-Actin is required for cytoskeletal maintenance but not development

Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 07/2009; 106(24):9703-8. DOI: 10.1073/pnas.0900221106
Source: PubMed
ABSTRACT
Beta(cyto)-actin and gamma(cyto)-actin are ubiquitous proteins thought to be essential building blocks of the cytoskeleton in all non-muscle cells. Despite this widely held supposition, we show that gamma(cyto)-actin null mice (Actg1(-/-)) are viable. However, they suffer increased mortality and show progressive hearing loss during adulthood despite compensatory up-regulation of beta(cyto)-actin. The surprising viability and normal hearing of young Actg1(-/-) mice means that beta(cyto)-actin can likely build all essential non-muscle actin-based cytoskeletal structures including mechanosensory stereocilia of hair cells that are necessary for hearing. Although gamma(cyto)-actin-deficient stereocilia form normally, we found that they cannot maintain the integrity of the stereocilia actin core. In the wild-type, gamma(cyto)-actin localizes along the length of stereocilia but re-distributes to sites of F-actin core disruptions resulting from animal exposure to damaging noise. In Actg1(-/-) stereocilia similar disruptions are observed even without noise exposure. We conclude that gamma(cyto)-actin is required for reinforcement and long-term stability of F-actin-based structures but is not an essential building block of the developing cytoskeleton.

Full-text

Available from: Edward J Walsh, Oct 16, 2014
-Actin is required for cytoskeletal maintenance
but not development
Inna A. Belyantseva
a,1
, Benjamin J. Perrin
b,1
, Kevin J. Sonnemann
b,1
, Mei Zhu
c
, Ruben Stepanyan
d
, JoAnn McGee
e
,
Gregory I. Frolenkov
d,f
, Edward J. Walsh
e
, Karen H. Friderici
c
, Thomas B. Friedman
a
, and James M. Ervasti
b,2
a
Laboratory of Molecular Genetics, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850;
b
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN 55455;
c
Microbiology and Molecular Genetics,
Michigan State University, East Lansing, MI 48824;
d
Department of Physiology, University of Kentucky, Lexington, KY 40536;
f
Molecular Biology and
Genetics Section, National Institute on Deafness and Other Communication Disorders/National Institutes of Health, Rockville, MD 20850;
and
e
Developmental 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
essential building blocks of the cytoskeleton in all non-muscle cells.
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
T
here are six genes encoding six vertebrate actins that are
classified 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 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 av ian 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 phenot ypes including severe development al mal-
for mations and bilateral deafness (7), humans carrying a variety
of dominant missense mutations in ACTG1 develop postlingual
nonsyndromic progressive hearing loss (DFNA20, OMIM
604717) (8–11).
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 c ochlea 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 sur face
of a hair cell is topped with actin-rich microvilli-derived protr u-
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 c onsists of a paracrys-
t alline array of unidirectionally oriented actin filaments (Fig. 2C)
(13–15).
In the mammalian organ of Corti, the precise architecture of
stereocilia is pre served for the life of the organism. Meanwhile, the
stereocilia actin core is reported to undergo renewal by continuous
actin polymerization at filament barbed ends and depolymerization
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
(2, 3, 18). Hair cells and their stereocilia are thus an attractive model
to study the structural consequence s of perturbing actin isoform
composition.
cyto
- and
cyto
-Actin are among the most abundant proteins
in every mammalian cell, leading to the common assumption that
both cytoplasmic actins are essential for function and viability.
To test this supposition and to unc over the unique function of
cyto
-actin, we generated a whole-body
cyto
-actin k nockout
mouse (Actg1
/
). We show here that mice completely lacking
cyto
-actin can survive to adulthood. Interestingly, Actg1
/
mice
in itially have normal hearing but develop progressive hearing
loss during adulthood that is characterized by stereocilia actin
c ore 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
c ytoskeleton.
Results
cyto
-Actin Null Mice Are Viable. To determine whether there is a
un ique 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.
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.
1
I.A.B., B.J.P., and K.J.S. contributed equally to this work.
2
To 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.
www.pnas.orgcgidoi10.1073pnas.0900221106 PNAS
June 16, 2009
vol. 106
no. 24
9703–9708
CELL BIOLOGY
Page 1
surviving Actg1
/
mice appeared largely normal, their body
weight was 20% lower than wild-type (Actg1
/
) and heteroz y-
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
ex pression 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
Actg1
/
mice.
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-nor mal 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
/
litter mates, which exhibited w ild-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 prev ious 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
str uctures in Actg1
/
hair cells (Fig. 2F), demonstrating the
specificit y 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 GP). We observed
cyto
-actin in auditory hair cell stereocilia as soon as they appear
around E16.5 (Fig. 2 GI) in the basal turn of the cochlea. The
first appearance of
cyto
-actin within stereocilia was detected
af ter stereocilia emerged at approximately E18.5 (Fig. 2 OP).
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 import ant for cytoskeleton
maintenance and/or reinforcement.
A lthough both actins are found in mouse stereocilia, we
observed differential localization within the stereocilia, again
c onsistent with
cyto
- and
cyto
-actin having disparate functions.
In the adult wild-t ype mouse stereocilia,
cyto
-actin staining
overlapped c ompletely with rhodamine-phalloidin st aining,
whereas
cyto
-actin was concentrated more toward the periphery
of the stereocilia actin core, often only partially overlapping with
rhodamine-phalloidin staining (Fig. 2 QT).
Phalloidin-Negative Gaps in F-Actin Stereocilia Cores Contain Core
Components. In the course of characterizing the localization of
the cytoplasmic actins, we observed occasional gaps in phalloidin
st aining of F-actin cores of vestibular hair cell stereocilia (Fig. 3
AC). 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 recogn ize 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-
ogn izes only filamentous actin (Fig. 3 AD). Usually gap staining
was much more intense relative to that along the length of
stereocilium (Figs. 3 AD and 3 FM), which may be caused by
enhanced antibody accessibility within the gaps. Alternatively,
intense gap staining could be partially ex plained 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 st aining 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 st aining was also observed for DNase I (Fig. 3E), a marker for
A
B
C
Frequency (kHz)
Level (dB SPL)
46101625
0
20
40
60
80
100
120
Actg1
+/+
6 weeks
Actg1
+/+
16 weeks
Actg1
+/+
24 weeks
Actg1
-
/
-
6 weeks
Actg1
-
/
-
16 weeks
Actg1
-
/
-
24 weeks
D
0 50 100 150 200 250 300
0
10
15
20
25
30
35
40
Mass (grams)
Age (days)
Actg1
-/-
Actg1
+/-
Actg1
+/+
Actg1
-/-
Actg1
+/-
γ
cyto
-actin
β
cyto
-actin
total actin
α-tubulin
γ
cyto
-actin
β
cyto
-actin
total actin
Actg1
-/-
Actg1
+/-
0
50
100
150
200
Protein level
(% of wild-type)
Actg1
+/+
Actg1
+/+
0 100 200 300
0
20
40
60
80
100
Survival (%)
Age (days)
Actg1
+/-
Actg1
-/-
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
/
,18Actg1
/
,11Actg1
/
,
mean SEM). (B) Kaplan-Meier survival curve of Actg1
/
and Actg1
/
mice
from P0 to P350, (n 31 for each genotype). (C) Representative immunoblots
of SDS extracts from Actg1
/
, Actg1
/
and Actg1
/
cochlear extracts
probed with antibodies specific for
cyto
-actin,
cyto
-actin, pan-actin, or tubulin
antibody. Protein levels were quantified and are expressed relative to the
wild-type level (mean SEM). (D) Actg1
/
mice develop progressive hearing
loss. Auditory brainstem response (ABR) thresholds were determined for
Actg1
/
and Actg1
/
mice at 6, 16, and 24 weeks of age using stimulus fre-
quencies from 4 to 22 kHz, presented at half-octave steps (n 5, mean SEM).
9704
www.pnas.orgcgidoi10.1073pnas.0900221106 Belyantseva et al.
Page 2
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 c omponents 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, c ofilin, 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
str uctural arrangement than the stereocilia actin core, (ii) gaps
are enriched for
cyto
- and
cyto
-actin along with other core
c omponents, and (iii) cofilin may mediate ongoing actin remod-
eling in the gap to facilitate repair of local damage of the F-actin
c ore.
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 af ter noise damage
(24), suggesting that gaps develop in response to stereocilia
damage. To assess whether damage-induced gaps are also en-
A
B
C
D
E
F
G
HI
J
KL
MN
O
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
and IHC stereocilia. (F) Absence of
cyto
-actin (green) in 6-week-old Actg1
/
OC. (GL) At E16.5,
cyto
-actin immunoreactivity follows rhodamine-phalloidin
labeling in wt hair cells (GI), whereas
cyto
-actin is detected in supporting cells
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).
(QT)
cyto
-Actin immunoreactivity (Q, R) overlaps with rhodamine-phalloidin
staining, whereas
cyto
-actin (S, T) is concentrated toward the periphery of the
IHC stereocilia F-actin core in adult wt mice. Scale bars (B, QT), 2
m; scale bars
(DP), 5
m.
Fig. 3.
cyto
-Actin concentrates at the sites of stereocilia core disruptions.
(AC)
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). (IK)
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.
(NP) Espin concentrates in gaps of Actg1
/
VHC stereocilia. Scale bars, 2
m.
Belyantseva et al. PNAS
June 16, 2009
vol. 106
no. 24
9705
CELL BIOLOGY
Page 3
riched in
cyto
-actin, we compared
cyto
-actin localization in
stereocilia from c ontrol and noise-damaged guinea pigs. Con-
sistent with previous studies (2, 18),
cyto
-actin was distributed
along the length of control stereocilia (Fig. 3G). Af ter a dam-
aging noise ex posure,
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 HK). In the same bundle, some stereocilia that appeared
unaf fected by noise still had
cyto
-actin evenly distributed along
their length and did not have an accumulation of
cyto
-actin at the
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 NP). The staining of
cyto
-actin within gaps of Actg
/
stereocilia was so intense that we had to turn down the gain on
the c onfocal 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 detect able level (compare Fig. 3M and Fig. S3A with
Fig. S3B).
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-deficienc y, we characterized the
morphology of auditory hair cells. We examined outer hair cells
of 6-week-old Actg1
/
mice by scann ing electron microscopy
and found that
cyto
-actin deficient stereocilia were indistin-
guishable f rom OHC stereocilia of wild-type littermates (Fig. 4
AD). However, Actg1
/
stereocilia deteriorated progressively
as the animals aged (Fig. 4 EH). 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.
Discussion
Actg1
/
mice survive to birth and beyond, demonstrating that
cyto
-actin is not strictly required for mammalian development or
viability. Our observations of stereocilia f rom Actg1
/
mice
instead indicate that
cyto
-actin is necessary to maintain cytoskel-
et al 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 EK), 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 f rom 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 c ore. 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
ac oustical stimulation. Furthermore, damaging noise induces the
appearance of
cyto
-actin–enriched, phalloidin-negative gaps in
the stereocilia c ore of wild-type rodent hair cells. These gaps
were not present in c ontrol stereocilia but were observed in
Actg1
/
mouse auditory stereocilia indicating that stereocilia
c ore 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-
t ain 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
tot al actin level was equivalent in Actg1
/
and wild-type mice
I
0
20
40
60
80
100
120
Number of stereocilia
per hair cell
*
Actg1
+/+
Actg1
-
/
-
Actg1
+/+
Actg1
-
/
-
6 weeks 16 weeks
Actg1
+/+
Actg1
-/-
B
6 weeks old
AB
CD
CD
16 weeks old
EF
GH
Actg1
+/+
Actg1
-/-
J
K
Fig. 4. Morphology of stereocilia bundles in adult wild-type (Actg
/
) and
cyto
-actin deficient (Actg1
/
) mice. (AD) 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, 5
th
–95
th
percentile;
line, mean) of the number of individual stereocilia in individual OHC
bundles from Actg1
/
or Actg1
/
mice at 6 and 16 weeks of age, *P
0.005. (JK) 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.
9706
www.pnas.orgcgidoi10.1073pnas.0900221106 Belyantseva et al.
Page 4
(Fig. 1C). However,
cyto
-actin–deficient stereocilia prog res-
sively deteriorated despite the localization of
cyto
-actin to gaps
in the F-actin c ore 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
c ompensated 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 w ith dif ferent functions of
cyto
- and
cyto
-actin,
we observed distinct localization patterns for each protein
w ithin w ild-type auditory stereocilia.
cyto
-Actin localized to
stereocilia c ores, exactly overlaying with phalloidin stain ing,
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 c ores. Alter-
natively, phalloidin-negative actin may still be filamentous but
unable to bind phalloidin because of a particular F-actin
c onfor mation, which was observed in nuclear actin as prev i-
ously rev iewed (25), dif ferent paracrystal filament pack ing
that excludes phalloidin, or mask ing by actin binding proteins.
In any case, the peripheral population of
cyto
-actin is distinct
and may be used for stereocilia c ore 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 rate s or polymer stability, which protect stereocilia
from 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 of
noise-damaged animals and untreated Actg1
/
mice (24) (Fig.
3) resemble discontinuities in actin-rich developing Drosophila
bristles (26, 27). In these str uctures, gaps are observed both
during formation, as short modules of F-actin are cross-linked to
for m 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 depoly merizes actin filaments, generat-
ing gaps similar to those that occur during modular disassembly
of Drosophila bristles (28).
The ac cumulation of
cyto
-actin at sites of damage in wild-type
hair cell stereocilia after noise exposure (Figs. 3 HK), together
with the disassembly and subsequent loss of individual stereocilia
in Actg1
/
hair cells (Fig. 4 GI), 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
nor mal 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 prev iously characterized a muscle-specific
cyto
-actin
k nockout mouse that was generated precisely because a whole-
body knockout was w idely 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 c ontrac-
tilit y or electromotilit y, respectively. Although other wise 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
maintenance.
Additional
cyto
-actin deficiency-based cytoskeletal patho-
logic conditions may exist in other organs and tissues of Actg1
/
mice that could affect long-ter m function. Indeed, the lower
body mass of Actg1
/
mice and their oc casional premature
death suggests a hidden, slowly developing or partially c ompen-
sated pathologic condition. We conclude that
cyto
-actin is not
necessary to build actin cytoskeletal str uctures required for
organogenesis and development but, instead, functions primarily
to reinforce and/or repair the actin c ytoskeleton.
Methods
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 N
10
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
powder, boiled in buffer (1% SDS, EGTA, PMSF, benzamidine, leupeptin), and
centrifuged to remove insoluble material. Protein concentration in the result-
ing lysate was determined with either a colorimetric assay (DC assay, BioRad)
or by A
280
measurement. Equal amounts of protein were separated by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred
to nitrocellulose membranes, and probed with the indicated antibodies (
cyto
-
actin; mAb 2–4 or pAb7577 (29);
cyto
-actin, AC15 (Sigma); pan-actin, C4, gift
of J. Lessard, University of Cincinnati;
smooth
-actin, B4 (J. Lessard, University of
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.
Ervasti as described, and the specificity was verified (29). The second anti-
cyto
-
actin polyclonal antibody was a gift from C. Bulinski and was characterized
previously (31). The third anti-
cyto
-actin polyclonal antibody was developed in
the laboratory of K. Friderici by immunizing rabbits with a peptide of the
N-terminal 15 residues (Princeton Biomolecules) and affinity purified as de-
scribed in the SI Text. The immunostaining is described in the SI Text. All animal
care and experimental procedures were approved by the NINDS/NIDCD ACUC.
Animals and Noise Exposure. Noise exposure methods are described in the
SI Text.
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
1 mM CaCl
2
, 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
were postfixed in 1% aqueous osmium tetroxide. Specimens were dehydrated
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
9707
CELL BIOLOGY
Page 5
ACKNOWLEDGMENTS. We thank J. Bartles and C. Bulinski for providing
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-
inary analysis of Actg1
/
mice. The work was supported by National Institutes
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.).
1. Herman IM (1993) Actin isoforms. Curr Opin Cell Biol 5:48–55.
2. Furness DN, Katori Y, Mahendrasingam S, Hackney CM (2005) Differential distribution
of beta- and gamma-actin in guinea-pig cochlear sensory and supporting cells. Hear
Res 207:22–34.
3. Hofer D, Ness W, Drenckhahn D (1997) Sorting of actin isoforms in chicken auditory hair
cells. J Cell Sci 110:765–770.
4. Slepecky NB, Savage JE (1994) Expression of actin isoforms in the guinea pig organ of
Corti: Muscle isoforms are not detected. Hear Res 73:16–26.
5. Yao X, Chaponnier C, Gabbiani G, Forte JG (1995) Polarized distribution of actin
isoforms in gastric parietal cells. Mol Biol Cell 6:541–557.
6. Karakozova M, et al. (2006) Arginylation of beta-actin regulates actin cytoskeleton and
cell motility. Science 313:192–196.
7. Procaccio V, et al. (2006) A mutation of beta-actin that alters depolymerization
dynamics is associated with autosomal dominant developmental malformations, deaf-
ness, and dystonia. Am J Hum Genet 78:947–960.
8. Morell RJ, et al. (2000) A new locus for late-onset, progressive, hereditary hearing loss
DFNA20 maps to 17q25. Genomics 63:1– 6.
9. Rendtorff ND, et al. (2006) A novel missense mutation in ACTG1 causes dominant
deafness in a Norwegian DFNA20/26 family, but ACTG1 mutations are not frequent
among families with hereditary hearing impairment. Eur J Hum Genet 14:1097–1105.
10. van Wijk E, et al. (2003) A mutation in the gamma actin 1 (ACTG1) gene causes
autosomal dominant hearing loss (DFNA20/26). J Med Genet 40:879 884.
11. Zhu M, et al. (2003) Mutations in the gamma-actin gene (ACTG1) are associated with
dominant progressive deafness (DFNA20/26). Am J Hum Genet 73:1082–1091.
12. Dallos P (1992) The active cochlea. J Neurosci 12:4575–4585.
13. DeRosier DJ, Tilney LG, Egelman E (1980) Actin in the inner ear: The remarkable
structure of the stereocilium. Nature 287:291–296.
14. Flock A, Cheung HC (1977) Actin filaments in sensory hairs of inner ear receptor cells.
J Cell Biol 75:339 –343.
15. Tilney LG, Derosier DJ, Mulroy MJ (1980) The organization of actin filaments in the
stereocilia of cochlear hair cells. J Cell Biol 86:244–259.
16. Schneider ME, Belyantseva IA, Azevedo RB, Kachar B (2002) Rapid renewal of auditory
hair bundles. Nature 418:837–838.
17. Rzadzinska AK, Schneider ME, Davies C, Riordan GP, Kachar B (2004) Anactinmolecular
treadmill and myosins maintain stereocilia functional architecture and self-renewal.
J Cell Biol 164:887– 897.
18. Furness DN, Mahendrasingam S, Ohashi M, Fettiplace R, Hackney CM (2008) The
dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells:
Comparison between high- and low-frequency cells and evidence for a connection to
the lateral membrane. J Neurosci 28:6342– 6353.
19. Biacabe B, Chevallier JM, Avan P, Bonfils P (2001) Functional anatomy of auditory
brainstem nuclei: Application to the anatomical basis of brainstem auditory evoked
potentials. Auris Nasus Larynx 28:85–94.
20. Liberman MC, et al. (2002) Prestin is required for electromotility of the outer hair cell
and for the cochlear amplifier. Nature 419:300 –304.
21. Mannherz HG, Leigh JB, Leberman R, Pfrang H (1975) A specific 1:1 G-actin:DNAase i
complex formed by the action of DNAase I on F-actin. FEBS Lett 60:34–38.
22. Loomis PA, et al. (2003) Espin cross-links cause the elongation of microvillus-type
parallel actin bundles in vivo. J Cell Biol 163:1045–1055.
23. Andrianantoandro E, Pollard TD (2006) Mechanism of actin filament turnover by
severing and nucleation at different concentrations of ADF/cofilin. Mol Cell 24:13–23.
24. Avinash GB, Nuttall AL, Raphael Y(1993)3-D analysis of F-actin in stereocilia of cochlear
hair cells after loud noise exposure. Hear Res 67:139–146.
25. Jockusch BM, Schoenenberger CA, Stetefeld J, Aebi U (2006) Tracking down the
different forms of nuclear actin. Trends Cell Biol 16:391–396.
26. Guild GM, Connelly PS, Ruggiero L, Vranich KA, Tilney LG (2003) Long continuous actin
bundles in Drosophila bristles are constructed by overlapping short filaments. J Cell
Biol 162:1069 –1077.
27. Tilney LG, Connelly P, Smith S, Guild GM (1996) F-actin bundles in Drosophila bristles
are assembled from modules composed of short filaments. J Cell Biol 135:1291–1308.
28. Tilney LG, Connelly PS, Ruggiero L, Vranich KA, Guild GM (2003) Actin filament
turnover regulated by cross-linking accounts for the size, shape, location, and number
of actin bundles in Drosophila bristles. Mol Biol Cell 14:3953–3966.
29. Sonnemann KJ, et al. (2006) Cytoplasmic gamma-actin is not required for skeletal
muscle development but its absence leads to a progressive myopathy. Dev Cell 11:387–
397.
30. McGee J, et al. (2006) The very large G-protein-coupled receptor VLGR1: A component
of the ankle link complex required for the normal development of auditory hair
bundles. J Neurosci 26:6543– 6553.
31. Otey CA, Kalnoski MH, Lessard JL, Bulinski JC (1986) Immunolocalization of the gamma
isoform of nonmuscle actin in cultured cells. J Cell Biol 102:1726 –1737.
9708
www.pnas.orgcgidoi10.1073pnas.0900221106 Belyantseva et al.
Page 6
  • Source
    • "In two-year-old mice, the amount of gactin in vestibular HC was dramatically reduced when compared with b-actin. Perhaps this decrease also occurs in cochlear HC and could be related to stereocilia modifications and concomitant progressive hearing loss in aging animals , as pointed out by g-actin mutant mice [Belyantseva et al. 2009]. Yet, point mutations on chromosome 17q25.3 in ACTG1 gene in humans cause autosomal dominant progressive sensorineural hearing loss (DFNA20/26) [Morell et al., 2000; DeWan et al., 2003; van Wijk et al., 2003; Rendtorff et al., 2006]. "
    [Show abstract] [Hide abstract] ABSTRACT: Cytoplasmic actin isoforms beta (β-) and gamma (γ-) perform crucial physiological roles in inner ear hair cells (HC). The stereocilium, which is structured by parallel actin filaments composed of both isoforms, is the responsive organelle to mechanical stimuli such as sound, gravity and head movements. Modifications in isoform proportions affect the function of the stereocilia as previously shown in genetic studies of mutant mice. Here, immunogold labeling TEM studies in mice showed that both β- and γ-actin isoforms colocalize throughout stereocilia actin filaments, adherens junctions and cuticular plates as early as embryonic stage 16.5. Gold-particle quantification indicated that there was 40% more γ- actin than β-actin at E16.5. In contrast, β- and γ-actin were equally concentrated in adult stereocilia of cochlear and vestibular HC. Interestingly, all actin-based structures presented almost five-fold more β-actin than γ-actin in 22 month- old mice, suggesting that γ-actin is probably under-expressed during the aging process. These data provide evidence of dynamic modifications of the actin isoforms in stereocilia, cuticular plates and cell junctions during the whole HC life. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.
    Full-text · Article · May 2015 · Cytoskeleton
  • Source
    • "Stereocilia proteins displayed a remarkably low turnover rate, with less than 50% replacement in 2 months, and there was no evidence of replacement of actin by treadmilling. Thus, there is not an obvious mechanism to repair breaks caused by noise trauma or aging (Belyantseva et al., 2009 ). It may be that actin filaments polymerize and depolymerize at multiple locations along the length of a stereocilium (Zhang et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Hair cells of the inner ear are mechanoreceptors for hearing and balance, and proteins highly enriched in hair cells may have specific roles in the development and maintenance of the mechanotransduction apparatus. We identified XIRP2/mXinβ as an enriched protein likely to be essential for hair cells. We found that different isoforms of this protein are expressed and differentially located: short splice forms (also called XEPLIN) are targeted more to stereocilia, whereas two long isoforms containing a XIN-repeat domain are in both stereocilia and cuticular plates. Mice lacking the Xirp2 gene developed normal stereocilia bundles, but these degenerated with time: stereocilia were lost and long membranous protrusions emanated from the nearby apical surfaces. At an ultrastructural level, the paracrystalline actin filaments became disorganized. XIRP2 is apparently involved in the maintenance of actin structures in stereocilia and cuticular plates of hair cells, and perhaps in other organs where it is expressed. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Mar 2015 · Cell Reports
  • Source
    • "There are studies describing both actin isoforms’ silencing and overexpression (Peckham et al. 2001; Schevzov et al. 1992; Shmerling et al. 2005; Belyantseva et al. 2009; Bunnell and Ervasti 2010), but they did not give a clear answer to the question of their functional diversification. In cited publications, either only one isoform was knocked down (Belyantseva et al. 2009; Bunnell and Ervasti 2010) or overexpressed (Peckham et al. 2001), or the studies focused on normal cells (Dugina et al. 2009; Schevzov et al. 1992). Because of that we decided to trigger overexpression of the β- or γ-actin isoform in the human colon cancer cell line BE, representing the mesenchymal mode of motility, and to observe its effects on cell migration and invasion capacities. "
    [Show abstract] [Hide abstract] ABSTRACT: Actins are eukaryotic proteins, which are involved in diverse cellular functions including muscle contraction, cell motility, adhesion and maintenance of cell shape. Cytoplasmic actin isoforms β and γ are ubiquitously expressed and essential for cell functioning. However, their unique contributions are not very well understood. The aim of this study was to determine the effect of β- and γ-actin overexpression on the migration capacity and actin cytoskeleton organization of human colon adenocarcinoma BE cells. In cells overexpressing β- or γ-actin, distinct cytoskeletal actin rearrangements were observed under the laser scanning confocal microscope. Overexpressed actins localized at the submembranous region of the cell body, especially near to the leading edge and on the tips of pseudopodia. The cells transfected with plasmids containing cDNA for β- or γ-actin were characterized by increased migration and invasion capacities. However, the migration velocity was statistically significantly higher only in the case of γ-actin overexpressing cells. In conclusion, the increased level of β- or γ-actin leads to actin cytoskeletal remodeling followed by an increase in migration and invasion capacities of human colon BE cells. These data suggest that expression of both actin isoforms has an impact on cancer cell motility, with the subtle predominance of γ-actin, and may influence invasiveness of human colon cancer.
    Full-text · Article · Feb 2014 · Histochemie
Show more