?-Actin 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;
andeDevelopmental 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 be
Despite this widely held supposition, we show that ?cyto-actin null
mice (Actg1?/?) are viable. However, they suffer increased mor-
tality and show progressive hearing loss during adulthood despite
compensatory up-regulation of ?cyto-actin. The surprising viability
and normal hearing of young Actg1?/?mice means that ?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 ?cyto-actin–deficient stereo-
cilia form normally, we found that they cannot maintain the
integrity of the stereocilia actin core. In the wild-type, ?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 ?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.
actin ? cytoskeleton ? hearing
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 expressed
in non-muscle cells, as reviewed elsewhere (1). Athough ?cyto-
actin and ?cyto-actin differ at only four biochemically similar
amino acid residues in their N-termini, several lines of evidence
suggest that each protein is functionally distinct. The amino acid
sequences of ?cyto- and ?cyto-actin are each exactly conserved
among 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 associated
with syndromic phenotypes including severe developmental mal-
formations and bilateral deafness (7), humans carrying a variety
of dominant missense mutations in ACTG1 develop postlingual
nonsyndromic progressive hearing loss (DFNA20, OMIM
?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
hair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig.
2A). IHCs function as auditory receptors, converting sound
energy into neuronal signals, whereas OHCs enhance sensitivity
to sound stimuli, as reviewed elsewhere (12). The apical surface
of a hair cell is topped with actin-rich microvilli-derived protru-
sions termed stereocilia, which deflect in response to sound
stimuli, 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-
here are six genes encoding six vertebrate actins that are
classified according to where they are predominately ex-
In the mammalian organ of Corti, the precise architecture of
stereocilia is preserved for the life of the organism. Meanwhile, the
stereocilia actin core is reported to undergo renewal by continuous
at pointed ends, which is precisely coupled to maintain stereocilia
length (15, 16). The speed of stereocilia treadmilling is reported to
be the same for all stereocilia of the same row and is proportional
to stereocilia length (17). Immuno-electron microscopy shows that
in wild-type hair cells ?cyto-actin is largely restricted to stereocilia,
their rootlets, and the cuticular plate (2, 3, 18), whereas ?cyto-actin
is reported to have more broad localization, including hair cell
stereocilia and their rootlets, the cuticular plate in which stereocilia
are anchored, adherens junctions, and outer hair cell lateral walls
to study the structural consequences of perturbing actin isoform
?cyto- and ?cyto-Actin are among the most abundant proteins
both 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 knockout
mouse (Actg1?/?). We show here that mice completely lacking
?cyto-actin can survive to adulthood. Interestingly, Actg1?/?mice
initially have normal hearing but develop progressive hearing
loss during adulthood that is characterized by stereocilia actin
core disruptions and stereocilia degradation. These findings led
us to conclude that ?cyto-actin is not necessary for the formation
of actin-based structures required for organogenesis and devel-
opment, but is essential for maintenance of the hair cell actin
?cyto-Actin Null Mice Are Viable. To determine whether there is a
unique function of ?cyto-actin that cannot be compensated by the
other actin family members, we generated a ?cyto-actin null
(Actg1?/?) mouse. Mice entirely devoid of ?cyto-actin were
viable, but born at one-third of the expected Mendelian ratio,
indicating that the absence of ?cyto-actin caused some embryonic
or 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.
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: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
June 16, 2009 ?
vol. 106 ?
no. 24 ?
surviving Actg1?/?mice appeared largely normal, their body
weight 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). To
investigate whether the observed effects were caused by a
general depletion of cellular actin, we analyzed actin isoform
expression 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 family
members 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 in
?cyto-Actin Null Mice Show Progressive Loss of Hearing. We assessed
hearing in wild-type and Actg1?/?mice by measuring auditory
brainstem response (ABR) thresholds. ABR objectively mea-
sures synchronous electrical activity generated by the neurons in
the ascending auditory system and can be recorded from scalp
electrodes by averaging responses to short tone bursts (19, 20).
We found that Actg1?/?mice up to 6 weeks of age had
near-normal ABR thresholds (Fig. 1D). However, 16-week-old
Actg1?/?mice demonstrated a marked hearing impairment at
each frequency tested, and by 24 weeks of age were profoundly
deaf (Fig. 1D). This progressive hearing loss was not found in
Actg1?/?littermates, which exhibited wild-type ABR thresholds
up to 24 weeks of age (Fig. S2) despite expressing only 50% of
wild-type levels of ?cyto-actin (Fig. 1C).
Differential Localization of ?cyto- and ?cyto-Actin in Developing and
Adult Mouse Hair Cells Revealed Delayed Appearance of ?cyto-Actin in
Stereocilia. Consistent with previous reports in postnatal chicken
and mature guinea pig or rat, both ?cyto- and ?cyto-actin were
detected in stereocilia (Fig. 2 D and E) and the cuticular plate
of adult wild-type mouse hair cells. The three independently
generated ?cyto-actin-specific antibodies used did not stain any
structures in Actg1?/?hair cells (Fig. 2F), demonstrating the
specificity of these antisera for ?cyto-actin. We found that during
embryonic development of wild-type mice, ?cyto-actin appeared
in the body of hair cells and subsequently in stereocilia earlier
than ?cyto-actin, which accumulated first in supporting cells and
only later appeared in hair cells (Fig. 2 G–P). We observed
?cyto-actin in auditory hair cell stereocilia as soon as they appear
around E16.5 (Fig. 2 G–I) in the basal turn of the cochlea. The
first appearance of ?cyto-actin within stereocilia was detected
after stereocilia emerged at approximately E18.5 (Fig. 2 O–P).
These data are consistent with ?cyto-actin primarily contributing
to the formation of the actin cytoskeleton of developing stere-
ocilia, whereas ?cyto-actin may be important for cytoskeleton
maintenance and/or reinforcement.
Although both actins are found in mouse stereocilia, we
observed differential localization within the stereocilia, again
consistent with ?cyto- and ?cyto-actin having disparate functions.
In the adult wild-type mouse stereocilia, ?cyto-actin staining
overlapped completely with rhodamine-phalloidin staining,
whereas ?cyto-actin was concentrated more toward the periphery
of the stereocilia actin core, often only partially overlapping with
rhodamine-phalloidin staining (Fig. 2 Q–T).
Phalloidin-Negative Gaps in F-Actin Stereocilia Cores Contain Core
Components. In the course of characterizing the localization of
staining of F-actin cores of vestibular hair cell stereocilia (Fig. 3
A–C). The gaps were most frequently observed at the base and
along the length of stereocilia in the tallest row (Fig. 3D). Using
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
likely to be predominantly monomeric, because phalloidin rec-
ognizes only filamentous actin (Fig. 3 A–D). Usually gap staining
was much more intense relative to that along the length of
stereocilium (Figs. 3 A–D and 3 F–M), which may be caused by
enhanced 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 available
non-filamentous actin within a stereocilium. A similar redistri-
bution to F-actin gaps was also seen for ?cyto-actin (Fig. S3). It
is likely that ?cyto-actin is also recruited to the gaps from a pool
of non-filamentous actin, as staining intensity along a stereoci-
lium with a gap was not different from the intensity of staining
along a stereocilium without gaps (Fig. S3B). The same pattern
of staining was also observed for DNase I (Fig. 3E), a marker for
Level (dB SPL)
Actg1+/+ 6 weeks
Actg1+/+ 16 weeks
Actg1+/+ 24 weeks
Actg1-/- 6 weeks
Actg1-/- 16 weeks
Actg1-/- 24 weeks
0 50 100
150 200250 300
(% of wild-type)
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?/?mice
from P0 to P350, (n ? 31 for each genotype). (C) Representative immunoblots
of SDS extracts from Actg1?/?, Actg1?/?and Actg1?/?cochlear extracts
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 fre-
quencies from 4 to 22 kHz, presented at half-octave steps (n ? 5, mean ? SEM).
www.pnas.org?cgi?doi?10.1073?pnas.0900221106Belyantseva et al.
monomeric actin (G-actin) (21), and espin (Fig. 3F), an actin
bundling protein essential for stereocilia formation, which is
reported to have both F- and G-actin binding sites (22). Inter-
estingly, only proteins that are either actin core components or
directly involved in actin turnover were found to accumulate in
the 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 and
nucleation of F-actin (23), selectively accumulates in stereocilia
gaps (Fig. S4).
Together, these data suggest that (i) gaps have a different
structural arrangement than the stereocilia actin core, (ii) gaps
are enriched for ?cyto- and ?cyto-actin along with other core
components, and (iii) cofilin may mediate ongoing actin remod-
eling in the gap to facilitate repair of local damage of the F-actin
?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 reported
in guinea pig auditory hair cell stereocilia after noise damage
(24), suggesting that gaps develop in response to stereocilia
damage. To assess whether damage-induced gaps are also en-
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
and IHC stereocilia. (F) Absence of ?cyto-actin (green) in 6-week-old Actg1?/?
but not in hair cells (J–L). (M–P) At E18.5, ?cyto-actin is present in all stereocilia
of hair cells throughout the cochlea (M, N), whereas ?cyto-actin begins to
appear only in stereocilia of more developed basal turn of the cochlea (O, P).
(Q–T) ?cyto-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidin
(D–P), 5 ?m.
Differential localization of ?cyto- and ?cyto-actin in the mouse organ
(A–C) ?cyto-Actin antibody highlights gaps (segments of F-actin depolymeriza-
tion; 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) ?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 ?cyto-actin along adult
guinea pig IHC stereocilia not exposed to damaging noise. (H) Redistribution
of ?cyto-actin in noise-damaged guinea pig IHC stereocilia. ?cyto-Actin absent
from the tips and evenly distributed along stereocilia which appear unaf-
fected (inset: second and fifth stereocilium from the left). (I–K) ?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) ?cyto-Actin
concentrates in the F-actin gap of Actg1?/?IHC stereocilium. ?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 ?m.
?cyto-Actin concentrates at the sites of stereocilia core disruptions.
Belyantseva et al.PNAS ?
June 16, 2009 ?
vol. 106 ?
no. 24 ?
riched in ?cyto-actin, we compared ?cyto-actin localization in
stereocilia from control and noise-damaged guinea pigs. Con-
sistent with previous studies (2, 18), ?cyto-actin was distributed
along the length of control stereocilia (Fig. 3G). After a dam-
aging noise exposure, ?cyto-actin was enriched in both the tips
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
(Fig. 3 H–K). In the same bundle, some stereocilia that appeared
unaffected by noise still had ?cyto-actin evenly distributed along
tips (Fig. 3H, inset).
Immunofluorescence Confocal Microscopy and Scanning Electron Mi-
croscopy Analyses Reveal an Unexpected Pattern of Degeneration of
Actg1?/?Stereocilia. Interestingly, F-actin gaps were occasionally
observed in auditory hair cell stereocilia of Actg1?/?mice
without 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 on
the confocal microscope so that the signal within gaps was not
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
The presence of F-actin gaps in the auditory hair cell stere-
ocilia of hearing-impaired, ?cyto-actin–deficient mice led us to
investigate whether a stereocilia maintenance/repair mechanism
is defective in Actg1?/?mice. To define the structural changes
associated with a ?cyto-actin-deficiency, we characterized the
morphology of auditory hair cells. We examined outer hair cells
of 6-week-old Actg1?/?mice by scanning electron microscopy
and found that ?cyto-actin deficient stereocilia were indistin-
guishable from OHC stereocilia of wild-type littermates (Fig. 4
A–D). However, Actg1?/?stereocilia deteriorated progressively
as the animals aged (Fig. 4 E–H). By 16 weeks of age, ?50% of
stereocilia within a hair bundle were degraded or absent in
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?/?mice survive to birth and beyond, demonstrating that
?cyto-actin is not strictly required for mammalian development or
viability. Our observations of stereocilia from Actg1?/?mice
etal integrity and function. The phenotype of Actg1?/?stereo-
cilia is unique; we observed stereocilia defects ranging from
simple shortening to complete loss of individual stereocilia
across all three rows within the hair bundle indicating selected
stereocilia disassembly (Fig. 4 E–K), whereas the remaining
stereocilia within the same bundle appeared intact. The appar-
ent independent nature of this phenomenon (affected stereocilia
surrounded by normal stereocilia) indicates that the disassembly
process is initiated and/or regulated at the level of an individual
stereocilium. 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/or
is 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 hearing
function, perhaps preparing stereocilia to withstand the rigors of
appearance of ?cyto-actin–enriched, phalloidin-negative gaps in
the stereocilia core of wild-type rodent hair cells. These gaps
were not present in control stereocilia but were observed in
Actg1?/?mouse auditory stereocilia indicating that stereocilia
core damage was more frequent or more slowly repaired in the
absence of ?cyto-actin. Finally, Actg1?/?stereocilia progressively
deteriorated, demonstrating that ?cyto-actin is required to main-
tain these structures.
?cyto- and ?cyto-Actin are nearly identical, featuring only four
biochemically similar residue substitutions in the N terminus,
suggesting likely compensation between these proteins. Indeed,
?cyto-actin protein levels were elevated in Actg1?/?mice and the
total actin level was equivalent in Actg1?/?and wild-type mice
Number of stereocilia
per hair cell
6 weeks 16 weeks
6 weeks old
16 weeks old
?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, 5th–95thpercentile;
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 ?m; scale bars (B, D,
F, H, J, and K), 1 ?m.
Morphology of stereocilia bundles in adult wild-type (Actg?/?) and
www.pnas.org?cgi?doi?10.1073?pnas.0900221106 Belyantseva et al.
(Fig. 1C). However, ?cyto-actin–deficient stereocilia progres-
sively deteriorated despite the localization of ?cyto-actin to gaps
in the F-actin core of Actg1?/?mouse auditory stereocilia (Fig.
3M and Fig. S3). This surprising result indicates that ?cyto-actin
has at least some functions that are unique and cannot be
compensated for by ?cyto-actin. One possibility is that ?cyto-actin
brings to the site of damage a unique ?cyto-actin protein partner
that 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 protein
within wild-type auditory stereocilia. ?cyto-Actin localized to
stereocilia cores, exactly overlaying with phalloidin staining,
whereas ?cyto-actin was concentrated toward the periphery of
stereocilia cores. Because the ?cyto-actin antibody detects both
monomeric and filamentous actin whereas phalloidin detects
only 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 but
unable to bind phalloidin because of a particular F-actin
conformation, which was observed in nuclear actin as previ-
ously reviewed (25), different paracrystal filament packing
that excludes phalloidin, or masking by actin binding proteins.
In any case, the peripheral population of ?cyto-actin is distinct
and may be used for stereocilia core remodeling and repair,
perhaps redistributing to F-actin gaps that form as a result of
stereocilia core damage.
Based on ?cyto-actin localization and Actg1?/?stereocilia
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
on 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 different
polymerization rates or polymer stability, which protect stereocilia
from mechanical stress. Deficient repair and/or diminished struc-
Interestingly, the gaps observed in auditory stereocilia of
noise-damaged animals and untreated Actg1?/?mice (24) (Fig.
3) resemble discontinuities in actin-rich developing Drosophila
bristles (26, 27). In these structures, gaps are observed both
during formation, as short modules of F-actin are cross-linked to
form fibers, and during disassembly, as the fibers are broken
down into the original modules (26). Although mammalian
stereocilia are not thought to be composed of cross-linked
F-actin modules, elements of Drosophila actin regulation may
nonetheless be conserved in mammalian stereocilia. Stereocilia
gaps may arise through physical damage that cause F-actin
bundle breakage or through the action of a protein that senses
damage and severs and depolymerizes actin filaments, generat-
ing gaps similar to those that occur during modular disassembly
of Drosophila bristles (28).
The accumulation of ?cyto-actin at sites of damage in wild-type
hair cell stereocilia after noise exposure (Figs. 3 H–K), together
in Actg1?/?hair cells (Fig. 4 G–I), are consistent with ?cyto-actin
being required for maintenance and/or repair of stereocilia in
adult hair cells. However, ?cyto-actin seems to be entirely dis-
pensable for the proper development and functional maturation
of hair cells. Indeed, the viability of Actg1?/?embryos and the
normal lifespan of at least one-third of all live-born Actg1?/?
mice demonstrate that ?cyto-actin is not crucial for general
organogenesis and thus is not necessary for the formation of
actin-based structures in general. Consistent with this idea,
wild-type and Actg1?/?mice intestinal brush border microvilli
are morphologically indistinguishable (Fig. S5).
We previously characterized a muscle-specific ?cyto-actin
knockout mouse that was generated precisely because a whole-
body knockout was widely presumed to be unviable. This murine
model exhibited normal muscle development followed by pro-
gressive myopathy and muscle cell necrosis (29). Both muscle
cells and outer hair cells must resist force generated by contrac-
tility or electromotility, respectively. Although otherwise clearly
disparate in structure and function, these mechanically chal-
lenged cells seem to have a particularly evident requirement for
the specialized properties of ?cyto-actin necessary for cellular
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 lower
body mass of Actg1?/?mice and their occasional premature
death suggests a hidden, slowly developing or partially compen-
sated pathologic condition. We conclude that ?cyto-actin is not
necessary to build actin cytoskeletal structures required for
organogenesis and development but, instead, functions primarily
to reinforce and/or repair the actin cytoskeleton.
Generation of Actg1-Null Mice. A targeting vector in which loxP sites flank
exons 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. All
genotypes were determined as previously described (29). Animals were
housed and treated in accordance with the standards set by the University of
Minnesota Institutional Animal Care and Use Committee.
Immunoblot Analysis. Brain, lung, kidney, and cochlea were dissected from
mice of the indicated genotypes, frozen in liquid nitrogen, ground into
ing lysate was determined with either a colorimetric assay (DC assay, BioRad)
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred
actin; mAb 2–4 or pAb7577 (29); ?cyto-actin, AC15 (Sigma); pan-actin, C4, gift
Cincinnati); ?smooth-actin, 1A4 (Sigma); ?-tubulin B512 (Sigma). Fluorescently
labeled secondary antibodies were detected and quantified from three sep-
arate experiments blotted in triplicate using an Odyssey infrared scanner and
software (Li-Cor Biosciences).
Auditory Brainstem Responses. ABRs were collected as previously described
(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.
Antibody Validation and Immunostaining. Polyclonal antibody pAb7577
against cytoplasmic ?cyto-actin (ACTG1) was generated in the laboratory of J.
actin polyclonal antibody was a gift from C. Bulinski and was characterized
the laboratory of K. Friderici by immunizing rabbits with a peptide of the
N-terminal 15 residues (Princeton Biomolecules) and affinity purified as de-
Animals and Noise Exposure. Noise exposure methods are described in the
Scanning Electron Microscopy. Cochlea were rapidly dissected and fixed by
1 mM CaCl2, through the round window, followed by immersion in the same
solution 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 and
in ethanol, critical point dried, sputter coated, and imaged using a cold field
emission gun scanning electron microscope (Hitachi S-4700).
Belyantseva et al.PNAS ?
June 16, 2009 ?
vol. 106 ?
no. 24 ?
ACKNOWLEDGMENTS. We thank J. Bartles and C. Bulinski for providing Download full-text
anti-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-
of Health (NIH) intramural funds 1 Z01 DC000048–11 LMG (to T.B.F.), NIH
intramural funds Z01-DC-000060 (to Andrew J. Griffith), funds from the DRF
and NOHR Foundation (to G.I.F.), and NIH grants DC004568 (to K.H.F.), F32
DC009539 (to B.J.P.), and a R01 AR049899 (to J.M.E.).
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