© 2015. Published by The Company of Biologists Ltd.
Short report Title: Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells
Running Title: IHC exocytosis operates without Munc13s
Journal of Cell Science
Accepted manuscript
Authors: Christian Vogl1,*, Benjamin H. Cooper2, Jakob Neef1, Sonja M. Wojcik2, Kerstin Reim2, Ellen Reisinger3, Nils Brose2,4,5, Jeong-Seop Rhee6, Tobias Moser1,4,5,* and Carolin Wichmann1,4,7,* 1
Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany 2 Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany 3 Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology, University Medical Center Göttingen, 37075 Göttingen, Germany 4 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany 5 Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany 6 Neurophysiology Group, Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany 7 Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany *
corresponding authors
Pages: 18 Figures: 4, Tables: 0 Number of words: Abstract 180/180 Total number of words: 3185/3000 Keywords: ribbon synapse, priming, tether, Munc13, CAPS, otoferlin Corresponding authors: Christian Vogl Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University of Göttingen Medical Center, 37099 Göttingen, Germany Tel.: +49-551-39-22604 Fax.: +49-551-39-12950 [email protected] Tobias Moser Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University of Göttingen Medical Center, 37099 Göttingen, Germany Tel.: +49-551-39-8968, -22803, -22837 Fax.: +49-551-39-12950 [email protected]
JCS Advance Online Article. Posted on 20 January 2015
Carolin Wichmann Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University Medical Centre, 37099 Göttingen, Germany Tel.: +49-551-39-22604 Fax.: +49-551-39-12950 [email protected]
Author Contributions: C.V., C.W., and T.M. designed the study. C.V. performed electrophysiology. C.V. and B.H.C
performed mouse mutagenesis and provided discussion and input into the manuscript preparation. C.V., J.N., and C.W. analyzed the data. C.V., C.W. and T.M. prepared the manuscript.
Conflict of Interest: The authors declare no competing financial interests.
Journal of Cell Science
Accepted manuscript
performed immunohistochemistry, C.W. EM tomography. E.R., S.M.W., K.R. and N.B.
2
Accepted manuscript Journal of Cell Science
1
Abstract
2
Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to
3
indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle
4
replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13) and Ca2+-
5
dependent activator proteins for secretion (CAPS) families, which prime vesicles for
6
exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in
7
mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC
8
patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms.
9
Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones
10
(AZs) and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in
11
Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein
12
critical for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of
13
the presynaptic AZ. Electron tomography of otoferlin-deficient IHC synapses revealed a
14
reduction of short tethers holding vesicles at the AZ, which might be a structural correlate of
15
impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an
16
unconventional priming machinery that involves otoferlin.
17 18
Introduction
19
The mechanisms that establish fusion-competence of synaptic vesicles are classically defined
20
as tethering, docking and priming. In this framework, vesicles are first loosely tethered to the
21
presynaptic AZ membrane, then closely attach to the membrane upon docking and finally
22
undergo further maturation steps to gain full fusion-competence. Recent high-resolution
23
ultrastructural work has indicated that such clear distinctions of morphological and functional
24
preparatory steps in vesicle fusion may have been too simple. Instead, protein tethers of
25
different lengths and numbers have been proposed to establish vesicular fusion-competence
26
(Fernández-Busnadiego et al., 2010, 2013; Siksou et al., 2009). In neurons, neuroendocrine, 3
Accepted manuscript Journal of Cell Science
1
immune, and airway epithelial cells, this process employs Munc13/CAPS priming factors
2
(Dudenhöffer-Pfeifer et al., 2013; Imig et al., 2014; Speidel et al., 2005; Zhu et al., 2008). The
3
Munc13 protein family includes the neuronal isoforms Munc13-1, Munc13-2, Munc13-3 and
4
brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3), as well as the non-
5
neuronal Munc13-4 isoform, while CAPS1 and CAPS2 constitute the CAPS protein family
6
(Ann et al., 1997; Augustin et al., 2001; Betz et al., 2001; Brose et al., 1995; Koch et al.,
7
2000; Shiratsuchi et al., 1998; Speidel et al., 2003). Munc13s and CAPSs are evolutionarily
8
conserved (i.e. UNC-13/UNC-31 in C. elegans, dUNC-13/dCAPS in Drosophila;
9
(Aravamudan et al., 1999; Renden et al., 2001; Richmond et al., 1999), and genetic deletion
10
causes dramatic defects, ranging from severe reduction to complete loss of the readily
11
releasable pool of synaptic vesicles (RRP) and total arrest of spontaneous and evoked
12
neurotransmission in several cell types (Augustin et al., 1999; Jockusch et al., 2007; Liu et al.,
13
2010; Varoqueaux et al., 2002).
14
Replenishment
of
release-ready
vesicles
is
likely
rate-limiting
for
tonic
15
neurotransmitter release at ribbon synapses. Governed by receptor potentials, each IHC AZ
16
transmits acoustic information via graded release of up to hundreds of vesicles per second.
17
For this challenging task, IHC synapses must employ mechanisms of vesicle replenishment,
18
that involve otoferlin, a multi-C2 domain protein critical for exocytosis in vestibular hair cells
19
and cochlear IHCs (Dulon et al., 2009; Pangršič et al., 2012). Otoferlin is required for hearing
20
(Roux et al., 2006; Yasunaga et al., 1999) and thought to act as a priming factor and vesicular
21
Ca2+-sensor for release (Johnson and Chapman, 2010; Pangršič et al., 2010; Roux et al.,
22
2006). However, which other proteins contribute to establishing vesicular fusion-competence
23
in IHCs remains to be determined. Here, we combined functional and morphological
24
approaches to investigate the roles of Munc13-like priming factors in IHCs. Our data indicate
25
that the conventional Munc13- and CAPS-dependent priming machinery of CNS synapses
26
does not operate in exocytosis at IHC ribbon synapses. 4
Accepted manuscript Journal of Cell Science
1
Results and Discussion
2
Hearing is unaffected in mouse mutants lacking Munc13/CAPS priming factors
3
To assess the impact of genetic disruption of Munc13 and CAPS proteins on auditory
4
function, we recorded ABRs evoked by short tone bursts and clicks in KO mice for Munc13-
5
1,-2,-3,-4 and Baiap3 as well as CAPS1/2. Since genetic deletion of Munc13-1 and CAPS1
6
results in perinatal lethality, we recorded ABRs from heterozygous mice for these genes. We
7
did neither observe alterations of ABR thresholds nor changes in amplitudes or latencies of
8
ABR wave I, reporting the compound action potential of the spiral ganglion, in any of the
9
mutants when compared to WT littermates (Figure 1 and Supp. Figure1). Moreover, we
10
recorded distortion product otoacoustic emissions to evaluate outer hair cell function, but did
11
not detect a statistically significant change for any of the mutant mouse strains suggesting
12
intact cochlear amplification (data not shown). Therefore, disruption of Munc13 and CAPS
13
does not seem to affect sound encoding in the cochlea. However, we note that testing the
14
effect of complete deletion of Munc13-1 and CAPS1 in IHCs will require future experiments
15
on conditional KO mice, as the heterozygous state tested here might provide protein copy
16
numbers that still support normal functionality (Augustin et al., 1999).
17 18
Loss of Munc13/CAPS priming factors does not alter Ca2+-currents and exocytosis of
19
IHCs
20
To clarify the contributions of the main Munc13 and CAPS isoforms to IHCs presynaptic
21
function, we analyzed presynaptic Ca2+-currents and exocytosis in respective deletion mutant
22
mice. We used an organotypic culture approach to investigate the effect of genetic deletion of
23
CAPS1/2 or Munc13-1/2 on Ca2+-driven exocytosis in IHCs in vitro (Figure 2). Cultured
24
organs of Corti appear to mature analogously to the in vivo situation (Sobkowicz et al., 1982)
25
and are suitable for patch-clamp recordings of presynaptic function (Nouvian et al., 2011;
26
Reisinger et al., 2011). After a week in culture, overall organ of Corti morphology was 5
Accepted manuscript Journal of Cell Science
1
preserved and IHCs abundantly expressed otoferlin (Figure 2A). When comparing IHC Ca2+-
2
currents from CAPS1/2-DKO and Munc13-1/2-DKO with data from WT and Otof-KO mice,
3
we did not detect differences in voltage-dependence, amplitude (Figure 2B; maximal
4
amplitudes WT: 292 ± 25 pA; Otof-KO: 291 ± 31 pA; CAPS1/2-DKO: 306 ± 30 pA;
5
Munc13-1/2-DKO: 286 ± 20 pA; p>0.05 between all groups), or kinetics (Figure 2C).
6
Exocytosis was monitored as changes in membrane capacitance (∆Cm) in response to maximal
7
Ca2+-influx elicited by depolarization of varying durations (Figure 2D-G). While ∆Cm were
8
indistinguishable between WT, CAPS1/2-DKO and Munc13-1/2-DKO IHCs, those of Otof-
9
KO exhibited dramatically reduced exocytosis, consistent with previous reports using acute
10
preparations (Beurg et al., 2010; Roux et al., 2006). Since exocytosis in IHCs acquires
11
otoferlin-dependence around P4 in vivo (Beurg et al., 2010), our findings indicate a functional
12
maturation of IHCs in organotypic culture and further validate this approach for studying
13
IHCs of perinatally lethal mutant mice. Detailed analysis of ∆C m values for stimuli of 2–50
14
ms duration by exponential fitting revealed comparable RRP size and depletion kinetics in
15
CAPS1/2-DKO and Munc13-1/2-DKO (Figure 2H; WT: 26.8 ± 4.2 fF; CAPS1/2-DKO: 20.9
16
± 5.4 fF; Munc13-1/2-DKO: 31.9 ± 5.5 fF; p>0.05 between all groups), while the strongly
17
reduced exocytosis of Otof-KO prohibited such analysis. Moreover, we tested presynaptic
18
IHC function in mice lacking Munc13-4 and Baiap3, but did not detect any changes in Ca2+
19
currents or exocytosis (Supp. Figure 1B). We conclude that Munc13-like priming factors are
20
dispensable for IHC presynaptic function.
21 22
Cochlear inner hair cells apparently lack Munc13-like priming factors
23
Previous reports indicated absence of Munc13-1 protein from chicken cochlear extracts
24
(Uthaiah and Hudspeth, 2010), however, the expression patterns of the remaining Munc13
25
and CAPS isoforms in the cochlea remain to be established. Therefore, we performed
26
immunostainings with extensively tested antibodies for all Munc13 and CAPS isoforms 6
Accepted manuscript Journal of Cell Science
1
(Cooper et al., 2012) on acutely dissected organs of Corti from hearing p16-17 WT mice
2
(Figure 3). Consistent with our electrophysiological data arguing against a functional role of
3
these proteins in presynaptic IHC function, we did not detect specific Munc13 or CAPS
4
immunofluorescence within IHCs. Rather, immunoreactivities of all tested proteins, including
5
Munc13-1 but not Munc13-2 were restricted to presynaptic terminals of efferent olivocochlear
6
neurons, as evident from colocalization with the neuronal synaptic vesicle marker
7
synapsin1/2, which served as internal positive controls in these experiments. We did not find a
8
specific immunolabeling for Munc13-4 in organs of Corti, i.e. a labeling that was absent from
9
knock-out tissue, with currently available antibodies. While we cannot exclude Munc13-4
10
expression in IHCs, we conclude, based on our analysis of presynaptic function (Supp. Fig
11
1B), that this isoform plays a minor – if any – role in vesicular release from IHC AZs. In the
12
present study, we could establish that IHCs seem to operate without Munc13s or CAPS
13
proteins, a finding that is in line with the notion that their interacting partners neuronal soluble
14
N-ethylmaleimide-sensitive-factor attachment receptors (SNAREs) appear to be absent from
15
IHCs (Nouvian et al., 2011). Based on our findings, we propose that the priming machinery of
16
IHCs is molecularly distinct from conventional neuronal AZs and likely involves the synaptic
17
ribbon and/or bassoon (Frank et al., 2010; Snellman et al., 2011) and otoferlin (Pangršič et al.,
18
2010).
19 20
Otoferlin is enriched in IHC AZ membranes and regulates vesicular tether formation
21
Since IHC exocytosis seems to operate without the classical priming factors, we aimed to
22
further investigate mechanism by which otoferlin facilitates vesicle replenishment. Our
23
confocal data revealed an enrichment of otoferlin at the AZ membrane (Figure 4A), a
24
localization analogous to neuronal Munc13/CAPS priming factors. In these experiments, we
25
employed an antibody that recognizes an intraluminal C-terminal epitope of otoferlin and
26
observed otoferlin clustering in the AZ membrane at the base of the ribbon that could also be 7
Accepted manuscript Journal of Cell Science
1
found with immunogold labelings (data not shown). Otoferlin enrichment at the release site
2
might indicate an involvement in vesicle tethering, which has been implicated as an
3
ultrastructural correlate of vesicular fusion competence (Fernández-Busnadiego et al., 2010;
4
Frank et al., 2010). Therefore, we used electron tomography of IHC synapses to investigate
5
vesicular tethers at AZs of WT and otoferlin-deficient IHCs, instantaneously frozen following
6
stimulation with high K+ to analyze ongoing synaptic activity (Figures 4B-G). We focused our
7
analysis on filamentous tethers connecting membrane-proximal vesicles to the presynaptic
8
density and AZ membrane, as this population was proposed to represent readily-releasable
9
vesicles (Fernández-Busnadiego et al., 2010; Frank et al., 2010). Interestingly, we failed to
10
detect tethers
Short report Title: Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells
Running Title: IHC exocytosis operates without Munc13s
Journal of Cell Science
Accepted manuscript
Authors: Christian Vogl1,*, Benjamin H. Cooper2, Jakob Neef1, Sonja M. Wojcik2, Kerstin Reim2, Ellen Reisinger3, Nils Brose2,4,5, Jeong-Seop Rhee6, Tobias Moser1,4,5,* and Carolin Wichmann1,4,7,* 1
Institute for Auditory Neuroscience and InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, 37099 Göttingen, Germany 2 Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany 3 Molecular Biology of Cochlear Neurotransmission Group, Department of Otolaryngology, University Medical Center Göttingen, 37075 Göttingen, Germany 4 Collaborative Research Center 889, University of Göttingen, Göttingen, Germany 5 Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany 6 Neurophysiology Group, Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany 7 Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, 37099 Göttingen, Germany *
corresponding authors
Pages: 18 Figures: 4, Tables: 0 Number of words: Abstract 180/180 Total number of words: 3185/3000 Keywords: ribbon synapse, priming, tether, Munc13, CAPS, otoferlin Corresponding authors: Christian Vogl Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University of Göttingen Medical Center, 37099 Göttingen, Germany Tel.: +49-551-39-22604 Fax.: +49-551-39-12950 [email protected] Tobias Moser Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University of Göttingen Medical Center, 37099 Göttingen, Germany Tel.: +49-551-39-8968, -22803, -22837 Fax.: +49-551-39-12950 [email protected]
JCS Advance Online Article. Posted on 20 January 2015
Carolin Wichmann Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience, InnerEarLab and Collaborative Research Center 889, University Medical Centre, 37099 Göttingen, Germany Tel.: +49-551-39-22604 Fax.: +49-551-39-12950 [email protected]
Author Contributions: C.V., C.W., and T.M. designed the study. C.V. performed electrophysiology. C.V. and B.H.C
performed mouse mutagenesis and provided discussion and input into the manuscript preparation. C.V., J.N., and C.W. analyzed the data. C.V., C.W. and T.M. prepared the manuscript.
Conflict of Interest: The authors declare no competing financial interests.
Journal of Cell Science
Accepted manuscript
performed immunohistochemistry, C.W. EM tomography. E.R., S.M.W., K.R. and N.B.
2
Accepted manuscript Journal of Cell Science
1
Abstract
2
Ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle replenishment to
3
indefatigably encode sound. In neurons, neuroendocrine and immune cells, vesicle
4
replenishment depends on proteins of the mammalian uncoordinated 13 (Munc13) and Ca2+-
5
dependent activator proteins for secretion (CAPS) families, which prime vesicles for
6
exocytosis. Here, we tested whether Munc13 and CAPS proteins also regulate exocytosis in
7
mouse IHCs by combining immunohistochemistry with auditory systems physiology and IHC
8
patch-clamp recordings of exocytosis in mice lacking Munc13 and CAPS isoforms.
9
Surprisingly, we did not detect Munc13 or CAPS proteins at IHC presynaptic active zones
10
(AZs) and found normal IHC exocytosis as well as auditory brainstem responses (ABRs) in
11
Munc13 and CAPS deletion mutants. Instead, we show that otoferlin, a C2-domain protein
12
critical for vesicular fusion and replenishment in IHCs, clusters at the plasma membrane of
13
the presynaptic AZ. Electron tomography of otoferlin-deficient IHC synapses revealed a
14
reduction of short tethers holding vesicles at the AZ, which might be a structural correlate of
15
impaired vesicle priming in otoferlin-deficient IHCs. We conclude that IHCs use an
16
unconventional priming machinery that involves otoferlin.
17 18
Introduction
19
The mechanisms that establish fusion-competence of synaptic vesicles are classically defined
20
as tethering, docking and priming. In this framework, vesicles are first loosely tethered to the
21
presynaptic AZ membrane, then closely attach to the membrane upon docking and finally
22
undergo further maturation steps to gain full fusion-competence. Recent high-resolution
23
ultrastructural work has indicated that such clear distinctions of morphological and functional
24
preparatory steps in vesicle fusion may have been too simple. Instead, protein tethers of
25
different lengths and numbers have been proposed to establish vesicular fusion-competence
26
(Fernández-Busnadiego et al., 2010, 2013; Siksou et al., 2009). In neurons, neuroendocrine, 3
Accepted manuscript Journal of Cell Science
1
immune, and airway epithelial cells, this process employs Munc13/CAPS priming factors
2
(Dudenhöffer-Pfeifer et al., 2013; Imig et al., 2014; Speidel et al., 2005; Zhu et al., 2008). The
3
Munc13 protein family includes the neuronal isoforms Munc13-1, Munc13-2, Munc13-3 and
4
brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3), as well as the non-
5
neuronal Munc13-4 isoform, while CAPS1 and CAPS2 constitute the CAPS protein family
6
(Ann et al., 1997; Augustin et al., 2001; Betz et al., 2001; Brose et al., 1995; Koch et al.,
7
2000; Shiratsuchi et al., 1998; Speidel et al., 2003). Munc13s and CAPSs are evolutionarily
8
conserved (i.e. UNC-13/UNC-31 in C. elegans, dUNC-13/dCAPS in Drosophila;
9
(Aravamudan et al., 1999; Renden et al., 2001; Richmond et al., 1999), and genetic deletion
10
causes dramatic defects, ranging from severe reduction to complete loss of the readily
11
releasable pool of synaptic vesicles (RRP) and total arrest of spontaneous and evoked
12
neurotransmission in several cell types (Augustin et al., 1999; Jockusch et al., 2007; Liu et al.,
13
2010; Varoqueaux et al., 2002).
14
Replenishment
of
release-ready
vesicles
is
likely
rate-limiting
for
tonic
15
neurotransmitter release at ribbon synapses. Governed by receptor potentials, each IHC AZ
16
transmits acoustic information via graded release of up to hundreds of vesicles per second.
17
For this challenging task, IHC synapses must employ mechanisms of vesicle replenishment,
18
that involve otoferlin, a multi-C2 domain protein critical for exocytosis in vestibular hair cells
19
and cochlear IHCs (Dulon et al., 2009; Pangršič et al., 2012). Otoferlin is required for hearing
20
(Roux et al., 2006; Yasunaga et al., 1999) and thought to act as a priming factor and vesicular
21
Ca2+-sensor for release (Johnson and Chapman, 2010; Pangršič et al., 2010; Roux et al.,
22
2006). However, which other proteins contribute to establishing vesicular fusion-competence
23
in IHCs remains to be determined. Here, we combined functional and morphological
24
approaches to investigate the roles of Munc13-like priming factors in IHCs. Our data indicate
25
that the conventional Munc13- and CAPS-dependent priming machinery of CNS synapses
26
does not operate in exocytosis at IHC ribbon synapses. 4
Accepted manuscript Journal of Cell Science
1
Results and Discussion
2
Hearing is unaffected in mouse mutants lacking Munc13/CAPS priming factors
3
To assess the impact of genetic disruption of Munc13 and CAPS proteins on auditory
4
function, we recorded ABRs evoked by short tone bursts and clicks in KO mice for Munc13-
5
1,-2,-3,-4 and Baiap3 as well as CAPS1/2. Since genetic deletion of Munc13-1 and CAPS1
6
results in perinatal lethality, we recorded ABRs from heterozygous mice for these genes. We
7
did neither observe alterations of ABR thresholds nor changes in amplitudes or latencies of
8
ABR wave I, reporting the compound action potential of the spiral ganglion, in any of the
9
mutants when compared to WT littermates (Figure 1 and Supp. Figure1). Moreover, we
10
recorded distortion product otoacoustic emissions to evaluate outer hair cell function, but did
11
not detect a statistically significant change for any of the mutant mouse strains suggesting
12
intact cochlear amplification (data not shown). Therefore, disruption of Munc13 and CAPS
13
does not seem to affect sound encoding in the cochlea. However, we note that testing the
14
effect of complete deletion of Munc13-1 and CAPS1 in IHCs will require future experiments
15
on conditional KO mice, as the heterozygous state tested here might provide protein copy
16
numbers that still support normal functionality (Augustin et al., 1999).
17 18
Loss of Munc13/CAPS priming factors does not alter Ca2+-currents and exocytosis of
19
IHCs
20
To clarify the contributions of the main Munc13 and CAPS isoforms to IHCs presynaptic
21
function, we analyzed presynaptic Ca2+-currents and exocytosis in respective deletion mutant
22
mice. We used an organotypic culture approach to investigate the effect of genetic deletion of
23
CAPS1/2 or Munc13-1/2 on Ca2+-driven exocytosis in IHCs in vitro (Figure 2). Cultured
24
organs of Corti appear to mature analogously to the in vivo situation (Sobkowicz et al., 1982)
25
and are suitable for patch-clamp recordings of presynaptic function (Nouvian et al., 2011;
26
Reisinger et al., 2011). After a week in culture, overall organ of Corti morphology was 5
Accepted manuscript Journal of Cell Science
1
preserved and IHCs abundantly expressed otoferlin (Figure 2A). When comparing IHC Ca2+-
2
currents from CAPS1/2-DKO and Munc13-1/2-DKO with data from WT and Otof-KO mice,
3
we did not detect differences in voltage-dependence, amplitude (Figure 2B; maximal
4
amplitudes WT: 292 ± 25 pA; Otof-KO: 291 ± 31 pA; CAPS1/2-DKO: 306 ± 30 pA;
5
Munc13-1/2-DKO: 286 ± 20 pA; p>0.05 between all groups), or kinetics (Figure 2C).
6
Exocytosis was monitored as changes in membrane capacitance (∆Cm) in response to maximal
7
Ca2+-influx elicited by depolarization of varying durations (Figure 2D-G). While ∆Cm were
8
indistinguishable between WT, CAPS1/2-DKO and Munc13-1/2-DKO IHCs, those of Otof-
9
KO exhibited dramatically reduced exocytosis, consistent with previous reports using acute
10
preparations (Beurg et al., 2010; Roux et al., 2006). Since exocytosis in IHCs acquires
11
otoferlin-dependence around P4 in vivo (Beurg et al., 2010), our findings indicate a functional
12
maturation of IHCs in organotypic culture and further validate this approach for studying
13
IHCs of perinatally lethal mutant mice. Detailed analysis of ∆C m values for stimuli of 2–50
14
ms duration by exponential fitting revealed comparable RRP size and depletion kinetics in
15
CAPS1/2-DKO and Munc13-1/2-DKO (Figure 2H; WT: 26.8 ± 4.2 fF; CAPS1/2-DKO: 20.9
16
± 5.4 fF; Munc13-1/2-DKO: 31.9 ± 5.5 fF; p>0.05 between all groups), while the strongly
17
reduced exocytosis of Otof-KO prohibited such analysis. Moreover, we tested presynaptic
18
IHC function in mice lacking Munc13-4 and Baiap3, but did not detect any changes in Ca2+
19
currents or exocytosis (Supp. Figure 1B). We conclude that Munc13-like priming factors are
20
dispensable for IHC presynaptic function.
21 22
Cochlear inner hair cells apparently lack Munc13-like priming factors
23
Previous reports indicated absence of Munc13-1 protein from chicken cochlear extracts
24
(Uthaiah and Hudspeth, 2010), however, the expression patterns of the remaining Munc13
25
and CAPS isoforms in the cochlea remain to be established. Therefore, we performed
26
immunostainings with extensively tested antibodies for all Munc13 and CAPS isoforms 6
Accepted manuscript Journal of Cell Science
1
(Cooper et al., 2012) on acutely dissected organs of Corti from hearing p16-17 WT mice
2
(Figure 3). Consistent with our electrophysiological data arguing against a functional role of
3
these proteins in presynaptic IHC function, we did not detect specific Munc13 or CAPS
4
immunofluorescence within IHCs. Rather, immunoreactivities of all tested proteins, including
5
Munc13-1 but not Munc13-2 were restricted to presynaptic terminals of efferent olivocochlear
6
neurons, as evident from colocalization with the neuronal synaptic vesicle marker
7
synapsin1/2, which served as internal positive controls in these experiments. We did not find a
8
specific immunolabeling for Munc13-4 in organs of Corti, i.e. a labeling that was absent from
9
knock-out tissue, with currently available antibodies. While we cannot exclude Munc13-4
10
expression in IHCs, we conclude, based on our analysis of presynaptic function (Supp. Fig
11
1B), that this isoform plays a minor – if any – role in vesicular release from IHC AZs. In the
12
present study, we could establish that IHCs seem to operate without Munc13s or CAPS
13
proteins, a finding that is in line with the notion that their interacting partners neuronal soluble
14
N-ethylmaleimide-sensitive-factor attachment receptors (SNAREs) appear to be absent from
15
IHCs (Nouvian et al., 2011). Based on our findings, we propose that the priming machinery of
16
IHCs is molecularly distinct from conventional neuronal AZs and likely involves the synaptic
17
ribbon and/or bassoon (Frank et al., 2010; Snellman et al., 2011) and otoferlin (Pangršič et al.,
18
2010).
19 20
Otoferlin is enriched in IHC AZ membranes and regulates vesicular tether formation
21
Since IHC exocytosis seems to operate without the classical priming factors, we aimed to
22
further investigate mechanism by which otoferlin facilitates vesicle replenishment. Our
23
confocal data revealed an enrichment of otoferlin at the AZ membrane (Figure 4A), a
24
localization analogous to neuronal Munc13/CAPS priming factors. In these experiments, we
25
employed an antibody that recognizes an intraluminal C-terminal epitope of otoferlin and
26
observed otoferlin clustering in the AZ membrane at the base of the ribbon that could also be 7
Accepted manuscript Journal of Cell Science
1
found with immunogold labelings (data not shown). Otoferlin enrichment at the release site
2
might indicate an involvement in vesicle tethering, which has been implicated as an
3
ultrastructural correlate of vesicular fusion competence (Fernández-Busnadiego et al., 2010;
4
Frank et al., 2010). Therefore, we used electron tomography of IHC synapses to investigate
5
vesicular tethers at AZs of WT and otoferlin-deficient IHCs, instantaneously frozen following
6
stimulation with high K+ to analyze ongoing synaptic activity (Figures 4B-G). We focused our
7
analysis on filamentous tethers connecting membrane-proximal vesicles to the presynaptic
8
density and AZ membrane, as this population was proposed to represent readily-releasable
9
vesicles (Fernández-Busnadiego et al., 2010; Frank et al., 2010). Interestingly, we failed to
10
detect tethers
