�-Actin is required for cytoskeletal maintenancebut not developmentInna 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 andGenetics 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 proteins thought to beessential building blocks of the cytoskeleton in all non-muscle cells.Despite this widely held supposition, we show that �cyto-actin nullmice (Actg1�/�) are viable. However, they suffer increased mor-tality and show progressive hearing loss during adulthood despitecompensatory up-regulation of �cyto-actin. The surprising viabilityand normal hearing of young Actg1�/� mice means that �cyto-actincan likely build all essential non-muscle actin-based cytoskeletalstructures including mechanosensory stereocilia of hair cells thatare necessary for hearing. Although �cyto-actin–deficient stereo-cilia form normally, we found that they cannot maintain theintegrity of the stereocilia actin core. In the wild-type, �cyto-actinlocalizes along the length of stereocilia but re-distributes to sitesof F-actin core disruptions resulting from animal exposure todamaging noise. In Actg1�/� stereocilia similar disruptions areobserved even without noise exposure. We conclude that �cyto-actin is required for reinforcement and long-term stability ofF-actin–based structures but is not an essential building block ofthe developing cytoskeleton.

actin � cytoskeleton � hearing

There are six genes encoding six vertebrate actins that areclassified according to where they are predominately ex-

pressed. �skeletal-Actin, �smooth-actin, �cardiac-actin, and �smooth-actin are primarily found in muscle cells, whereas cytoplasmic�cyto-actin and �cyto-actin are ubiquitously and highly expressedin non-muscle cells, as reviewed elsewhere (1). Athough �cyto-actin and �cyto-actin differ at only four biochemically similaramino acid residues in their N-termini, several lines of evidencesuggest that each protein is functionally distinct. The amino acidsequences of �cyto- and �cyto-actin are each exactly conservedamong avian and mammalian species. In addition, �cyto- and�cyto-actin proteins are differentially localized (2–5) and post-translationally modified (6). Finally, although dominant mis-sense mutations in ACTB encoding �cyto-actin are associatedwith syndromic phenotypes including severe developmental mal-formations and bilateral deafness (7), humans carrying a varietyof dominant missense mutations in ACTG1 develop postlingualnonsyndromic progressive hearing loss (DFNA20, OMIM604717) (8–11).

�cyto-Actin is widely expressed in the inner ear sensory epi-thelial cells on which mammalian hearing depends. These cellsare organized in rows along the cochlea length: one row of innerhair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig.2A). IHCs function as auditory receptors, converting soundenergy into neuronal signals, whereas OHCs enhance sensitivityto sound stimuli, as reviewed elsewhere (12). The apical surfaceof a hair cell is topped with actin-rich microvilli-derived protru-sions termed stereocilia, which deflect in response to soundstimuli, initiating mechanoelectrical transduction (Fig. 2B).�cyto- and �cyto-Actin are both thought to be essential compo-nents of the stereocilia core (2–4), which consists of a paracrys-

talline array of unidirectionally oriented actin filaments (Fig. 2C)(13–15).

In the mammalian organ of Corti, the precise architecture ofstereocilia is preserved for the life of the organism. Meanwhile, thestereocilia actin core is reported to undergo renewal by continuousactin polymerization at filament barbed ends and depolymerizationat pointed ends, which is precisely coupled to maintain stereocilialength (15, 16). The speed of stereocilia treadmilling is reported tobe the same for all stereocilia of the same row and is proportionalto stereocilia length (17). Immuno-electron microscopy shows thatin wild-type hair cells �cyto-actin is largely restricted to stereocilia,their rootlets, and the cuticular plate (2, 3, 18), whereas �cyto-actinis reported to have more broad localization, including hair cellstereocilia and their rootlets, the cuticular plate in which stereociliaare anchored, adherens junctions, and outer hair cell lateral walls(2, 3, 18). Hair cells and their stereocilia are thus an attractive modelto study the structural consequences of perturbing actin isoformcomposition.

�cyto- and �cyto-Actin are among the most abundant proteinsin every mammalian cell, leading to the common assumption thatboth cytoplasmic actins are essential for function and viability.To test this supposition and to uncover the unique function of�cyto-actin, we generated a whole-body �cyto-actin knockoutmouse (Actg1�/�). We show here that mice completely lacking�cyto-actin can survive to adulthood. Interestingly, Actg1�/� miceinitially have normal hearing but develop progressive hearingloss during adulthood that is characterized by stereocilia actincore disruptions and stereocilia degradation. These findings ledus to conclude that �cyto-actin is not necessary for the formationof actin-based structures required for organogenesis and devel-opment, but is essential for maintenance of the hair cell actincytoskeleton.

Results�cyto-Actin Null Mice Are Viable. To determine whether there is aunique function of �cyto-actin that cannot be compensated by theother actin family members, we generated a �cyto-actin null(Actg1�/�) mouse. Mice entirely devoid of �cyto-actin wereviable, but born at one-third of the expected Mendelian ratio,indicating that the absence of �cyto-actin caused some embryonicor perinatal lethality. Although the overall development of

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.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.,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.,B.J.P., K.J.S., G.I.F., K.H.F., T.B.F., and J.M.E. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1I.A.B., B.J.P., and K.J.S. contributed equally to this work.

2To whom correspondence should be addressed. E-mail: jervasti@umn.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0900221106/DCSupplemental.

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surviving Actg1�/� mice appeared largely normal, their bodyweight was �20% lower than wild-type (Actg1�/�) and heterozy-gous (Actg1�/�) littermates (Fig. 1A). In addition, some Actg1�/�

mice died prematurely of unknown cause(s) (Fig. 1B). Toinvestigate whether the observed effects were caused by ageneral depletion of cellular actin, we analyzed actin isoformexpression in wild-type, Actg1�/� and Actg1�/� tissues by West-ern blot. We observed gene dose-dependent expression of�cyto-actin and compensatory up-regulation of other actin familymembers to maintain the total actin level in all tissues examined(Fig. 1C and [supporting information (SI) Fig. S1]). Therefore,the actin composition, but not the concentration, was altered inActg1�/� mice.

�cyto-Actin Null Mice Show Progressive Loss of Hearing. We assessedhearing in wild-type and Actg1�/� mice by measuring auditorybrainstem response (ABR) thresholds. ABR objectively mea-sures synchronous electrical activity generated by the neurons inthe ascending auditory system and can be recorded from scalpelectrodes by averaging responses to short tone bursts (19, 20).We found that Actg1�/� mice up to 6 weeks of age hadnear-normal ABR thresholds (Fig. 1D). However, 16-week-oldActg1�/� mice demonstrated a marked hearing impairment ateach frequency tested, and by 24 weeks of age were profoundlydeaf (Fig. 1D). This progressive hearing loss was not found inActg1�/� littermates, which exhibited wild-type ABR thresholdsup to 24 weeks of age (Fig. S2) despite expressing only 50% ofwild-type levels of �cyto-actin (Fig. 1C).

Differential Localization of �cyto- and �cyto-Actin in Developing andAdult Mouse Hair Cells Revealed Delayed Appearance of �cyto-Actin inStereocilia. Consistent with previous reports in postnatal chickenand mature guinea pig or rat, both �cyto- and �cyto-actin weredetected in stereocilia (Fig. 2 D and E) and the cuticular plateof adult wild-type mouse hair cells. The three independentlygenerated �cyto-actin-specific antibodies used did not stain anystructures in Actg1�/� hair cells (Fig. 2F), demonstrating thespecificity of these antisera for �cyto-actin. We found that duringembryonic development of wild-type mice, �cyto-actin appearedin the body of hair cells and subsequently in stereocilia earlierthan �cyto-actin, which accumulated first in supporting cells andonly later appeared in hair cells (Fig. 2 G–P). We observed�cyto-actin in auditory hair cell stereocilia as soon as they appeararound E16.5 (Fig. 2 G–I) in the basal turn of the cochlea. Thefirst appearance of �cyto-actin within stereocilia was detectedafter stereocilia emerged at approximately E18.5 (Fig. 2 O–P).These data are consistent with �cyto-actin primarily contributingto the formation of the actin cytoskeleton of developing stere-ocilia, whereas �cyto-actin may be important for cytoskeletonmaintenance and/or reinforcement.

Although both actins are found in mouse stereocilia, weobserved differential localization within the stereocilia, againconsistent with �cyto- and �cyto-actin having disparate functions.In the adult wild-type mouse stereocilia, �cyto-actin stainingoverlapped completely with rhodamine-phalloidin staining,whereas �cyto-actin was concentrated more toward the peripheryof the stereocilia actin core, often only partially overlapping withrhodamine-phalloidin staining (Fig. 2 Q–T).

Phalloidin-Negative Gaps in F-Actin Stereocilia Cores Contain CoreComponents. In the course of characterizing the localization ofthe cytoplasmic actins, we observed occasional gaps in phalloidinstaining of F-actin cores of vestibular hair cell stereocilia (Fig. 3A–C). The gaps were most frequently observed at the base andalong the length of stereocilia in the tallest row (Fig. 3D). Usingour �cyto-actin specific antibodies, which recognize both globular(G) and filamentous (F) forms of actin (see SI Text), we foundthat gaps were enriched in �cyto-actin. This actin population islikely to be predominantly monomeric, because phalloidin rec-ognizes only filamentous actin (Fig. 3 A–D). Usually gap stainingwas much more intense relative to that along the length ofstereocilium (Figs. 3 A–D and 3 F–M), which may be caused byenhanced antibody accessibility within the gaps. Alternatively,intense gap staining could be partially explained by the redis-tribution of �cyto-actin to F-actin gaps from a pool of availablenon-filamentous actin within a stereocilium. A similar redistri-bution to F-actin gaps was also seen for �cyto-actin (Fig. S3). Itis likely that �cyto-actin is also recruited to the gaps from a poolof non-filamentous actin, as staining intensity along a stereoci-lium with a gap was not different from the intensity of stainingalong a stereocilium without gaps (Fig. S3B). The same patternof staining was also observed for DNase I (Fig. 3E), a marker for

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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 trian-gles) mice from P28 until P300 (n � 12 Actg1�/�, 18 Actg1�/�, 11 Actg1�/�,mean � SEM). (B) Kaplan-Meier survival curve of Actg1�/� and Actg1�/� micefrom P0 to P350, (n � 31 for each genotype). (C) Representative immunoblotsof SDS extracts from Actg1�/�, Actg1�/� and Actg1�/� cochlear extractsprobed with antibodies specific for �cyto-actin, �cyto-actin, pan-actin, or tubulinantibody. Protein levels were quantified and are expressed relative to thewild-type level (mean � SEM). (D) Actg1�/� mice develop progressive hearingloss. Auditory brainstem response (ABR) thresholds were determined forActg1�/� and Actg1�/� mice at 6, 16, and 24 weeks of age using stimulus fre-quencies from 4 to 22 kHz, presented at half-octave steps (n � 5, mean � SEM).

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monomeric actin (G-actin) (21), and espin (Fig. 3F), an actinbundling protein essential for stereocilia formation, which isreported to have both F- and G-actin binding sites (22). Inter-estingly, only proteins that are either actin core components ordirectly involved in actin turnover were found to accumulate inthe phalloidin-negative gaps. For example, actin-associated pro-teins cadherin-23, protocadherin-15-CD1, myosin-VIIa, and my-osin-XVa are not present in gaps (Fig. S4 and data not shown).In contrast, cofilin, which was implicated in both severing andnucleation of F-actin (23), selectively accumulates in stereociliagaps (Fig. S4).

Together, these data suggest that (i) gaps have a differentstructural arrangement than the stereocilia actin core, (ii) gapsare enriched for �cyto- and �cyto-actin along with other corecomponents, and (iii) cofilin may mediate ongoing actin remod-eling in the gap to facilitate repair of local damage of the F-actincore.

�cyto-Actin Localizes to Phalloidin-Negative Gaps That Form in Re-sponse to Damage. In contrast to vestibular hair cell stereocilia,gaps were not observed in undamaged auditory hair cell stere-ocilia of wild-type mice. Previously, F-actin gaps were reportedin guinea pig auditory hair cell stereocilia after noise damage(24), suggesting that gaps develop in response to stereociliadamage. To assess whether damage-induced gaps are also en-

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Fig. 2. Differential localization of �cyto- and �cyto-actin in the mouse organof Corti (OC). (A) The OC has three rows of outer hair cells (OHCs) and one rowof inner hair cells (IHCs). Each hair cell is surrounded by non-sensory support-ing cells. (B) Scanning electron microscopy images of OHC and IHC stereociliabundles. (C) Stereocilium core consists of tightly packed unidirectional actinfilaments (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) OHCand IHC stereocilia. (F) Absence of �cyto-actin (green) in 6-week-old Actg1�/�

OC. (G–L) At E16.5, �cyto-actin immunoreactivity follows rhodamine-phalloidinlabeling in wt hair cells (G–I), whereas �cyto-actin is detected in supporting cellsbut not in hair cells (J–L). (M–P) At E18.5, �cyto-actin is present in all stereociliaof hair cells throughout the cochlea (M, N), whereas �cyto-actin begins toappear only in stereocilia of more developed basal turn of the cochlea (O, P).(Q–T) �cyto-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidinstaining, whereas �cyto-actin (S, T) is concentrated toward the periphery of theIHC stereocilia F-actin core in adult wt mice. Scale bars (B, Q–T), 2 �m; scale bars(D–P), 5 �m.

Fig. 3. �cyto-Actin concentrates at the sites of stereocilia core disruptions.(A–C) �cyto-Actin antibody highlights gaps (segments of F-actin depolymeriza-tion; arrows) in wild-type (wt) mouse vestibular hair cell (VHC) stereocilia. Inall panels, rhodamine-phalloidin highlights F-actin in red, and labeling withantibodies is in green. (D) �cyto-Actin at the base and within the F-actin gapsof longest stereocilia in wt mouse VHC (arrows). (E) DNase I stains globularactin within F-actin gaps of VHC stereocilia (arrows). (F) Espin concentrates ingaps of wt VHC stereocilia. (G) Uniform distribution of �cyto-actin along adultguinea pig IHC stereocilia not exposed to damaging noise. (H) Redistributionof �cyto-actin in noise-damaged guinea pig IHC stereocilia. �cyto-Actin absentfrom the tips and evenly distributed along stereocilia which appear unaf-fected (inset: second and fifth stereocilium from the left). (I–K) �cyto-Actinconcentrates at sites of F-actin damage (arrows) and at tips of shortenedstereocilia (asterisks, inset in H) in a noise-damaged bundle from (H). (L) TheF-actin gaps in IHC stereocilia from Actg1�/� mouse (arrows). (M) �cyto-Actinconcentrates in the F-actin gap of Actg1�/� IHC stereocilium. �cyto-Actinstaining along stereocilia is barely visible because of intense gap staining.(N–P) Espin concentrates in gaps of Actg1�/� VHC stereocilia. Scale bars, 2 �m.

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riched in �cyto-actin, we compared �cyto-actin localization instereocilia from control and noise-damaged guinea pigs. Con-sistent with previous studies (2, 18), �cyto-actin was distributedalong the length of control stereocilia (Fig. 3G). After a dam-aging noise exposure, �cyto-actin was enriched in both the tipsand in phalloidin-negative gaps observed along the length ofnoise-damaged stereocilia, which were often abnormally shorterthan neighboring normal appearing stereocilia of the same row(Fig. 3 H–K). In the same bundle, some stereocilia that appearedunaffected by noise still had �cyto-actin evenly distributed alongtheir length and did not have an accumulation of �cyto-actin at thetips (Fig. 3H, inset).

Immunofluorescence Confocal Microscopy and Scanning Electron Mi-croscopy Analyses Reveal an Unexpected Pattern of Degeneration ofActg1�/� Stereocilia. Interestingly, F-actin gaps were occasionallyobserved in auditory hair cell stereocilia of Actg1�/� micewithout exposure to damaging noise. These gaps lacked �cyto-actin (Fig. 3L) but contained �cyto-actin (Fig. 3M) and espin (Fig.3 N–P). The staining of �cyto-actin within gaps of Actg�/�

stereocilia was so intense that we had to turn down the gain onthe confocal microscope so that the signal within gaps was notsaturated (Fig. 3M and Fig. S3A). As a consequence, the�cyto-actin signal along the lengths of stereocilia was reduced toa barely detectable level (compare Fig. 3M and Fig. S3A withFig. S3B).

The presence of F-actin gaps in the auditory hair cell stere-ocilia of hearing-impaired, �cyto-actin–deficient mice led us toinvestigate whether a stereocilia maintenance/repair mechanismis defective in Actg1�/� mice. To define the structural changesassociated with a �cyto-actin-deficiency, we characterized themorphology of auditory hair cells. We examined outer hair cellsof 6-week-old Actg1�/� mice by scanning electron microscopyand found that �cyto-actin deficient stereocilia were indistin-guishable from OHC stereocilia of wild-type littermates (Fig. 4A–D). However, Actg1�/� stereocilia deteriorated progressivelyas the animals aged (Fig. 4 E–H). By 16 weeks of age, �50% ofstereocilia within a hair bundle were degraded or absent inActg1�/� mice (Fig. 4I). Across all three rows within the hairbundle, we observed missing or shortened (Fig. 4 J and K)stereocilia, although the remaining stereocilia looked normal.

DiscussionActg1�/� mice survive to birth and beyond, demonstrating that�cyto-actin is not strictly required for mammalian development orviability. Our observations of stereocilia from Actg1�/� miceinstead indicate that �cyto-actin is necessary to maintain cytoskel-etal integrity and function. The phenotype of Actg1�/� stereo-cilia is unique; we observed stereocilia defects ranging fromsimple shortening to complete loss of individual stereociliaacross all three rows within the hair bundle indicating selectedstereocilia disassembly (Fig. 4 E–K), whereas the remainingstereocilia within the same bundle appeared intact. The appar-ent independent nature of this phenomenon (affected stereociliasurrounded by normal stereocilia) indicates that the disassemblyprocess is initiated and/or regulated at the level of an individualstereocilium. Therefore, this disassembly process appears dif-ferent from actin treadmilling that normally occurs simulta-neously in all stereocilia of the same row (17).

Rather, our results suggest that �cyto-actin strengthens stere-ocilia F-actin cores, preventing stereocilia core damage, and/oris required to repair the damaged core. Consistent with this view,in developing wild-type mice �cyto-actin appeared and accumu-lated in stereocilia a few days before the onset of hearingfunction, perhaps preparing stereocilia to withstand the rigors ofacoustical stimulation. Furthermore, damaging noise induces theappearance of �cyto-actin–enriched, phalloidin-negative gaps inthe stereocilia core of wild-type rodent hair cells. These gaps

were not present in control stereocilia but were observed inActg1�/� mouse auditory stereocilia indicating that stereociliacore damage was more frequent or more slowly repaired in theabsence of �cyto-actin. Finally, Actg1�/� stereocilia progressivelydeteriorated, demonstrating that �cyto-actin is required to main-tain these structures.

�cyto- and �cyto-Actin are nearly identical, featuring only fourbiochemically similar residue substitutions in the N terminus,suggesting likely compensation between these proteins. Indeed,�cyto-actin protein levels were elevated in Actg1�/� mice and thetotal actin level was equivalent in Actg1�/� and wild-type mice

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mice. (E–H) Scanning electron microscopy images of OHC stereocilia from16-week-old Actg1�/� (E, F) and 16-week-old Actg1�/� mice (G, H). There is aloss of individual stereocilia from all three rows of OHC hair bundle fromActg1�/� mice. Images are from the middle turn of the cochlea. (I) Box andwhisker plot (whiskers, maximum and minimum; box, 5th–95th percentile;line, mean) of the number of individual stereocilia in individual OHCbundles 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 missingand shortened stereocilia. Scale bars (A, C, E, and G), 5 �m; scale bars (B, D,F, H, J, and K), 1 �m.

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(Fig. 1C). However, �cyto-actin–deficient stereocilia progres-sively deteriorated despite the localization of �cyto-actin to gapsin the F-actin core of Actg1�/� mouse auditory stereocilia (Fig.3M and Fig. S3). This surprising result indicates that �cyto-actinhas at least some functions that are unique and cannot becompensated for by �cyto-actin. One possibility is that �cyto-actinbrings to the site of damage a unique �cyto-actin protein partnerthat is necessary for �cyto-actin, �cyto-actin, or for �cyto- and�cyto-actin together, to repair damage to the core.

Consistent with different functions of �cyto- and �cyto-actin,we observed distinct localization patterns for each proteinwithin wild-type auditory stereocilia. �cyto-Actin localized tostereocilia cores, exactly overlaying with phalloidin staining,whereas �cyto-actin was concentrated toward the periphery ofstereocilia cores. Because the �cyto-actin antibody detects bothmonomeric and filamentous actin whereas phalloidin detectsonly filamentous actin, there appears to be a pool of mono-meric �cyto-actin at the periphery of stereocilia cores. Alter-natively, phalloidin-negative actin may still be filamentous butunable to bind phalloidin because of a particular F-actinconformation, which was observed in nuclear actin as previ-ously reviewed (25), different paracrystal filament packingthat excludes phalloidin, or masking by actin binding proteins.In any case, the peripheral population of �cyto-actin is distinctand may be used for stereocilia core remodeling and repair,perhaps redistributing to F-actin gaps that form as a result ofstereocilia core damage.

Based on �cyto-actin localization and Actg1�/� stereociliadegradation, we envision two models of �cyto-actin function.First, �cyto-actin may have a specific role involving annealing ofbroken filaments or de novo polymerization, perhaps dependingon an unknown actin-binding protein with specificity for �cyto-actin. Alternatively, �cyto-actin-containing filaments may have dis-tinct biophysical and biochemical properties, such as differentpolymerization rates or polymer stability, which protect stereociliafrom mechanical stress. Deficient repair and/or diminished struc-tural integrity then result in the eventual loss of Actg1�/� stereocilia.

Interestingly, the gaps observed in auditory stereocilia ofnoise-damaged animals and untreated Actg1�/� mice (24) (Fig.3) resemble discontinuities in actin-rich developing Drosophilabristles (26, 27). In these structures, gaps are observed bothduring formation, as short modules of F-actin are cross-linked toform fibers, and during disassembly, as the fibers are brokendown into the original modules (26). Although mammalianstereocilia are not thought to be composed of cross-linkedF-actin modules, elements of Drosophila actin regulation maynonetheless be conserved in mammalian stereocilia. Stereociliagaps may arise through physical damage that cause F-actinbundle breakage or through the action of a protein that sensesdamage and severs and depolymerizes actin filaments, generat-ing gaps similar to those that occur during modular disassemblyof Drosophila bristles (28).

The accumulation of �cyto-actin at sites of damage in wild-typehair cell stereocilia after noise exposure (Figs. 3 H–K), togetherwith the disassembly and subsequent loss of individual stereociliain Actg1�/� hair cells (Fig. 4 G–I), are consistent with �cyto-actinbeing required for maintenance and/or repair of stereocilia inadult hair cells. However, �cyto-actin seems to be entirely dis-pensable for the proper development and functional maturationof hair cells. Indeed, the viability of Actg1�/� embryos and thenormal lifespan of at least one-third of all live-born Actg1�/�

mice demonstrate that �cyto-actin is not crucial for generalorganogenesis and thus is not necessary for the formation ofactin-based structures in general. Consistent with this idea,wild-type and Actg1�/� mice intestinal brush border microvilliare morphologically indistinguishable (Fig. S5).

We previously characterized a muscle-specific �cyto-actinknockout mouse that was generated precisely because a whole-

body knockout was widely presumed to be unviable. This murinemodel exhibited normal muscle development followed by pro-gressive myopathy and muscle cell necrosis (29). Both musclecells and outer hair cells must resist force generated by contrac-tility or electromotility, respectively. Although otherwise clearlydisparate in structure and function, these mechanically chal-lenged cells seem to have a particularly evident requirement forthe specialized properties of �cyto-actin necessary for cellularmaintenance.

Additional �cyto-actin deficiency-based cytoskeletal patho-logic conditions may exist in other organs and tissues of Actg1�/�

mice that could affect long-term function. Indeed, the lowerbody mass of Actg1�/� mice and their occasional prematuredeath suggests a hidden, slowly developing or partially compen-sated pathologic condition. We conclude that �cyto-actin is notnecessary to build actin cytoskeletal structures required fororganogenesis and development but, instead, functions primarilyto reinforce and/or repair the actin cytoskeleton.

MethodsGeneration of Actg1-Null Mice. A targeting vector in which loxP sites flankexons 2 and 3 of the murine Actg1 gene was described previously (29).Embryonic stem cell targeting, screening, blastocyst injections, and subse-quent EIIa-cre breeding were performed to generate Actg1�/� mice (29).Actg1�/� mice were backcrossed to C57BI/6 for 10 generations before N10

Actg1�/� X Actg1�/� breedings were arranged to obtain Actg1�/� mice. Allgenotypes were determined as previously described (29). Animals werehoused and treated in accordance with the standards set by the University ofMinnesota Institutional Animal Care and Use Committee.

Immunoblot Analysis. Brain, lung, kidney, and cochlea were dissected frommice of the indicated genotypes, frozen in liquid nitrogen, ground intopowder, boiled in buffer (1% SDS, EGTA, PMSF, benzamidine, leupeptin), andcentrifuged to remove insoluble material. Protein concentration in the result-ing lysate was determined with either a colorimetric assay (DC assay, BioRad)or by A280 measurement. Equal amounts of protein were separated by sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferredto nitrocellulose membranes, and probed with the indicated antibodies (�cyto-actin; mAb 2–4 or pAb7577 (29); �cyto-actin, AC15 (Sigma); pan-actin, C4, giftof J. Lessard, University of Cincinnati; �smooth-actin, B4 (J. Lessard, University ofCincinnati); �smooth-actin, 1A4 (Sigma); �-tubulin B512 (Sigma). Fluorescentlylabeled secondary antibodies were detected and quantified from three sep-arate experiments blotted in triplicate using an Odyssey infrared scanner andsoftware (Li-Cor Biosciences).

Auditory Brainstem Responses. ABRs were collected as previously described(30) or using a Tucker-Davis Technologies System3 to generate sound stimuliand to amplify and record brainstem potentials as described in the SI Text.

Antibody Validation and Immunostaining. Polyclonal antibody pAb7577against cytoplasmic �cyto-actin (ACTG1) was generated in the laboratory of J.Ervasti as described, and the specificity was verified (29). The second anti-�cyto-actin polyclonal antibody was a gift from C. Bulinski and was characterizedpreviously (31). The third anti-�cyto-actin polyclonal antibody was developed inthe laboratory of K. Friderici by immunizing rabbits with a peptide of theN-terminal 15 residues (Princeton Biomolecules) and affinity purified as de-scribed in the SI Text. The immunostaining is described in the SI Text. All animalcare and experimental procedures were approved by the NINDS/NIDCD ACUC.

Animals and Noise Exposure. Noise exposure methods are described in theSI Text.

Scanning Electron Microscopy. Cochlea were rapidly dissected and fixed byperfusing 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.3 with1 mM CaCl2, through the round window, followed by immersion in the samesolution for 2 hours. After microdissection to reveal hair cell stereocilia,cochlea were incubated in 2% arginine-HCl, glycine, glutamic acid, and su-crose followed by treatment with 2% tannic acid and 2% guanidine-HCl andwere postfixed in 1% aqueous osmium tetroxide. Specimens were dehydratedin ethanol, critical point dried, sputter coated, and imaged using a cold fieldemission gun scanning electron microscope (Hitachi S-4700).

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ACKNOWLEDGMENTS. We thank J. Bartles and C. Bulinski for providinganti-espin antibody and one of the anti-�cyto-actin antibodies, respectively; D.Catts and P. Diers for technical assistance; D. Drayna, R. Chadwick, N. Gavara,A. Griffith, J. Bird, and R. Morell for critically reading the manuscript; P.Belyantsev for Fig. 1 drawing; and K. Prins, S. Ikeda, and A. Ikeda for prelim-

inary analysis of Actg1�/� mice. The work was supported by National Institutesof Health (NIH) intramural funds 1 Z01 DC000048–11 LMG (to T.B.F.), NIHintramural funds Z01-DC-000060 (to Andrew J. Griffith), funds from the DRFand NOHR Foundation (to G.I.F.), and NIH grants DC004568 (to K.H.F.), F32DC009539 (to B.J.P.), and a R01 AR049899 (to J.M.E.).

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