Myosin VI is required for the proper maturation and function of inner hair cell ribbon synapses.

Abstract

The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active zone. In Snell's waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation proceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition, the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically immature. Ca2+-dependent exocytosis probed by capacitance measurement was markedly reduced despite normal Ca2+ currents and the large proportion of morphologically mature synapses, which suggests additional defects, such as loose Ca2+-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence that Myo6 and otoferlin, a putative Ca2+ sensor of synaptic exocytosis also involved in a genetic form of deafness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.

Full text

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Myosin VI is required for the proper maturation
and function of inner hair cell ribbon synapses
Isabelle Roux1,{, Suzanne Hosie2, Stuart L. Johnson2,{, Amel Bahloul1,3, Nade `ge Cayet4,
Sylvie Nouaille1,3, Corne ´ J. Kros2,?, Christine Petit1,3,5and Saaid Safieddine1,3,?
1Inserm UMRS587, Unite ´ de Ge ´ne ´tique et Physiologie de l’Audition, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris
cedex 15, France,2School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK,3UPMC Paris 06,
F75015 Paris, France,4Plate-Forme Microscopie E´lectronique, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris
Cedex 15, France and5Colle `ge de France, F75015 Paris, France
Received July 8, 2009; Revised and Accepted September 7, 2009
The ribbon synapses of auditory inner hair cells (IHCs) undergo morphological and electrophysiological
transitions during cochlear development. Here we report that myosin VI (Myo6), an actin-based motor protein
involved in genetic forms of deafness, is necessary for some of these changes to occur. By using post-
embedding immunogold electron microscopy, we showed that Myo6 is present at the IHC synaptic active
zone. In Snell’s waltzer mutant mice, which lack Myo6, IHC ionic currents and ribbon synapse maturation pro-
ceeded normally until at least post-natal day 6. In adult mutant mice, however, the IHCs displayed immature
potassium currents and still fired action potentials, as normally only observed in immature IHCs. In addition,
the number of ribbons per IHC was reduced by 30%, and 30% of the remaining ribbons were morphologically
immature. Ca21-dependent exocytosis probed by capacitance measurement was markedly reduced despite
normal Ca21currents and the large proportion of morphologically mature synapses, which suggests additional
defects, such as loose Ca21-exocytosis coupling or inefficient vesicular supply. Finally, we provide evidence
that Myo6 and otoferlin, a putative Ca21sensor of synaptic exocytosis also involved in a genetic form of deaf-
ness, interact at the IHC ribbon synapse, and we suggest that this interaction is involved in the recycling of
synaptic vesicles. Our findings thus uncover essential roles for Myo6 at the IHC ribbon synapse, in addition
to that proposed in membrane turnover and anchoring at the apical surface of the hair cells.
INTRODUCTION
Hearing relies primarily on the ability of cochlear inner hair
cells (IHCs) to encode sound information with an extreme sen-
sitivity and a high temporal precision (1). Sound-evoked
mechanical stimuli are transduced by the mechanoelectrical
transduction machinery of the IHC hair bundles into graded
membrane potential variations that drive neurotransmitter
release. This release can be maintained at high rate in response
to sustained stimulation (2). Each mature IHC has 10 to 20
synaptic active zones that are characterized by the presence
of an electron-dense structure of submicron diameter, the
synaptic ribbon, to which synaptic vesicles are tethered, and
which faces the dendrite of a bipolar auditory neuron (3,4).
During the first 2 post-natal weeks in the mouse, the IHCs
undergo morphological and electrophysiological changes.
The magnitudes of both IHC Ca2þcurrents and Ca2þ-
dependent exocytosis increase until post-natal day 6 (P6)
(5,6). The Ca2þcurrents then decline progressively during
IHC maturation, reaching their mature steady-state level by
P12, but leaving the size of Ca2þ-induced exocytosis almost
unaffected. In parallel, the number of ribbons per IHC
decreases, and the ribbon shape evolves from spherical to
plate-like (3,7). Concomitant with the onset of hearing
(P12), the IHCs also acquire fast membrane electrical proper-
ties, enabling phase locking of the auditory nerve firing pattern
†Present address: Department of Otolaryngology—Head and Neck Surgery, The Johns Hopkins, University School of Medicine, Baltimore, MD 21205,
USA.
‡Present address: Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.
?To whom correspondence should be addressed. Tel: þ33 145688892; Fax: þ33 140613442; saaid.safieddine@pasteur.fr (S.S.); c.j.kros@sussex.ac.uk
(C.J.K.)
# The Author 2009. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Human Molecular Genetics, 2009, Vol. 18, No. 23
doi:10.1093/hmg/ddp429
Advance Access published on September 10, 2009
4615–4628

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to the periodicity of the auditory stimulus up to several
kilohertz (kHz), mainly through the expression of the large
and fast Ca2þ-activated Kþcurrent, Ik,f(6,8). To date, little
is known about the molecular players that are involved in
these transitions.
Myosin VI (Myo6) defects underlie the dominant and reces-
sive forms of human hearing impairment, DFNA22 and
DFNB37 (9–12), and the recessive deafness of Snell’s
waltzer mice (Myo6sv/sv) (13,14). Myo6 can function either
as an actin-based transporter or as an anchoring protein (15).
This myosin is unique in that it moves toward the minus end
of actin filaments (16), which tend to be oriented toward the
cell center. Because Myo6 is abundant in the cuticular plate
and the pericuticular necklace of auditory hair cells (17),
and mutant mice (14) and zebrafish (18) lacking Myo6
display disorganized hair bundles, most of the structural and
functional analyses carried out so far have focused on the
role of this myosin in the apical part of the hair cells (19–
22). Myo6 is indeed essential for the structural integrity of
the hair bundle (19,20). Here, we report that Myo6 is involved
in the morphological and functional maturation of IHC ribbon
synapses and is required for efficient Ca2þ-dependent exocyto-
sis in mature IHC ribbon synapses. We also show that Myo6 is
necessary for the maturation of the electrophysiological mem-
brane properties of the IHCs to occur.
RESULTS
Myo6 is present at the IHC ribbon synapse
Two rabbit immune sera were raised against the His-tagged C-
terminal part of murine Myo6 (amino acids 1047–1253), and
the specific antibodies were affinity-purified against Myo6
globular tail produced as a glutathione S-transferase (GST)
fusion protein (GST-Myo6-gt, amino acids 981–1253). The
specificity of these antibodies was established by the immu-
noreactivity of the inner ear sensory epithelia observed in
the wild-type mice, but absent in Myo6sv/svmice. The labeling
was also absent when the primary antibody was omitted or
pre-adsorbed ontheantigen
Fig. S1c, f, g and h). In the inner ear of wild-type mice,
Myo6 immunoreactivity was restricted to the sensory cells
of the cochlea and vestibule, in agreement with previous
reports (14,17) (Fig. 1A, and Supplementary Material,
Fig. S1c and f). Immunolabeling experiments on whole-mount
preparations of the organ of Corti analyzed by confocal
microscopy at P6, P15 and P21, that is before and after the
onset of hearing, showed that Myo6 is present throughout
the hair cell soma (Fig. 1B). A strong labeling was observed
in the two IHC regions known for their intense vesicular traf-
ficking, namely the pericuticular necklace (23) and the baso-
lateral region, where the afferent boutons of the primary
auditory neurons are located (Fig. 1B) (24). We then carried
out post-embedding immunogold electron microscopy exper-
iments on sections of organs of Corti from P10 and P15
mice. In the IHCs, the gold particles were not only associated
with the cuticular plate and the peri-cuticular necklace as
reported (17,20), but were also seen in the synaptic region
of the cells (Fig. 1C–F). In all the ribbon-containing sections
examined (10 sections from 3 mice), most of the gold particles
(SupplementaryMaterial,
(n ¼ 73) were located at the edge of the active zone (48% of
the gold particles), whereas 16% of the particles were nearby
or associated with the ribbon, and 36% were seen within the
cytoplasm. Notably, many of the gold particles seen at the
active zone (31%) were associated with tubular features that
have been considered to be endocytotic structures (Fig. 1F)
(25), thus suggesting that Myo6 is involved in synaptic
vesicle recycling. The presence of the protein throughout the
IHC soma, just like other key synaptic proteins including
SNAP25, syntaxin1, otoferlin and VGLUT3 (26–28), may
reflect the intense synaptic vesicular trafficking of the IHCs.
Calcium-dependent exocytosis is impaired in mature
IHCs lacking Myo6
To investigate a possible role of Myo6 in the synaptic exocy-
totic process, we studied IHC Ca2þ-dependent exocytosis in
Myo6sv/svmutant mice that lack Myo6 expression. We moni-
tored voltage-gated Ca2þcurrents and membrane capacitance
changes (DCm) in P6-P7 (immature) and P26–P31 (mature)
cochlear IHCs (Fig. 2A–C). At both stages, the Ca2þcurrents
(Fig. 2A and B, middle panels) were not significantly different
between Myo6sv/svmice and their heterozygous littermates.
Current–voltage curve of peak ICa were fitted with the
equation:
ICa¼
gmaxðV ? VrevÞ
ð1 þ expððV1=2? VÞ=SÞÞ;
ð1Þ
where V is the membrane potential, Vrevthe reversal potential,
gmaxthe maximum chord conductance and V1/2the membrane
potential at which the conductance is half activated, and the
slope factor (S) describes the voltage sensitivity of activation.
Fits for P6–P7 Myo6þ/svIHCs gave values of gmax¼ 8.8 nS,
Vrev¼ 37 mV, V1/2¼ 229 mV and S ¼ 6.3 mV, whereas
for P6–P7 Myo6sv/svIHCs gmax¼ 8.7 nS, Vrev¼ 36 mV,
V1/2¼ 228 mV and S ¼ 6.1 mV (Fig. 2A, middle panel).
Fits for P26–P31 Myo6þ/svIHCs yielded gmax¼ 7.8 nS,
Vrev¼ 18 mV, V1/2¼ 233 mV, S ¼ 9.9 mV, and for P26–
P31
Myo6sv/sv
IHCs
gmax¼ 6.2 nS,
V1/2¼ 236 mV, S ¼ 8.9 mV (Fig. 2B, middle panel). Rever-
sal potentials in the mature IHCs were about 20 mV less
depolarized than in immature IHCs, most likely due to incom-
plete block of residual outward currents through the Kþchan-
nels (6). In P6–P7 Myo6sv/svIHCs, peak maximum inward ICa
was 2389+30 pA, n ¼ 4 (versus Myo6þ/sv2396+25 pA,
n ¼ 5), whereas in P262P31 Myo6sv/svIHCs maximum peak
ICa was 2184+15 pA, n ¼ 4 (versus Myo6þ/sv2232+
15 pA, n ¼ 5; P . 0.05 at both stages). The Ca2þcurrents
of Myo6þ/svand Myo6sv/svIHCs declined significantly with
age (P , 0.001 for both genotypes), indicating that the
normal developmental downregulation of Ca2þinflux did
occur in the IHCs from adult mutant mice (5,6). The Ca2þ-
induced exocytosis of IHCs from mature homozygous
mutant mice, however, was markedly reduced compared
with the heterozygotes (Fig. 2B, right panel): the maximum
DCmwas 11.3+4.1 fF, n ¼ 4, for Myo6sv/sv, versus 27.0+
2.8 fF, n ¼ 5, for Myo6þ/sv(P , 0.02). Such a difference
was not found in P6–P7 immature homozygous mutants
Vrev¼ 15 mV,
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(maximum DCm51.8+8.0 fF, n ¼ 4, for Myo6sv/sv, versus
59.3+13.1 fF, n ¼ 5, for Myo6þ/sv; P . 0.05) (Fig. 2A,
right panel). Next, we compared synaptic transfer functions
(6) that were obtained by plotting DCmagainst peak ICafor
100 ms voltage steps over a range of membrane potentials
from 281 mV up to 211 mV (Fig. 2C). Fitted lines are
according to the power function:
DCm/ IN
Ca;
ð2Þ
where the power N is a measure for the Ca2þ-dependence of
exocytosis. The synaptic transfer functions at P6–P7 and
P26–P31 show that mature Myo6þ/svIHCs increased their
Ca2þ-efficiency (DCm/ICa) over the range of their smaller
Ca2þcurrents and decreased their Ca2þ-dependence of exocy-
tosis (N ¼ 2.0 at P6–P7 and N ¼ 0.93 at P26–P31), just as in
normal development. The P26–P31 Myo6sv/svIHCs had an
even larger developmental reduction in Ca2þdependence
(N ¼ 2.1 at P6–P7 and N ¼ 0.51 at P26–P31, Fig. 2C). Ca2þ-
Figure 1. Myo6 immunodetection in the sensory cells of the mouse inner ear. (A) Low magnification image of an almost entire organ of Corti spiral. The labeling
is restricted to the one row of IHCs and the three rows of OHCs. Scale bar: 80 mm. (B) Confocal microscopy images of projections (upper panel) and unique plan
(middle and lower panels) of whole-mount organs of Corti at P6, P15 and P21, labeled for Myo6. A strong labeling can be seen in the pericuticular necklace and
in the basolateral region of the IHCs, where the afferent synaptic contacts (in brown on the scheme) are located. Scale bars: 10 mm. The inset depicts a schematic
illustration of an IHC ribbon synapse. The center of the active zone is marked by an ovoid structure, called ribbon (r), which faces the afferent bouton of a type I
auditory neuron (aff). Synaptic vesicles (in blue) are surrounding the ribbon. (C–F) Localization by immunogold electron microscopy of Myo6 in the synaptic
region of mouse IHCs. Gold particles can be seen on or nearby the ribbon (arrows in C–E), but the majority of gold particles are associated with the plasma
membrane and are mainly seen at the edge of the active zone (double-arrows in C–E). Gold particles (arrow) are also associated with plasma membrane tubular
structures (asterisk in F), which could correspond to endocytotic pits. Scale bars: 125 nm.
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efficiency at the maximum inward ICa(6), which occurred near
a membrane potential of 221 mV, in mature Myo6sv/svIHCs
(0.066 fF/pA) was about half that of IHCs of their heterozy-
gous littermates (0.119 fF/pA). For smaller depolarizations,
the effect became progressively less pronounced due to the
reduced Ca2þ-dependence, but it was maintained for Ca2þcur-
rents .20 pA, so it would affect neurotransmitter release for
physiologically relevant receptor potentials, spanning a mem-
brane potential range of about 260 to 220 mV (Fig. 2B and
C) (29). Taken together, these findings suggest that Myo6 is
essential for efficient synaptic vesicle exocytosis in IHCs of
the mature cochlea.
IHC ribbon synapse maturation is delayed
in Myo6sv/svmice
To investigate whether an abnormal structural development
could contribute to the observed exocytotic defect, we first
carried out quantitative analyses of the IHC ribbons in
mutant and wild-type mice at P6, P15 and P21. In the apical
part of the cochlea where exocytosis was studied, the
ribbons were visualized by using anti-CtBP2/ribeye antibodies
that label the ribbons and the cell nucleus (26). The ribbons
were quantified after 3D reconstruction of the IHC immunola-
beling analyzed by confocal microscopy (Fig. 3A). At P6, the
number of ribbons harbored by the IHCs of Myo6þ/þand
Figure 2. ICaandDCminIHCsofimmatureandmaturecochleasfromSnell’swaltzerheterozygousandhomozygousmutantmice.(AandB,leftpanels)Examplesof
currents (middle traces) and DCmrecordings (lower traces) in immature (A) or mature (B) Myo6þ/svand Myo6sv/svIHCs at the holding potential (281 or 291 mV,
respectively) and in response to a 100 ms voltage step to 221 mV. Top traces show voltage protocols, in which the sinusoid used to track Cmappears as a thick
line. P6 Myo6þ/svIHC: Cm8.2 pF, Rs5.2 MV, Gleak1.8 nS; P7 Myo6sv/svIHC: Cm7.8 pF, Rs4.9 MV, Gleak1.9 nS. P28 Myo6þ/svIHC: Cm8.1 pF, Rs4.5 MV,
Gleak0.45 nS; P29 Myo6sv/svIHC: Cm6.6 pF, Rs4.8 MV, Gleak1.48 nS. (Middle panels) Averaged peak ICa–V curves for five Myo6þ/svand four Myo6sv/svP6–P7
IHCs(A)andforfiveMyo6þ/svandfourMyo6sv/svP26–P31IHCs(B),withfittedlinesaccordingtoEq.(1).(Rightpanels)AveragedDCm–Vcurvesforcorresponding
cellsinmiddlepanels.(C)Synaptictransferfunctions,relatingDCmandpeakICarecordedatdifferentmembranepotentialsfromtheholdingpotentialuptothemaximum
amplitude for ICa. Dotted lines according to Eq. (2) fit P6–P7 (open symbols), continuous lines P26–P31 (filled symbols) IHCs.
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Myo6sv/svmice was similar (27.3+0.8, n ¼ 31, versus 24.7+
0.7, n ¼ 29, respectively; P . 0.05). Likewise, we did not
observe a significant difference in the number of ribbons per
IHC between the two genotypes at P15 (10.9+0.5, n ¼ 71,
versus 12.3+0.3, n ¼ 65, respectively; P . 0.05). This
result shows that the normal developmental downregulation
of the number of ribbons (3) occurred also in the mutant
mice. At P21, however, a moderate but statistically significant
reduction in the number of ribbons was found in the IHCs of
Myo6sv/svmice (13.4+0.3 ribbons per IHC, n ¼ 70 from 3
Myo6þ/þcochleas, versus 8.9+0.3 ribbons per IHC, n ¼
91 from 3 Myo6sv/svcochleas; P , 0.05) (Fig. 3A). Ultrastruc-
tural analysis showed that at P6, the IHC synapses of Myo6þ/þ
and Myo6sv/svmice were indistinguishable (Fig. 3B and C).
The same analysis carried out at P21 confirmed our immuno-
fluorescence results, showing that the number of ribbon
synapses in Myo6sv/svIHCs was lower than that in the wild-
type mice (8+0.7 per IHC of Myo6þ/þ, n ¼ 10; versus
4.5+0.4 per IHC of Myo6sv/sv, n ¼ 21; P , 0.05). Notably,
70% of the ribbons encountered in P21 Myo6sv/svIHCs (n ¼
57) exhibited a mature shape and were decorated with synaptic
vesicles (Fig. 3H). However, 30% of the ribbons still had an
immature shape, indicating a delay in the ribbon maturation
process. The immature phenotype of the ribbons in Myo6sv/sv
Figure 3. Impaired IHC ribbon synapse morphological maturation in the Myo6sv/svmice. (A) Top panel shows examples of confocal microscopy images of whole
mounts of the organ of Corti used for ribbon quantification. The P6, P15 and P21 IHCs are labeled with the anti-CtBP2/ribeye antibody that labels the ribbons
(arrowhead), and the nuclei (n). Scale bars: 10 mm. The histogram summarizes the results of the quantitative analysis in the Myo6þ/þand Myo6sv/svmice IHCs.
(B–H) Ultrastructural analysis of IHC ribbon synapses from P6, P15 and P21 Myo6þ/þand Myo6sv/svmice. Typical spherical ribbons facing an afferent bouton
(aff) are present at the active zone in both Myo6þ/þ(B) and Myo6sv/sv(C) P6 mice. At P15, the IHC synapse of Myo6sv/svmice (E) still has a round-shaped ribbon
compared with the wild-type synapse (D), whose ribbon is more elongated. Note the persistence of the two rootlets linking the ribbon to the active zone in the
Myo6sv/svIHC (arrowheads). (F and G) In the IHC of the P15 Myo6sv/svmouse (G), some efferent nerve fibers (eff) still synapse on the IHC somata (asterisk),
instead of the afferent bouton (aff and arrowhead), as seen in the P15 Myo6þ/þmice IHC (F). (H) Example of a mature ribbon from Myo6sv/svIHC. Scale bars:
125 nm.
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IHCs was even more pronounced at P15, at which stage most
of the synapses had round-shaped ribbons anchored to the
active zone through two tubular rodlets (Fig. 3E). In P15 wild-
type mice, 46 of 51 ribbons examined (91%) had a plate-like
shape characteristic of mature synapses (Fig. 3D), whereas in
the mutant mice, such mature ribbons were rarely encountered
(11 out 83 ribbons examined in 20 IHCs from 4 cochleas, i.e.
13%). Nevertheless, the immature ribbons (87%) had matured
beyond the P6–P8 stage, as their shape was comparable with
that of wild-type P10–P12 ribbons (3). Notably, many P15
Myo6sv/svIHCs were still contacted by efferent nerve fibers,
an immature feature that was not observed in the wild-type
IHCs of the same stage (Fig. 3F and G). These results show
that Myo6 is not necessary for the formation of the IHC
ribbon synapse, but is required for its proper maturation and
maintenance. This is consistent with what has been reported
in neuronal synapses. In cultured hippocampal neurons
derived from Myo6sv/svmice, synapses seemed to develop nor-
mally, but were also 21% fewer than in cultures from wild-
type mice (30,31). Notably, the loss is even greater when
Myo6 was acutely disrupted (30,31).
IHCs of adult Myo6sv/svmice exhibit immature
electrophysiological membrane properties
At the onset of hearing (P12), the mature IHCs display the IK,f
current, also called BK current, which enables the cells to
switch from spiking pacemakers to high-frequency signal
transducers (8,32,33).
The presence of evoked and spontaneous spiking in the
absence of Myo6 was studied by recording voltage responses
to current injection in three IHCs from three P5–P6 Myo6sv/sv
mice. Spontaneous and evoked spiking activity was recorded
in all three cells (Supplementary Material, Fig. S2), indicating
that at least up to P6, the IHC basolateral currents develop nor-
mally in Myo6sv/svmice, which is consistent with our morpho-
logical observations. Surprisingly, we found that IHCs from
adult Myo6sv/svmice also fired action potentials (Fig. 4A
right), just like immature IHCs (8). This contrasts with the
normal graded voltage responses recorded in the mature
Myo6þ/svIHCs (Fig. 4A left). The resting potentials of
mature Myo6sv/svIHCs were hyperpolarized compared with
those of their heterozygous littermates (275.2+2.2 mV,
n ¼ 6, versus
suggested that mutant and heterozygote IHCs have differences
in membrane currents, which we studied under voltage clamp.
Myo6sv/svIHCs lacked IK,frecorded in IHCs of mature hetero-
zygous littermates (IK,fmeasured 3 ms following a voltage
step to 225 mV: Myo6þ/sv2.27+0.37 nA, n ¼ 6, versus
Myo6sv/sv–0.10+0.02 nA, n ¼ 6; P , 0.0001) (Fig. 4B). In
addition, the delayed rectifier potassium current IK,s(8,33)
recorded in P21–P31 Myo6sv/svIHCs was smaller than that
recorded in IHCs from heterozygotes (IK,s, measured by sub-
tracting IK,ffrom the total current at the end of a voltage
step to 225 mV: Myo6sv/sv1.74+0.14 nA, n ¼ 5, versus
Myo6þ/sv
2.82+0.31 nA, n ¼ 5, respectively; P , 0.01)
(Fig. 4C). Notably, Myo6sv/svIHCs also lacked the deactivat-
ing inward Kþcurrent IK,n, characterized by a very negative
activation range, which appears in mature wild-type IHCs
(34,35) and was found in Myo6þ/svmice. The amplitude of
262.7+2.2 mV, n ¼ 5; P , 0.01). This
the IK,n current recorded was measured as the difference
between the peak inward current and the steady-state current
in response to a voltage step to 2124 mV from a holding
potential of 284 mV (36), and was 294+24 pA (n ¼ 3).
The inward current in the Myo6sv/svIHCs was distinctly differ-
ent from that of the Myo6þ/svmice. Currents increased rather
than deactivated during hyperpolarizing voltage steps in the
Myo6sv/svIHCs (Fig. 4D), thus resembling those of the
inward rectifier IK1found in immature IHCs. IHCs normally
increase in size during development, which is reflected as an
increase in resting membrane capacitance (Cm) (34). This
increase failed to occur in Myo6sv/svIHCs. Cmwas 8.0+
0.1 pF in immature Myo6þ/svIHCs (P4–P6, n ¼ 17), increas-
ing to 9.9+0.4 pF upon maturation (P25–P31, n ¼ 17). In
Myo6sv/svIHCs, Cmwas 7.5+0.2 pF (P4–P7, n ¼ 17) early
in development, and 7.4+0.3 pF later on (P21–P31, n ¼
15). Mature Myo6þ/svIHCs had a significantly larger Cm
than the other groups (all P , 0.001). In conclusion, although
the electrophysiological development of Myo6þ/sv
appeared entirely normal, as investigated in normal CD-1
mice (32,34), the Myo6sv/svIHCs retained immature electro-
physiological properties.
IHCs
Myo6 and otoferlin interact in vivo
By using a yeast two-hybrid assay and molecular and bio-
chemical analyses, we have identified Myo6 as a potential
otoferlin-interacting protein. Otoferlin, a six C2-domain trans-
membrane protein of synaptic vesicles (Fig. 5A), is respon-
sible, when mutated, for a recessive form of deafness
(DFNB9) and has been proposed to be a major Ca2þsensor
at IHC ribbon synapses (26). The otoferlin N-terminal frag-
ment, containing the first three C2 domains (amino acids 1–
761), was used as a bait to screen a library constructed from
P3–P5 mouse inner-ear sensory epithelia (Fig. 5A) (37). The
Myo6 globular tail (Fig. 5A), a domain regarded as a cargo-
binding region (22), was among the preys identified. The
direct interaction between otoferlin and Myo6 was confirmed
by in vitro binding assays. In addition to Myo6 full length
(Myo6-fl), two different fragments of Myo6, its globular tail
(Myo6-gt amino acids 981–1253) and the protein without its
globular tail (Myo6-Dgt amino acids 1–981) (Fig. 5A), pro-
duced in vitro and labeled with35S-methionine, were incu-
bated with the N-terminal fragment of otoferlin produced as
a GST fusion protein. Myo6 full-length and its globular tail
domain did bind to the otoferlin fragment, whereas Myo6
deleted from its globular tail domain did not (Fig. 5B). We
then assessed the co-localization of otoferlin and Myo6 in
IHCs. Whole-mount preparations of the organ of Corti from
P21 mice labeled for otoferlin and Myo6 were analyzed by
confocal microscopy. Myo6 and otoferlin labelings largely
overlapped in the IHCs: in particular, many co-labeled varic-
osities were visible in the basolateral region of these cells
(Fig. 5C). We next carried out double-immunogold electron
microscopy on sections of the organ of Corti, which showed
the presence of the two proteins at the IHC active zones, but
with slightly different distributions (Fig. 5D). Indeed, Myo6
was preferentially detected at the edge of the active zone
(Fig. 1C–F), whereas otoferlin was mainly localized around
the ribbon, as described previously (Fig. 5D) (26). In some
4620Human Molecular Genetics, 2009, Vol. 18, No. 23

Page 7

Figure 4. Defective electrophysiological maturation in Myo6sv/svIHCs. (A) Voltage responses to current injection from a P25 Myo6þ/svIHC [resting membrane
capacitance (Cm): 11.4 pF, Rs: 2.1 MV, resting potential (Vm): 263 mV] and a P28 Myo6sv/svIHC (Cm: 8.1 pF, Rs: 5.2 MV, Vm: 267 mV). The voltage
responses of the Myo6þ/svIHC show graded increases in response to increasing current injection (left panel). The Myo6sv/svIHC responses to current injection
are markedly different (right panel). Current injection of 100 pA results in fluctuations in the membrane potential, resembling spiking activity seen in IHCs of
neonatal mice. Injection of larger currents (1000 pA) results in large depolarizations and is characterized by a single peak followed by a decline to a steady level
where the cell remains until current injection ceases. (B) Representative current traces elicited from a series of depolarizing voltage steps from a –84 mV holding
potential in 10 mV increments in a P25 Myo6þ/svIHC-Cm: 13.5 pF, Rs: 0.86 MV, zero-current potential (Vz): 270 mV, Gleak: 4.2 nS) and a P21 Myo6sv/svIHC
(Cm: 8.7 pF, Rs: 2.5 MV, Vz: 274 mV, Gleak: 1.3 nS). Fast time scale emphasizes the contribution of the fast potassium current IK,f, which is absent in the Myo6
sv/svIHC (B, right panel). (C) Currents from the same cells as panels (B) on a longer time scale to show the contribution of the slow Kþcurrent IK,s, which
remains present in the Myo6sv/svIHC (C, right panel). (D) Currents elicited from a series of hyperpolarizing voltage steps in 10 mV increments from a
holding potential of 284 mV for a P27 Myo6þ/svIHC (Cm: 11.5 pF, Rs: 0.74 MV, Vz: 264 mV) and a P21 Myo6sv/svIHC (Cm: 6.9 pF, Rs: 0.95 MV, Vz:
265 mV). The deactivating IK,ncurrent was not detected in the Myo6sv/svcell (right panel), which had an inward rectifier IK1current instead.
Human Molecular Genetics, 2009, Vol. 18, No. 23 4621

Page 8

Figure 5. Myo6 and otoferlin co-localize at the IHC synaptic active zone and directly interact. (A) Schematic illustration of otoferlin and Myo6 structures. Upper
panel depicts otoferlin with its six predicted C2 domains (C2A–F) and its transmembrane (TM) domain. The bait used in the yeast two-hybrid screening is
indicated by a red horizontal line (Otof-Nt). Lower panel represents Myo6 and its alternative splice forms, which may or may not include a large insertion
(LI) immediately preceding the globular tail (in blue), and a second, smaller insertion (SI) within the globular tail. The overlapping sequences of the two
preys (red bar) identified in the yeast two-hybrid screening map to the globular tail of Myo6, which lacks the SI. Note that the full-length Myo6 (Myo6-fl,
1253 amino acids) (see Materials and Methods), cloned from our mouse cochlear cDNA library, lacks both inserts. (B) In vitro-translated35S-methionine-labeled
full-length Myo6 (Myo6-fl) or its truncated fragments, namely the globular tail (Myo6-gt) and the protein deleted from its globular tail (Myo6-Dgt), were incu-
bated with GST-Otof-Nt fusion protein or GST alone. Myo6-fl and Myo6-gt, but not Myo6-Dgt, directly interact with the N-terminal fragment of otoferlin. (C,
left panel) Scheme of an IHC and its basolateral region, where the afferent boutons (in brown) are located. (Middle panel) Transverse section of a P21 IHC after
3D reconstruction from a cochlear whole-mount preparation labeled for otoferlin and Myo6 (in red and green, respectively). Isolated varicosities of the baso-
lateral area of the IHC (arrow heads) are immunoreactive for both antibodies. (Right panel) Close-up view of a section through the basolateral region of three
IHCs. Scale bar: 10 mm. (D) Co-localization by immunogold electron microscopy of Myo6 and otoferlin in the mouse IHC. Arrowheads point to juxtaposed 10
and 5 nm gold particles, reflecting otoferlin and Myo6 immunoreactivity, respectively. The Myo6 labeling is especially abundant at the edge of the active zone
(double-arrows), whereas otoferlin is more abundant around the ribbon (arrow). Scale bar: 125 nm. (E) Extracts from transfected HEK-293 cells expressing
Myo6 alone (1) or with otoferlin (2) were subjected to immunoprecipitation (IP) with the anti-otoferlin antibody. Myo6 is co-immunoprecipitated with otoferlin.
(F) Co-immunoprecipitation experiment carried out with protein extracts from mouse cochlear sensory epithelium. The antibody directed against otoferlin immu-
noprecipites Myo6 with otoferlin in addition to syntaxin1 and SNAP25, whereas only otoferlin and Myo6 are co-immunopreciptated by the antibody directed
against Myo6. (G) Equal amounts of Myo6-ct (Input) were incubated with Glutathione Sepharose 4B beads alone (R) or pre-incubated with GST, GST-C2A,
GST-C2B or GST-C2C in the presence of 2 mM EDTA or of 1 mM free Ca2þconcentration. Red Ponceau-staining (upper panel) was used to assess the levels of
GST fusion proteins bound to the resin. Only the C2B domain is able to bind to Myo6-ct (lower panel).
4622Human Molecular Genetics, 2009, Vol. 18, No. 23

Page 9

locations, the two types of gold particles were found just a few
nanometers apart, including at the edge of the active zone
(Fig.5D), suggestingthat
physicallyinteract
in vivo.
co-immunoprecipitation experiments carried out on protein
extracts fromtransfected
(HEK)-293 cells producing otoferlin and Myo6 (Fig. 5E),
and on protein extracts from inner ears of P15 mice
(Fig. 5F). The anti-otoferlin antibody immunoprecipitated
not only Myo6, but also syntaxin1 and SNAP25, two
members of the SNARE complex (26). The anti-Myo6 anti-
body, however, immunoprecipitated only otoferlin. This
suggests that the SNARE proteins do not physically interact
with Myo6, and that otoferlin interaction with Myo6 is inde-
pendent of the interaction between otoferlin and the SNARE
proteins. To determine which otoferlin domain(s) is involved
in the binding to Myo6, the precise boundaries of each of
the C2 domains present in the otoferlin N-terminal fragment,
namely, C2A-C, were determined. This was achieved by com-
bining programs of secondary elements search (HCA and PSI)
and multiple sequence alignment (Dialign and Clustal W), fol-
lowed by in vitro expression. The C2A, C2B and C2C
domains were produced as GST-fusion proteins and purified
by affinity chromatography. These proteins were then incu-
bated with Myo6-ct (amino acids 835–1253) or Myo6-gt.
Only C2B domain was able to specifically bind to Myo6
carboxy terminal tail (Fig. 5G and data not shown). To deter-
mine whether this interaction could be modulated by Ca2þ
ions, we repeated this test in the presence of 2 mM EDTA or
1 mM free Ca2þ. Modifying the free Ca2þconcentration did
not affect the binding of C2B to Myo6 carboxy terminal tail
(Fig. 5G). Indeed, C2B sequence lacks most of the residues
known to be involved in Ca2þ-binding in other C2 domains
(38,39). It is noteworthy that a different otoferlin–Myo6 inter-
action has recently been reported, which involves the otoferlin
C2D domain (40) that can bind to Ca2þions (26). The Ca2þ-
dependence of this interaction, however, remains to be
determined.
the
This
two
was
proteins
confirmed
can
by
humanembryonic kidney
DISCUSSION
We found that Myo6 is present at the IHC synaptic active zone
and that its absence prevents the IHC ribbon synapse matu-
ration from proceeding normally. We also found that Myo6
defect leads to a markedly reduced synaptic exocytosis and
to the lack of several Kþcurrents in adult IHCs. Finally, we
provide evidence that Myo6 and otoferlin, a putative Ca2þ
sensor of synaptic exocytosis also involved in a genetic form
of deafness, DFNB9, interact at the IHC ribbon synapse.
The implication of Myo6 in IHC exocytosis is consistent
with several studies showing that this myosin is involved
both in endocytotic and exocytotic membrane-trafficking path-
ways (41). This was shown not only in non-neuronal cells, in
which Myo6 was found to be required for efficient secretion
and for the maintenance of the structure of the Golgi apparatus
(42), but also in synaptic vesicle exocytosis at hippocampal
synapses (30,31). In hippocampal neurons, both spontaneous
and evoked synaptic vesicle exocytosis were reduced by
35% in Myo6sv/svmice (31,41). The more dramatic reduction
of synaptic exocytosis reported here (?60%) may reflect a
specific requirement of the IHC ribbon synapse for a high
rate of exocytosis and recycling, and/or the inability of
mutant IHCs to develop compensatory mechanisms as effi-
cient as hippocampal neurons do in the absence of Myo6.
The marked decrease of IHC synaptic exocytosis observed
in mature Myo6sv/svmice could, in principle, result from a
failure of IHC synaptic development, a vesicular trafficking
defect, an inefficient Ca2þ-exocytosis coupling, or a combi-
nation of these defects.
The finding that the IHCs of adult Myo6sv/svmice harbor
morphologically mature ribbon synapses excludes a major
ultrastructural abnormality as the sole cause of the exocytosis
defect. Likewise, the moderate reduction of the number of
ribbons (30%) is unlikely to fully account for the dramatic
decrease of IHC exocytosis, since in the bassoon mutant
mice that have over 90% of ribbon loss, IHC exocytosis is
largely unaffected (4).
Part of the IHC exocytosis defect of Myo6sv/svmice could be
the outcome of the delayed maturation of the synapse we
observed, which ultimately leads to IHCs in which mature
and immature ribbon synapses coexist. Such a configuration
may hinder IHC transmitter release, as the two types of
synapses have different dependences on local Ca2þconcen-
tration. It is generally believed that during development,
IHC exocytosis switches from a mode of Ca2þ-dependence
in which each release event requires the cooperative action
of overlapping Ca2þmicrodomains, to a nanodomain configur-
ation in which very few channels are required for the release
of a single vesicle (6,43,44). It has been suggested that the
more efficient coupling between Ca2þentry and vesicle exocy-
tosis in the mature IHCs arises from a developmental tighten-
ing between synaptic vesicles and the Ca2þchannels, which
would compensate the lower Ca2þinflux in mature IHCs
(44–46). Therefore, the immature ribbon synapses may not
function properly in IHCs that display smaller adult-type
Ca2þcurrents suitable for a release machinery operating in a
nanodomain configuration.
Insufficient supply of synaptic vesicles due to an endocyto-
sis defect could also contribute to the observed reduction of
exocytosis. Indeed, Myo6 has been found to be associated
with clathrin-coated pits/vesicles and to be able to modulate
endocytosis in the kidney (42), a mechanism that is likely to
also take place at glutamatergic synapses during synaptic exo-
cytosis and the recycling of AMPA receptors (30,31). Myo6 is
also required for the efficient transportation of nascent endocy-
totic vesicles from the actin-rich periphery of the retinal
pigment epithelium cell to supply the early endosome com-
partment with endocytotic vesicles (47). In the IHC, endocyto-
sis at the synaptic active zone and at the peri-cuticular
necklace has been proposed to be the main source for IHC
synaptic vesicle pool replenishment (24,25). The abundance
of Myo6 in these two regions, its association with tubular
structures, likely endocytotic structures, at the edge of the
active zone, and the increasing evidence that the protein is
involved inendocytotic membrane-trafficking
suggest that Myo6 is implicated in the transport of vesicles
on actin filaments from the IHC apical region to the ribbon
synapse and/or in the retrieval of IHC synaptic vesicles after
exocytosis (41,48,49). Our results suggest that at the IHC
together
Human Molecular Genetics, 2009, Vol. 18, No. 234623

Page 10

ribbon synapse, Myo6 could achieve such a task through its
binding to the vesicular membrane protein otoferlin. Two
findings support this proposal. First, Myo6 and otoferlin are
co-localized at the edge of the synaptic active zone, where
IHCs endocytosis is suggested to take place (24,25). Second,
the amount of otoferlin is apparently reduced in the
Myo6sv/svIHCs (40).
In addition to its role at the synapse, we report that Myo6 is
necessary for the maturation of the electrophysiological mem-
brane properties of the IHCs. Mature Myo6sv/svmice lack IK,f
and IK,n, whereas they exhibit a reduced amplitude of the
delayed rectifier potassium current IK,s. IK,fcurrent not only
prevents the mature IHCs from firing action potentials, but
also lowers their membrane electrical time constant to well
under 1 ms (8), enabling phase-locking of transmitter release
for sound frequencies up to a few kHz. In the absence of
this current in Myo6sv/svIHCs, the membrane time constant
is expected to be abnormally large, which will prevent phase-
locking of action potentials in the auditory nerve for sound fre-
quencies above a few tens of Hz. Mature Myo6sv/svmice are
therefore expected to have defects in the precision of spike
timing in the auditory nerve, as reported for BK knockout
mice which lack IK,f(50). Somewhat unexpectedly, the imma-
ture electrophysiological membrane properties of the IHCs
recorded in adult mutant mice did not include the synaptic
Ca2þcurrents, suggesting that maturation of Kþand Ca2þcur-
rents in IHCs proceed independently. The absence of adult-
type Kþcurrents in the Myo6sv/svIHCs could in principle
result from a gene expression defect or from a mistargeting
of the corresponding channels. The latter possibility is the
most likely, since a missorting of the BK channel a-subunit
has recently been reported in the Myo6sv/svIHCs (40).
Finally, it is worthy of note that although otoferlin and
Myo6 directly interact, otoferlin and Myo6 mutants display
major differences in their phenotypes. The lack of otoferlin
does not interfere with IHC ribbon or Kþcurrents maturation,
whereas it leads to an almost completely abolished Ca2þ-
induced exocytosis, despite normal Ca2þcurrents (26). More-
over, this failure of Ca2þ-induced exocytosis is already present
at P6 (26), whereas in the absence of Myo6, the defect of
Ca2þ-induced exocytosis is partial and only appears in
mature IHCs. This suggests that the retrieval of synaptic ves-
icles, where otoferlin may act as a cargo adapter, is just part
of the variety of cellular functions wherein Myo6 is
involved (51).
Together, our results broaden the roles of Myo6 in hair cells
and uncover an unexpected and essential role of this protein in
the maturation of the IHC electrophysiological membrane
properties and in the morphological and functional maturation
of the ribbon synapse.
MATERIALS AND METHODS
Animals
Snell’s waltzer mice (Myo6sv/sv), which have an intragenic
deletion of Myo6 leading to a null allele (13,14), were bred
with C57BL/6J mice. Genotyping at the Myo6 locus was con-
ducted by PCR as described in what follows. The day of birth
was denoted P0. All experiments reported were carried out
according to INSERM, Institut Pasteur and UK Home Office
welfare guidelines.
Genotyping
Genomic DNAs from Myo6þ/svinter-cross litters were ampli-
fied using primers (50-TGGTGAAAAGAGTCAACCTGTG-30
and 50-GCTTCAGCTCGATATTTTATT-30), flanking the del-
etion in the Myo6svallele (14). The reaction mixture contained
200 ng of genomic DNA, LA PCR Buffer II with 2.5 mM
MgCl2, 0.4 mM of each dNTP, 500 nM of each primer and
0.05 unit/ml of reaction mixture of TaKaRa LA Taq polymer-
ase (TaKaRa Bio Inc). PCR was performed as follows: 5 min
denaturation at 958C, followed by 30 cycles of 1 min denatura-
tion at 948C, 1 min annealing at 608C, 2.5 min extension at
728C and finally an additional 10 min at 728C. Wild-type
mice displayed a PCR product of 2255 bp, whereas Myo6sv/
svmice displayed a 1245 bp fragment, and DNA from
Myo6þ/svmice generated both fragments (Supplementary
Material, Fig. S2).
Yeast two-hybrid screening
We used the yeast two-hybrid cDNA library constructed from
a P3–P5 inner-ear sensory epithelia previously reported (37).
Yeast two-hybrid screenings were carried out as reported
(52,53), using otoferlin amino terminal region (Otof-Nt,
amino acids 1–761) as bait (Fig. 5A). The interacting ‘prey’
fragments of the positive clones were PCR-amplified and
sequenced. The resulting sequences were used to identify the
corresponding gene in the GenBank database (NCBI) using
a fully automated procedure.
DNA constructs
The Myo6 full-length cDNA (NM_001039546) was cloned by
three successive rounds of RACE-PCR/subcloning on a
BALB/c mouse cochlear cDNA library. The predicted
protein sequence was 1253 amino acid long and lacks both
inserts described in the Myo6 C-terminal domain (Fig. 5A).
The full-length Myo6 cDNA was then cloned into the
plasmid vectorspCDNA3.1-V5
cDNAs encoding different Myo6 fragments (Myo6-ct: amino
acid 835–1253, Myo6-gt: 981–1253 and Myo6-Dgt: 1–981)
were then PCR-amplified and subcloned into pGST parallel
1 [modified from GEX-4T1, glutathione-S-transferase (GST)
tag, Amersham Biosciences] and pCDNA3.1-V5 His.
The mouse cDNA encoding otoferlin amplified from the
same cDNA library (26) was used as a template to subclone
cDNAs encoding otoferlin full-length or truncated forms into
pcDNA (No tag, Invitrogen) and pGST parallel 1. All con-
structs were verified by DNA sequencing.
His (Invitrogen).The
Protein expression in Escherichia coli
GST-tagged proteins were expressed in Escherichia coli
BL21-CodonPlus (DE3)-RIPL (Stratagene). Production was
made in 2YT medium (17 g of bacto-tryptone, 10 g bacto-yeast
extract and 5 g NaCl per liter) overnight at 148C, following
4624Human Molecular Genetics, 2009, Vol. 18, No. 23

Page 11

a 0.1 mM isopropyl-1-thio-beta-D-galactopyranoside induction
at optical density OD600nm¼ 0.6–0.7.
In vitro binding assays
35S-methionine-labeled Myo6 protein fragments were pro-
ducedby
invitro
transcription/translation
pCDNA3.1-V5 His constructs and the TNT-coupled reticulo-
cyte lysate system (Promega). The35S-labeled products were
equally divided between the samples and incubated with
GST alone or GST-Otof-Nt immobilized on Glutathione
Sepharose 4B beads (Pharmacia) at 48C on a rotating wheel
for 4 h in binding buffer [20 mM Tris–HCl, pH 7.4, 150 mM
NaCl, 0.5% Triton X-100, 5 mM b-mecaptoethanol, EDTA-
free protease inhibitor cocktail (Roche)], supplemented with
1 mg/ml bovine serum albumin (BSA). The beads were then
washed once with this buffer and three times in the binding
buffer without BSA. The bound proteins were separated by
SDS–PAGE. Levels of GST fusion proteins were assessed
by Coomassie blue labeling (Pharmacia Biotech). The
gel was then incubated in signal enhancement buffer
(Amplify, Amersham Biosciences), dried and processed for
autoradiography.
Otoferlin C2A, C2B and C2C domains (amino acid 1–127,
262–408, 423–593, respectively) were purified as GST fusion
proteins, treated with Benzonase (Merck) and washed with
high-salt buffers as described (54) to remove the bacterial con-
taminants that stick to the C2 domains and alter their proper-
ties (54), before being eluted from the resin using 20 mM
glutathione. The purified proteins were dialyzed overnight at
48C against buffer D (25 mM Tris–HCl, pH 7.4, 300 mM
NaCl, 0.1% Triton X-100, 5 mM b-mecaptoethanol) and then
ultracentrifugated for 30 min at 100 000g at 48C. Myo6-ct
and Myo6-gt were also purified as GST fusion proteins, then
cleaved from their GST tag with rTEV, ultracentrifugated
for 30 min at 100 000g at 48C, before being subjected to gel
filtration using a Superdex 200 in 25 mM Tris–HCl, pH 7.4,
300 mM NaCl, 1 mM 1,4-dithio-DL-threitol (DTT). The mono-
dispersity of Myo6 recombinant proteins was attested by
dynamic light scattering. About 8 mg of GST and GST-C2
domains were immobilized on Glutathione Sepharose 4B
beads equilibrated with buffer D supplemented with EDTA-
free protease inhibitor cocktail, 1 mg/ml BSA and either
2 mM EDTA to chelate free Ca2þions in solution or 2 mM
EDTA plus 3 mM CaCl2. About 8 mg of Myo6-ct or
Myo6-gt was then added to each sample and incubated for
5 h. Sepharose beads were then washed three times in the cor-
responding buffer without BSA before being processed for
SDS–PAGE. The proteins electrotransferred to nitrocellulose
sheets were stained with 0.2% Ponceau-S in 10% acetic acid
before being processed for immunodetection using antibody
directed against Myo6.
using
Cell culture, immunoprecipitation and immunoblotting
HEK-293 cells were cultured in high-glucose Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum, 2 mM of Glutamax-1 (Invitrogen),
50 units/ml penicillin, 50 mg/ml streptomycin. The cells
were transfected with otoferlin full length cDNA alone or in
combination with Myo6 full length (10 mg total DNA/10 cm
dish), using Lipofectamin with Plus reagents (Invitrogen)
according to the manufacturer’s instruction. After 36 h, cells
were rinsed in cold PBS, harvested and pelleted by centrifu-
gation. Cell extracts were prepared using 1% Triton X-100,
0.5% deoxycholate and 0.2% SDS, 5 mM DTT supplemented
with 3 mM ATP/MgCl2 and EDTA-free cocktail of protease
inhibitors (Roche). Immunoprecipitation was carried out
using the polyclonal antibody directed against Myo6 or the
anti-otoferlin monoclonal antibody,
protein G-agarose (Pharmacia) for 1.5 h at room temperature.
The resin was pelleted by centrifugation, washed twice with
50 mM Tris–HCl, pH 7.4, and 0.1% Triton X-100, and three
times in the same buffer complemented with 150 mM NaCl.
Proteins were transferred electrophoretically to nitrocellulose
sheets. Blots were blocked with 5% non-fat dry milk in Tris-
buffered saline containing 0.05% Tween-20 and probed with
the following antibodies: anti-Myo6 1:500, anti-otoferlin
1:500, anti-SNAP25 1:2500, anti-syntaxin1 1:2500. Horse-
radish peroxidase-conjugated goat anti-rabbit or anti-mouse
antibodies (BioRad) and the ECL chemiluminescence system
(Amersham) were used for detection.
pre-incubated with
Immunohistofluorescence
Cochlear tissue preparation and immunohistochemistry for
light microscopy were carried out as reported previously
(26). Cochlear whole-mount preparations were fixed with
4% paraformaldehyde (PFA) in PBS, permeabilized with
0.3% Triton X-100 in PBS containing 20% normal goat
serum for 1 h at room temperature and incubated with the
polyclonal antibody directed against Myo6 (1:400), otoferlin
(1:500) or CtBP2(Transduction
(1:100), overnight at 48C. After three washes in PBS, the
cochleas were incubated for 1 h with F(ab)02 fragment of
goat anti-rabbit IgG antibody conjugated with Alexa488 fluor-
escein (Interchim, France) alone or with goat anti-mouse IgG
antibody conjugated with Cy3 fluorophore (Jackson Immu-
noResearch Laboratories) diluted at 1:500 in PBS. Rhodamine
phalloidin (1:2000, Invitrogen) was used to label F-actin. The
preparations were then washed three times in PBS and finally
mounted in one drop of Fluorsave medium (Biochem Labora-
tories, France).
The samples were analyzed using a confocal laser scanning
microscope, LSM510 Meta (Zeiss, Pasteur Institute, Imageo-
ple). The images taken with a step size of 0.1 mm were used
to generate 3D reconstructions of the IHCs using Osirix soft-
ware (Antoine Rosset, Department of Radiology, Geneva Uni-
versity Hospital, Switzerland). The total number of ribbons
counted was divided by the number of IHCs analyzed. All
comparative images between wild-type and mutant mice
were done in the same conditions of preparation, acquisition
and analysis.
Laboratories,France)
Electron microscopy
Mouse cochleas were fixed as described (26). The organs of
Corti were microdissected and processed by the progressive
temperature-lowering technique as reported (26). Ultrathin
sections (70 nm) were cut with a Leica Ultracut S microtome
Human Molecular Genetics, 2009, Vol. 18, No. 234625

Page 12

and
Immunogold-labeling was carried out as described (26). The
sections were incubated overnight with the anti-Myo6 polyclo-
nal antibodies alone or in combination with the anti-otoferlin
monoclonal antibody, diluted at 1:200 and 1:100, respectively.
The sections were washed and then incubated for 2 h with
10 nM gold-conjugated goat anti-mouse and 5 nM gold-
conjugated goat anti-rabbit antibodies (1:50, Tebu, France).
The sections were then stained with uranyl acetate and lead
citrate and examined under a Jeol1200EX electron micro-
scope. Gold particle distribution in the synaptic region was
quantified compared with the ribbon considered as the center
of the active zone. A gold particle was counted as being at
the edge when it was associated with the presynaptic density
over 100 nm away from the ribbon. A gold particle was
counted as being nearby the ribbon when it was not associated
with the membrane but located within 50 nm from it. For mor-
phological analyses, cochleas were perfused with 4% PFA and
2% glutaraldehyde in PBS at pH 7.4 and immersed in the fixa-
tive solution for 2 h. They were then post-fixed by overnight
incubation in 1% osmium tetraoxide at 48C, dehydrated in
graded acetone concentrations and embedded in Spurr’s low-
viscosity epoxy resin hardened at 708C. Ultrathin sections
were transferred to formvar-coated single-slot grids, stained
with uranyl acetate and lead citrate and examined under a
Jeol1200EX electron microscope (Pasteur, Imageopole).
transferredtoformvar-coatedsingle-slot grids.
Electrophysiological recordings and capacitance tracking
Voltage- and current-clamp recordings were obtained from
apical-coil IHCs, using an Optopatch amplifier (Cairn
Research). Voltage-clamp experiments were done at room
temperature (20–248C), current-clamp and capacitance track-
ing close to body temperature (32–358C). The extracellular
solution contained (in mM): 135 NaCl, 5.8 KCl, 1.3 CaCl2,
0.9 MgCl2, 0.7 NaH2PO4, 2 Na pyruvate, 5.6 D-glucose, 10
HEPES–NaOH, amino acids and vitamins for Eagle’s
MEM. Osmolality was around 308 mOsm/kg and pH 7.5.
The pipette filling solution for voltage- and current-clamp
experiments was (mM) 131 KCl, 3 MgCl2, 5 Na2ATP, 1
EGTA–KOH, 5 HEPES–KOH (osmolality 282 mOsm/kg,
pH 7.3). Currents under voltage clamp were corrected offline
for linear leak conductance calculated from 10 mV steps
from the holding potential, usually 284 mV, including a
24 mV liquid junction potential correction. Membrane poten-
tials were corrected for voltage drop across the residual series
resistance (Rs) of 1.7+0.3 MV (n ¼ 12) after compensation
of around 80%. Sizes of the Kþcurrents IK,fand IK,swere
determined as reported before (8).
Real-time changes in membrane capacitance (DCm) were
studied using the Optopatch (6), applying a 2.5 kHz sine
wave of 13 or 18.5 mV amplitude around a holding potential
of 281 or 291 mV (including 211 mV correction for the
liquid junction potential), to avoid activating significant
voltage-dependent membrane currents, as accurate capaci-
tance tracking requires a membrane resistance (Rm) that is
relatively constant and high compared with Rs(6). For the
capacitance-tracking experiments, no Rscompensation could
be applied. Nevertheless, no offline correction of membrane
potentials for Rs(5.0+0.1 MV, n ¼ 18) was necessary, as
maximum voltage errors were in the order of 1 to 2 mV. Rm
did not differ significantly between the four groups of IHCs
(mature versus immature, heterozygote versus mutant) and
averaged 803+108 MV (n ¼ 18). The extracellular superfu-
sion solution for capacitance tracking and the associated
Ca-current recordings contained 30 mM TEA–Cl (Fluka) to
help block the large currents through the Kþchannels. Some
of the apparent inactivation of the inward Ca2þcurrents
(Fig. 2A and B, left panels) is likely to be due to residual
unblocked currents. We therefore measured Ca2þcurrent
size as that of the initial peak current. In the superfusion sol-
ution, amino acids and vitamins were omitted and NaCl was
reduced to 115 mM to keep osmolality near 308 mOsm/kg.
The intracellular solution was (mM) 140 Cs-glutamate,
3 MgCl2, 5 Na2ATP, 0.3 Na2GTP, 1 EGTA–CsOH, 5
Hepes–CsOH (osmolality around 282 mOsm/kg and pH 7.3).
Electrophysiological recordings used for the data analysis
were mostly in response to single presentations of the stimulus
protocol, but occasionally averaged from two successive pre-
sentations. All quantitative data are presented as mean+
SEM. Student’s t-test or one-way ANOVA followed by the
Tukey post-test was used to analyze statistical significance
(criterion P , 0.05).
SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.
ACKNOWLEDGEMENTS
We thank Dr Jean-Pierre Hardelin for critical reading of the
manuscript. We are grateful to Drs Elisabeth Verpy and
Michel Leibovici for the cochlear library, and to all staff
members of the intramural platforms for excellent technical
assistance.
Conflict of Interest statement. None declared.
FUNDING
This work was supported by grants from EMBO ALTF 952–
2006 to I.R., the French Ministry of Research and the
European Commission FP6 Integrated Project EuroHear
LSHG-CT-2004–512063 to C.P., Human Frontiers Science
Program and the Agence Nationale de la Recherche
(ANR-07-Neuro-036–01) to S.S. and a BBSRC Case student-
ship sponsored by Pfizer to S.H. and C.J.K.
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