VHC) stereocilia. In (Q–T) ␤cyto-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidin all panels, rhodamine-phalloidin highlights F-actin in red, and labeling with ␥ staining, whereas ␥cyto-actin (S, T) is concentrated toward the periphery of the antibodies is in green. (D) cyto-Actin at the base and within the F-actin gaps IHC stereocilia F-actin core in adult wt mice. Scale bars (B, Q–T), 2 ␮m; scale bars of longest stereocilia in wt mouse VHC (arrows). (E) DNase I stains globular (D–P), 5 ␮m. actin within F-actin gaps of VHC stereocilia (arrows). (F) concentrates in gaps of wt VHC stereocilia. (G) Uniform distribution of ␥cyto-actin along adult guinea pig IHC stereocilia not exposed to damaging noise. (H) Redistribution ␥ ␥ monomeric actin (G-actin) (21), and espin (Fig. 3F), an actin of cyto-actin in noise-damaged guinea pig IHC stereocilia. cyto-Actin absent bundling protein essential for stereocilia formation, which is from the tips and evenly distributed along stereocilia which appear unaf- ␥ reported to have both F- and G-actin binding sites (22). Inter- fected (inset: second and fifth stereocilium from the left). (I–K) cyto-Actin estingly, only proteins that are either actin core components or 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 directly involved in actin turnover were found to accumulate in Ϫ/Ϫ F-actin gaps in IHC stereocilia from Actg1 mouse (arrows). (M) ␤cyto-Actin Ϫ/Ϫ the phalloidin-negative gaps. For example, actin-associated pro- concentrates in the F-actin gap of Actg1 IHC stereocilium. ␤cyto-Actin teins cadherin-23, protocadherin-15-CD1, -VIIa, and my- staining along stereocilia is barely visible because of intense gap staining. osin-XVa are not present in gaps (Fig. S4 and data not shown). (N–P) Espin concentrates in gaps of Actg1Ϫ/Ϫ VHC stereocilia. Scale bars, 2 ␮m. In contrast, cofilin, which was implicated in both severing and nucleation of F-actin (23), selectively accumulates in stereocilia ␥ gaps (Fig. S4). cyto-Actin Localizes to Phalloidin-Negative Gaps That Form in Re- Together, these data suggest that (i) gaps have a different sponse to Damage. In contrast to vestibular hair cell stereocilia, structural arrangement than the stereocilia actin core, (ii) gaps gaps were not observed in undamaged auditory hair cell stere- are enriched for ␤cyto- and ␥cyto-actin along with other core ocilia of wild-type mice. Previously, F-actin gaps were reported components, and (iii) cofilin may mediate ongoing actin remod- in guinea pig auditory hair cell stereocilia after noise damage eling in the gap to facilitate repair of local damage of the F-actin (24), suggesting that gaps develop in response to stereocilia core. damage. To assess whether damage-induced gaps are also en-

Belyantseva et al. PNAS ͉ June 16, 2009 ͉ vol. 106 ͉ no. 24 ͉ 9705 Downloaded by guest on September 25, 2021 riched in ␥cyto-actin, we compared ␥cyto-actin localization in 6 weeks old stereocilia from control and noise-damaged guinea pigs. Con- B sistent with previous studies (2, 18), ␥cyto-actin was distributed AB along the length of control stereocilia (Fig. 3G). After a dam- aging noise exposure, ␥cyto-actin was enriched in both the tips Actg1+/+ and in phalloidin-negative gaps observed along the length of noise-damaged stereocilia, which were often abnormally shorter than neighboring normal appearing stereocilia of the same row CD (Fig. 3 H–K). In the same bundle, some stereocilia that appeared -/- unaffected by noise still had ␥cyto-actin evenly distributed along Actg1 their length and did not have an accumulation of ␥cyto-actin at the tips (Fig. 3H, inset). 16 weeks old Immunofluorescence Confocal Microscopy and Scanning Electron Mi- croscopy Analyses Reveal an Unexpected Pattern of Degeneration of CDEF Actg1؊/؊ Stereocilia. Interestingly, F-actin gaps were occasionally Ϫ/Ϫ observed in auditory hair cell stereocilia of Actg1 mice Actg1+/+ without exposure to damaging noise. These gaps lacked ␥cyto- actin (Fig. 3L) but contained ␤cyto-actin (Fig. 3M) and espin (Fig. Ϫ/Ϫ 3 N–P). The staining of ␤cyto-actin within gaps of Actg stereocilia was so intense that we had to turn down the gain on GH the confocal microscope so that the signal within gaps was not Actg1-/- saturated (Fig. 3M and Fig. S3A). As a consequence, the ␤cyto-actin signal along the lengths of stereocilia was reduced to a barely detectable level (compare Fig. 3M and Fig. S3A with Fig. S3B). J The presence of F-actin gaps in the auditory hair cell stere- I ocilia of hearing-impaired, ␥cyto-actin–deficient mice led us to 120 investigate whether a stereocilia maintenance/repair mechanism is defective in Actg1Ϫ/Ϫ mice. To define the structural changes 100 ␥ associated with a cyto-actin-deficiency, we characterized the 80 morphology of auditory hair cells. We examined outer hair cells of 6-week-old Actg1Ϫ/Ϫ mice by scanning electron microscopy 60 * ␥ and found that cyto-actin deficient stereocilia were indistin- 40

guishable from OHC stereocilia of wild-type littermates (Fig. 4 per hair cell K A–D). However, Actg1Ϫ/Ϫ stereocilia deteriorated progressively 20 as the animals aged (Fig. 4 E–H). By 16 weeks of age, Ϸ50% of Number of stereocilia 0

stereocilia within a hair bundle were degraded or absent in - - Ϫ Ϫ / / - - Actg1 / mice (Fig. 4I). Across all three rows within the hair +/+ +/+ bundle, we observed missing or shortened (Fig. 4 J and K)

stereocilia, although the remaining stereocilia looked normal. Actg1 Actg1 Actg1 Actg1 6 weeks 16 weeks Discussion Ϫ Ϫ Actg1 / mice survive to birth and beyond, demonstrating that Fig. 4. Morphology of stereocilia bundles in adult wild-type (Actgϩ/ϩ) and ␥ Ϫ/Ϫ ␥cyto-actin is not strictly required for mammalian development or cyto-actin deficient (Actg1 ) mice. (A–D) Scanning electron micrographs of viability. Our observations of stereocilia from Actg1Ϫ/Ϫ mice stereocilia from (A, B) 6-week-old Actg1ϩ/ϩ and (C, D) 6-week-old Actg1Ϫ/Ϫ instead indicate that ␥ -actin is necessary to maintain cytoskel- mice. (E–H) Scanning electron microscopy images of OHC stereocilia from cyto ϩ/ϩ Ϫ/Ϫ etal integrity and function. The phenotype of Actg1Ϫ/Ϫ stereo- 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 cilia is unique; we observed stereocilia defects ranging from Actg1Ϫ/Ϫ mice. Images are from the middle turn of the cochlea. (I) Box and simple shortening to complete loss of individual stereocilia whisker plot (whiskers, maximum and minimum; box, 5th–95th percentile; across all three rows within the hair bundle indicating selected line, mean) of the number of individual stereocilia in individual OHC stereocilia disassembly (Fig. 4 E–K), whereas the remaining bundles from Actg1ϩ/ϩ or Actg1Ϫ/Ϫ mice at 6 and 16 weeks of age, *P Ͻ stereocilia within the same bundle appeared intact. The appar- 0.005. (J–K) enlargements of image in (H) with arrows indicating missing ent independent nature of this phenomenon (affected stereocilia and shortened stereocilia. Scale bars (A, C, E, and G), 5 ␮m; scale bars (B, D, surrounded by normal stereocilia) indicates that the disassembly F, H, J, and K), 1 ␮m. process is initiated and/or regulated at the level of an individual stereocilium. Therefore, this disassembly process appears dif- were not present in control stereocilia but were observed in ferent from actin treadmilling that normally occurs simulta- Ϫ/Ϫ neously in all stereocilia of the same row (17). Actg1 mouse auditory stereocilia indicating that stereocilia Rather, our results suggest that ␥ -actin strengthens stere- core damage was more frequent or more slowly repaired in the cyto ␥ Ϫ/Ϫ ocilia F-actin cores, preventing stereocilia core damage, and/or absence of cyto-actin. Finally, Actg1 stereocilia progressively ␥ is required to repair the damaged core. Consistent with this view, deteriorated, demonstrating that cyto-actin is required to main- in developing wild-type mice ␥ -actin appeared and accumu- tain these structures. cyto ␥ ␤ lated in stereocilia a few days before the onset of hearing cyto- and cyto-Actin are nearly identical, featuring only four function, perhaps preparing stereocilia to withstand the rigors of biochemically similar residue substitutions in the N terminus, acoustical stimulation. Furthermore, damaging noise induces the suggesting likely compensation between these proteins. Indeed, ␤ Ϫ/Ϫ appearance of ␥cyto-actin–enriched, phalloidin-negative gaps in cyto-actin protein levels were elevated in Actg1 mice and the the stereocilia core of wild-type rodent hair cells. These gaps total actin level was equivalent in Actg1Ϫ/Ϫ and wild-type mice

9706 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0900221106 Belyantseva et al. Downloaded by guest on September 25, 2021 (Fig. 1C). However, ␥cyto-actin–deficient stereocilia progres- body knockout was widely presumed to be unviable. This murine sively deteriorated despite the localization of ␤cyto-actin to gaps model exhibited normal muscle development followed by pro- in the F-actin core of Actg1Ϫ/Ϫ mouse auditory stereocilia (Fig. gressive and necrosis (29). Both muscle 3M and Fig. S3). This surprising result indicates that ␥cyto-actin cells and outer hair cells must resist force generated by contrac- has at least some functions that are unique and cannot be tility or electromotility, respectively. Although otherwise clearly ␤ ␥ compensated for by cyto-actin. One possibility is that cyto-actin disparate in structure and function, these mechanically chal- ␥ brings to the site of damage a unique cyto-actin protein partner lenged cells seem to have a particularly evident requirement for ␤ ␥ ␤ that is necessary for cyto-actin, cyto-actin, or for cyto- and the specialized properties of ␥ -actin necessary for cellular ␥ cyto cyto-actin together, to repair damage to the core. maintenance. ␥ ␤ Consistent with different functions of cyto- and cyto-actin, Additional ␥cyto-actin deficiency-based cytoskeletal patho- we observed distinct localization patterns for each protein logic conditions may exist in other organs and tissues of Actg1Ϫ/Ϫ ␤ within wild-type auditory stereocilia. cyto-Actin localized to mice that could affect long-term function. Indeed, the lower stereocilia cores, exactly overlaying with phalloidin staining, body mass of Actg1Ϫ/Ϫ mice and their occasional premature ␥ whereas cyto-actin was concentrated toward the periphery of death suggests a hidden, slowly developing or partially compen- stereocilia cores. Because the ␥cyto-actin antibody detects both sated pathologic condition. We conclude that ␥cyto-actin is not monomeric and filamentous actin whereas phalloidin detects necessary to build actin cytoskeletal structures required for only filamentous actin, there appears to be a pool of mono- ␥ organogenesis and development but, instead, functions primarily meric cyto-actin at the periphery of stereocilia cores. Alter- to reinforce and/or repair the actin cytoskeleton. natively, phalloidin-negative actin may still be filamentous but unable to bind phalloidin because of a particular F-actin Methods conformation, which was observed in nuclear actin as previ- Generation of Actg1-Null Mice. A targeting vector in which loxP sites flank ously reviewed (25), different paracrystal filament packing exons 2 and 3 of the murine Actg1 gene was described previously (29). that excludes phalloidin, or masking by actin binding proteins. Embryonic stem cell targeting, screening, blastocyst injections, and subse- In any case, the peripheral population of ␥cyto-actin is distinct quent EIIa-cre breeding were performed to generate Actg1ϩ/Ϫ mice (29). ϩ/Ϫ and may be used for stereocilia core remodeling and repair, Actg1 mice were backcrossed to C57BI/6 for 10 generations before N10 perhaps redistributing to F-actin gaps that form as a result of Actg1ϩ/Ϫ X Actg1ϩ/Ϫ breedings were arranged to obtain Actg1Ϫ/Ϫ mice. All stereocilia core damage. genotypes were determined as previously described (29). Animals were Ϫ/Ϫ Based on ␥cyto-actin localization and Actg1 stereocilia housed and treated in accordance with the standards set by the University of Minnesota Institutional Animal Care and Use Committee. degradation, we envision two models of ␥cyto-actin function. First, ␥cyto-actin may have a specific role involving annealing of broken filaments or de novo polymerization, perhaps depending Immunoblot Analysis. Brain, lung, kidney, and cochlea were dissected from mice of the indicated genotypes, frozen in liquid nitrogen, ground into on an unknown actin-binding protein with specificity for ␥cyto- actin. Alternatively, ␥ -actin-containing filaments may have dis- powder, boiled in buffer (1% SDS, EGTA, PMSF, benzamidine, leupeptin), and cyto centrifuged to remove insoluble material. Protein concentration in the result- tinct biophysical and biochemical properties, such as different ing lysate was determined with either a colorimetric assay (DC assay, BioRad) polymerization rates or polymer stability, which protect stereocilia or by A280 measurement. Equal amounts of protein were separated by sodium from mechanical stress. Deficient repair and/or diminished struc- dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred Ϫ/Ϫ tural integrity then result in the eventual loss of Actg1 stereocilia. to nitrocellulose membranes, and probed with the indicated antibodies (␥cyto- Interestingly, the gaps observed in auditory stereocilia of actin; mAb 2–4 or pAb7577 (29); ␤cyto-actin, AC15 (Sigma); pan-actin, C4, gift Ϫ/Ϫ noise-damaged animals and untreated Actg1 mice (24) (Fig. of J. Lessard, University of Cincinnati; ␥smooth-actin, B4 (J. Lessard, University of 3) resemble discontinuities in actin-rich developing Drosophila Cincinnati); ␣smooth-actin, 1A4 (Sigma); ␣-tubulin B512 (Sigma). Fluorescently bristles (26, 27). In these structures, gaps are observed both labeled secondary antibodies were detected and quantified from three sep- during formation, as short modules of F-actin are cross-linked to arate experiments blotted in triplicate using an Odyssey infrared scanner and form fibers, and during disassembly, as the fibers are broken software (Li-Cor Biosciences). down into the original modules (26). Although mammalian CELL BIOLOGY stereocilia are not thought to be composed of cross-linked Auditory Brainstem Responses. ABRs were collected as previously described F-actin modules, elements of Drosophila actin regulation may (30) or using a Tucker-Davis Technologies System3 to generate sound stimuli and to amplify and record brainstem potentials as described in the SI Text. nonetheless be conserved in mammalian stereocilia. Stereocilia gaps may arise through physical damage that cause F-actin Antibody Validation and Immunostaining. Polyclonal antibody pAb7577 bundle breakage or through the action of a protein that senses against cytoplasmic ␥cyto-actin (ACTG1) was generated in the laboratory of J. damage and severs and depolymerizes actin filaments, generat- Ervasti as described, and the specificity was verified (29). The second anti-␥cyto- ing gaps similar to those that occur during modular disassembly actin polyclonal antibody was a gift from C. Bulinski and was characterized of Drosophila bristles (28). previously (31). The third anti-␥cyto-actin polyclonal antibody was developed in The accumulation of ␥cyto-actin at sites of damage in wild-type the laboratory of K. Friderici by immunizing rabbits with a peptide of the hair cell stereocilia after noise exposure (Figs. 3 H–K), together N-terminal 15 residues (Princeton Biomolecules) and affinity purified as de- with the disassembly and subsequent loss of individual stereocilia scribed in the SI Text. The immunostaining is described in the SI Text. All animal Ϫ/Ϫ in Actg1 hair cells (Fig. 4 G–I), are consistent with ␥cyto-actin care and experimental procedures were approved by the NINDS/NIDCD ACUC. being required for maintenance and/or repair of stereocilia in Animals and Noise Exposure. Noise exposure methods are described in the adult hair cells. However, ␥cyto-actin seems to be entirely dis- pensable for the proper development and functional maturation SI Text. of hair cells. Indeed, the viability of Actg1Ϫ/Ϫ embryos and the normal lifespan of at least one-third of all live-born Actg1Ϫ/Ϫ Scanning Electron Microscopy. Cochlea were rapidly dissected and fixed by perfusing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.3 with mice demonstrate that ␥cyto-actin is not crucial for general 1 mM CaCl2, through the round window, followed by immersion in the same organogenesis and thus is not necessary for the formation of solution for 2 hours. After microdissection to reveal hair cell stereocilia, actin-based structures in general. Consistent with this idea, cochlea were incubated in 2% arginine-HCl, , glutamic acid, and su- Ϫ/Ϫ wild-type and Actg1 mice intestinal brush border microvilli crose followed by treatment with 2% tannic acid and 2% guanidine-HCl and are morphologically indistinguishable (Fig. S5). were postfixed in 1% aqueous osmium tetroxide. Specimens were dehydrated We previously characterized a muscle-specific ␥cyto-actin in ethanol, critical point dried, sputter coated, and imaged using a cold field knockout mouse that was generated precisely because a whole- emission gun scanning electron microscope (Hitachi S-4700).

Belyantseva et al. PNAS ͉ June 16, 2009 ͉ vol. 106 ͉ no. 24 ͉ 9707 Downloaded by guest on September 25, 2021 ACKNOWLEDGMENTS. We thank J. Bartles and C. Bulinski for providing inary analysis of Actg1Ϫ/Ϫ mice. The work was supported by National Institutes anti-espin antibody and one of the anti-␥cyto-actin antibodies, respectively; D. of Health (NIH) intramural funds 1 Z01 DC000048–11 LMG (to T.B.F.), NIH Catts and P. Diers for technical assistance; D. Drayna, R. Chadwick, N. Gavara, intramural funds Z01-DC-000060 (to Andrew J. Griffith), funds from the DRF A. Griffith, J. Bird, and R. Morell for critically reading the manuscript; P. and NOHR Foundation (to G.I.F.), and NIH grants DC004568 (to K.H.F.), F32 Belyantsev for Fig. 1 drawing; and K. Prins, S. Ikeda, and A. Ikeda for prelim- DC009539 (to B.J.P.), and a R01 AR049899 (to J.M.E.).

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