To decipher the role of otoferlin in the synaptic vesicle cycle, we produced knock-in mice  with lower Ca2+-binding affinity of the C2C domain. The IHC ribbon synapse structure, synaptic Ca2+ currents, and otoferlin distribution were unaffected in these mutant mice, but auditory brainstem response wave-I amplitude was reduced. Lower Ca2+ sensitivity and delay of the fast and sustained components of synaptic exocytosis were revealed by membrane capacitance measurement upon modulations of intracellular Ca2+ concentration, by varying Ca2+ influx through voltage-gated Ca2+-channels or Ca2+ uncaging. Otoferlin thus functions as a Ca2+ sensor, setting the rates of primed vesicle fusion with the presynaptic plasma membrane and synaptic vesicle pool replenishment in the IHC active zone.Hearing relies on rapid, temporally precise, and sustained neurotransmitter release at the ribbon synapses of sensory cells, the inner hair cells (IHCs). This process requires otoferlin, a six C The extremely precise encoding of sound temporal features by the first synapse of the mammalian auditory system, that is, between the sensory inner hair cell (IHC) and the primary auditory neuron, is crucial for many perceptive tasks. It is involved in periodicity-pitch detection, prosody cue detection, and sound source localization required for voice and melody identification, speech perception, and auditory scene analysis, respectively . Sound-e2+ sensors for rapid, synchronous neurotransmitter release at central nervous system synapses  and two Fer domains in its cytoplasmic region (Otof-/- mice), despite normal Ca2+ currents and ribbon synapse morphogenesis  of the synaptic vesicle cycle otoferlin exerts its putative Ca2+ sensing role. The Pachanga mutant, a deaf mouse harboring a missense mutation in the otoferlin C2F domain, showed unaffected vesicle fusion but a major decrease of the sustained component of IHC synaptic exocytosis  of the synaptic vesicle cycle at which otoferlin may act as a Ca2+ sensors for fast exocytosis, and that Syt7 functions as the Ca2+ sensor for synaptic facilitation, at central nervous system synapses  P2] in vitro, suggesting a possible role in mediating the preferential membrane targeting of otoferlin  located on top loops 1 and 3 of the C2C domain. The substitution of two of these aspartic acid residues by alanine residues (Asp515Ala and Asp517Ala) is predicted to affect Ca2+ binding , by homologous recombination .We investigated the roles of otoferlin in the IHC synaptic vesicle cycle through a mutagenesis strategy similar to that previously used to demonstrate that Syt1 and Syt2 function as Casynapses . The Ca2residues . Substitlitation . The oto3–25 µM) . The C2Ctoferlin . In addio assays . These rtemplate , we cons binding . We therOtof+/+, OtofC2C/+, and OtofC2C/C2C mice. At the age of one month, ABR thresholds were similar in Otof +/+ (n = 5) and OtofC2C/+ (n = 4) mice (OtofC2C/C2C mice (n = 11), by about 5.4 ± 3.3 dB, on average, than in OtofC2C/+ mice (n = 9), for all frequencies tested (Otof⁄C2C C2Cmice (n = 7) than in OtofC2C/+ mice (n = 7), by a factor of 2.7 (OtofC2C/C2C mice (n = 7) were similar to those of Otof C2C/+ mice . From the age of three months onwards, ABR thresholds increased in OtofC2C/C2C mice (n = 5–6) to reach, on average, values 19 ± 5.8 dB higher than those in OtofC2C/+ mice . The peak amplitude of the ABR wave-I also decreased further by a factor of about 10  and OtofC2C/C2C (n = 5–8) mice, in terms of both threshold and amplitude. The cochlear amplification of sound stimuli was, thus, preserved in homozygous mutant mice (OtofC2C/C2C mice is consistent with the restriction of otoferlin dysfunction to IHCs (We first recorded auditory brainstem responses (ABRs), to monitor the electrical response of the primary auditory neurons and the successive neuronal relays of the central auditory pathway to brief sound stimuli, in  4) mice , p>0.3, r of 2.7  . All ribeye-immunoreactive ribbons in OtofC2C/+ and OtofC2C/C2C IHCs displayed Cav1.3 staining systematically apposed to the GluA2 subunit staining. The mean number of ribbons per IHC did not differ significantly between OtofC2C/+ mice (16.4 ± 0.2) and OtofC2C/C2C mice (16.2 ± 0.2) . In mature IHCs of OtofC2C/+ and OtofC2C/C2C mice, otoferlin was detected throughout the cytosol, with intense immunolabeling of the basolateral region containing the ribbon synapses, whereas it was undetectable in the IHCs of Otof-/- mice, as previously reported .We analyzed the IHC ribbon synapses, immunofluorescently labeled for ribeye (a core ribbon protein), the presynaptic L-type Caor GluA2 , by confreported  , and probably corresponding to the resolution of our imaging conditions. These results suggest that otoferlin associates correctly with synaptic vesicles in OtofC2C/C2C IHCs.We also compared the distributions of immunostaining for otoferlin and the vesicular glutamate transporter Vglut3, imaged at high resolution by stimulated emission depletion microscopy (STED) . In bothOtofC2C/+ and ten OtofC2C/C2C mice, on P17 (OtofC2C/C2C ribbons (n = 10), the RRP, RAP, and OP contained 14.0 ± 0.8, 42.4 ± 5.0, and 32.9 ± 3.6 synaptic vesicles per ribbon synapse, respectively . Estimated vesicle densities in the RAP and OP  were similar in OtofC2C/+ and OtofC2C/C2C IHCs (OtofC2C/+ (27.8 ± 1.2 nm) and OtofC2C/C2C IHCs (24.3 ± 1.4 nm) (OtofC2C/C2C IHCs (46.6 ± 1.5 nm) than in OtofC2C/+ IHCs (39.5 ± 1.7 nm)  ; p=0.1.  1.7 nm) , p<0.01.OtofC2C/C2C mice, by monitoring depolarization-evoked membrane capacitance changes (ΔCm) in the IHCs of P15-P18 mice, under various conditions of stimulation. We varied Ca2+ influx through the L-type voltage-gated Ca2+ channels by modifying IHC depolarization level, depolarization duration, or extracellular Ca2+ concentration.Deficient IHC synaptic exocytosis may be due to a failure of synaptic vesicle fusion, vesicle pool replenishment, or endocytosis and synaptic vesicle reformation. We sought to identify the steps of the IHC synaptic vesicle cycle affected in 2+ currents (ICa) and the corresponding ΔCm in response to depolarizations of various amplitudes , each lasting 20 ms, during which synaptic exocytosis mostly reflects the fusion of RRP vesicles  and OtofC2C/+ IHCs , and between OtofC2C/+ (n = 11) and Otof C2C/C2C IHCs . The ICa plots had the typical inverted bell shape, with a negative peak at around −10 mV . In comparisons of release in Otof C2C/C2C and Otof C2C/+ IHCs, the ΔCm evoked by stepwise depolarization from a holding membrane potential of −95 mV to potentials between −65 mV and −30 mV, characterized by a low opening probability for Ca2+-channels and a strong Ca2+ influx-driving force, was similar. However, for depolarization to −30 mV and beyond . A 1.6-fold decrease was observed for depolarization to −10 mV. Assuming a membrane capacitance of 45 aF for a single synaptic vesicle (OtofC2C/+ IHCs (21.7 vesicles/ms) and OtofC2C/C2C IHCs (13.4 vesicles/ms). We then assessed the Ca2+ efficiency of RRP release, by plotting ΔCm as a function of ICa amplitude for depolarization to membrane potentials of −65 mV to −10 mV, corresponding to the falling segment of the Ca2+ current amplitude-voltage (ICa/Vm) curve  in OtofC2C/C2C IHCs ((3.9 ± 0.7) x 10−2 fF.pA−1) than in OtofC2C/+ IHCs ((6.6 ± 0.4) x 10−2 fF.pA−1)  curve , in whicm elicited by brief depolarizations, of 2 to 50 ms duration, to −10 mV   were about a third (p=0.01) and a half (p=0.02) those in OtofC2C/+ mice (n = 11), respectively, indicating a direct effect of the C2C domain mutations on vesicle fusion. By fitting the average relationship between ΔCm and depolarization duration (Δt) for the shortest depolarizations, we found that the slope of this fit, taken as an approximation of the Ca2+ sensitivity of release, was lower (by a factor of 1.5) in OtofC2C/C2C IHCs (0.46 ± 0.04 fF.ms−1) than in OtofC2C/+ IHCs (0.69 ± 0.05 fF.ms−1)  and 5 mM (n = 9) was similar for depolarization durations between 2 and 10 ms, reflecting a tight coupling of Ca2+ channels to RRP vesicles . In OtofC2C/C2C IHCs, RRP release at intracellular EGTA concentrations of 0.5 mM (n = 16) and 5 mM (n = 10) was also similar for depolarization durations between 2 and 10 ms  . For depchannels   during depolarizations of the IHCs for 20 ms to −10 mV  to 268 ± 18 pA ([Ca2+]e = 10 mM) in OtofC2C/+ IHCs (n = 43) and from 40 ± 4 pA ([Ca2+]e = 0.5 mM) to 246 ± 9 pA ([Ca2+]e = 10 mM) in OtofC2C/C2C IHCs . However, for any given level of Ca2+ current, synaptic release in OtofC2C/C2C IHCs was only about half that in OtofC2C/+ IHCs. Moreover, the Ca2+ sensitivity of release, evaluated by fitting a linear function to the ΔCm/ICa curve, was lower (by a factor of 2.2) in OtofC2C/C2C IHCs ((2.2 ± 0.5) x 10−2 fF.pA−1) than in OtofC2C/+ IHCs ((4.8 ± 1.0) x 10−2 fF.pA−1)   and OtofC2C/C2C IHCs  than in OtofC2C/+ IHCs  than in OtofC2C/+ IHCs  , led to 2+ sensitivity of vesicle fusion in OtofC2C/+ and OtofC2C/C2C IHCs, by simultaneously monitoring ΔCm and intracellular Ca2+ variations in IHCs loaded with the low-affinity fluorescent Ca2+-indicator Oregon Green BAPTA 5N (OGB-5N) , no photolysis of DM-nitrophen being triggered by visible-light excitation at 488 nm  and OtofC2C/C2C IHCs  upon UV flash were similar (p=0.8), whereas the peak exocytosis rate occurred later in OtofC2C/C2C IHCs than in OtofC2C/+ IHCs in agreement with the first set of Ca2+ uncaging experiments  than that in OtofC2C/+ IHCs  . The uset 488 nm . In theseriments  and 7A. equation , left. NC2C IHCs  and the channels , these C2+-dependent thereafter  than in OtofC2C/+ IHCs (n = 9), for interpulse intervals of up to 500 ms . This result led us to conclude that the impairment of sustained release in OtofC2C/C2C IHCs is not merely a consequence of impaired RRP release; the recruitment of synaptic vesicles per se is also defective. However, the paired-pulse ratio recovered fully at interpulse intervals of about 1 s. We probed the response of OtofC2C/C2C IHCs (n = 8) to long depolarizations to −10 mV of up to 3 s and observed a much lower (by a factor of 5.3) rate of exocytosis than that measured in OtofC2C/+ IHCs  depolarizations to −10 mV, separated by 10 ms intervals  than obtained for OtofC2C/+ IHCs (~5.6 vesicles/ms/IHC). The time course of synaptic exocytosis in OtofC2C/+ IHCs during the train of successive depolarizations  than in Otof C2C/+ IHCs (n = 12), at 0.6 fF and 1.7 fF per depolarization step, respectively. The onset of the superlinear release component has been shown to be Ca2+-dependent . This almost total absence of superlinear release in OtofC2C/C2C IHCs was probably unlinked to intracellular Ca2+ diffusion defects, because the patterns of calcium entry and diffusion imaged by high-speed swept-field confocal microscopy in IHCs loaded with OGB-5N were similar in OtofC2C/+ (n = 10–12) and Otof C2C/C2C IHCs (n = 6–10). Thus, the C2C domain mutations did not affect the temporal and spatial dynamics of Ca2+ entry and diffusion around the synaptic ribbon  and a fast one with an exponential decline upon longer depolarizations  . Increasing the duration of depolarization to 200–500 ms increased this proportion to six of the ten recorded OtofC2C/C2C IHCs. We therefore compared the slow and rapid endocytotic components between OtofC2C/+ and OtofC2C/C2C IHCs with similar preceding exocytosis levels. In IHCs for which values of 20 to 150 fF had previously been recorded for exocytosis, the rate of the slow component was similar in Otof C2C/+  and OtofC2C/C2C IHCs  and OtofC2C/C2C IHCs  and OtofC2C/C2C  . In Otofobserved . Howeverescribed . In thes2+ influx during sustained stimulation  and Otof+/+ IHCs  recorded at IHC postsynaptic boutons is known to be independent of presynaptic membrane voltage and Camulation . We therescribed , elicitito 25 mM . EPSC fr+/+ IHCs . Howeveriquantal  or multiiquantal  — rather2+-sensing characteristics of otoferlin in RRP fusion and linear and superlinear release  recorded in OtofC2C/+ mice  and the rate of transition from the recycling pool to the RRP (2K) lower than those in OtofC2C/+ IHCs, by factors of 3.0 and 7.7, respectively (K3) was also markedly lower, but with a large confidence interval   .2+-binding properties of the otoferlin C2C domain, we were able to investigate the hypothetical Ca2+-sensor function(s) of the protein in the mature IHC synapse in vivo. By contrast to previously reported Otof mouse mutants . The lower Ca2+ sensitivity of vesicle fusion in OtofC2C/C2C IHCs was further established by rapid increases in intracellular Ca2+ concentration upon photolysis of caged Ca2+ that resulted in a delayed onset of exocytosis and a doubling of the time taken to reach the maximal rate of fusion. By simultaneously monitoring ΔCm and intracellular Ca2+ concentration, we were able to show that the C2C domain mutation, by reducing the Ca2+ affinity of otoferlin, affected the Ca2+ sensitivity of synaptic release but with no major effect on its cooperativity. Finally, the results of the simulation derived from the modified mass-action model  experiments, probing synaptic vesicle pool replenishment efficiency independently of fusion efficiency  and that for synaptic vesicle pool replenishment (K2) were decreased. The detrimental effect of C2C mutations on synaptic vesicle pool replenishment is unlikely to be caused by an impairment of endocytosis, because endocytosis rates and synaptic vesicle numbers were similar in OtofC2C/+ and Otof C2C/C2C IHCs. Finally, the RRP replenishment defect in OtofC2C/C2C IHCs was partially rescued, in terms of total synaptic release, by making Ca2+ available at high concentrations throughout the cytoplasm, as in Ca2+ uncaging experiments, suggesting that synaptic vesicle reformation is not affected in OtofC2C/C2C IHCs. The similar patterns of Ca2+ entry and diffusion in OtofC2C/+ and Otof C2C/C2C IHCs excluded a lower local Ca2+ concentration as the cause of the superlinear release suppression in OtofC2C/C2C IHCs, and provided additional evidence that the lack of superlinear sustained release is due to the abnormal Ca2+ sensing properties of the mutated otoferlin. We therefore conclude that the synaptic vesicle pool replenishment defect in OtofC2C/C2C IHCs is independent of the RRP fusion defect, and results from the slower recruitment of vesicles to the release site caused by the lower Ca2+ affinity of the mutated otoferlin. The molecular motor myosin VI, which has been shown to interact with otoferlin , resulting in few open Ca2+ channels but strong Ca2+ influx-driving forces, the Ca2+ dependence of ΔCm was similar in OtofC2C/C2C and OtofC2C/+ IHCs. At these membrane potentials, the Ca2+ sensor of the RRP vesicles closest to the few open Ca2+ channels is readily saturated with Ca2+, triggering vesicle fusion at similar levels in OtofC2C/C2C and OtofC2C/+ IHCs. Based on the proposed role of the C2A domain in Syt1  knock-in mouse mutant was generated by homologous recombination . The targeting construct, spanning exon 15 (ENSEMBL ENSMUSE00001209343) and containing the two missense mutations, was introduced by electroporation into embryonic stem cells from the 129S1/SvlmJ mouse strain. Stem cells carrying the desired construct were injected into blastocysts from C57BL/6J mice to obtain chimeric mice. After germline transmission, mice were crossed with C57BL/6J mice. Birth rates for all genotypes conformed to Mendelian ratios. Experiments were carried out on both male and female mice, mostly obtained by crossing heterozygous mice (OtofC2C/+) with homozygous mutant mice (OtofC2C/C2C), genotyped by PCR with the following primers: forward 5’-ATTACCTCTGCTGCTTTTGCACCTG-3’ and reverse 5’-CTCAGCAGGTGCTTCTGACCAC-3’, spanning the excised selection marker region in intron 15. As IHC synaptic exocytosis was similar in OtofC2C/+ and Otof+/+ (i.e. wild-type) mice, this breeding scheme was used to produce substantial numbers of OtofC2C/C2C homozygous mutant mice and OtofC2C/+ mice (used as controls) within a given mouse litter. For all experiences, except otoferlin immunofluorescence quantification, experimentalists were not blinded against the genotypes of the mice.Animal experiments were carried out in accordance with European Community Council Directive 2010/63/UE under authorizations 2012–028, 2012–038, and 2014–005 from the Institut Pasteur ethics committee for animal experimentation. The v1.3 antibody was used, the cochlea was fixed by incubation with 99% methanol for 20 min at −20°C. Cochlear whole-mount preparations were permeabilized by incubation with 0.3% Triton X-100 in PBS supplemented with 20% normal horse serum for one hour at room temperature. 4',6-diamidino-2-phenylindole (DAPI) was used to stain cell nuclei. The following antibodies were used: rabbit anti-otoferlin (1:250 dilution) , mouse anti-GluA2 , rabbit anti-Vglut3  and secondary Atto Fluor Cy5-conjugated anti-mouse, Alexa Fluor 488-conjugated anti-goat, and Atto Fluor 647-conjugated anti-rabbit IgG  antibodies. Anti-Ctbp2, anti-GluA2 and anti-Cav1.3 antibodies have been widely used and shown to be specific  equipped with a high-resolution objective . Images were acquired with a 4 x digital zoom in a 1024 × 1024 raster (pixel size = 0.036 μm in x and y) with 0.2 μm steps in z. Three OtofC2C/+ and five OtofC2C/C2C mice were examined for the analysis of otoferlin labeling. Four OtofC2C/+ and four OtofC2C/C2C mice were examined for determination of the number of ribbon synapses per IHC.The entire mouse inner ear was isolated from the surrounding bone, as previously described . For rapilution) , goat anspecific . After iOtofC2C/+ and OtofC2C/C2C littermates were blind-processed in parallel, using the same experimental settings from tissue fixation to confocal imaging. For each organ of Corti, fluorescence intensity was averaged in Gaussian volumes with standard deviations of 1 µm along the X, Y and Z axes, centered around points selected at the apex, the middle, and the base of 7–8 IHCs per confocal stack . In effect, the 3D images were blurred by convolution with a Gaussian of the prescribed standard deviations, and intensity values of the blurred images at the selected points were retrieved.For otoferlin immunofluorescence quantification, whole-mount preparations of organs of Corti from Dual-color STED microscopy was carried out with a custom-built system  using twWavelet denoising and deconvolution were applied to the images, as previously described, with a point spread function extracted from the images , and lin2O for 40 min, followed by Reynold’s lead citrate for 3 min. The sections were viewed in an FEI Tecnai G2 200kV transmission electron microscope, and single- or double-tilt series were acquired from approximately −65° to +65° with 1° increments, with FEI Xplore 3D software and a Gatan US 4000 camera. The acquired tilt series were processed with a wavelet-denoising algorithm implemented in Matlab (Mathworks) , which was then hardened at 70°C. For tomographic analysis, thick (200 or 250 nm) sections of the sensory epithelium (organ of Corti) were collected on 100-mesh parallel bar copper grids, and incubated, for 10 min on each side, with 15 nm protein gold particles . The grids were contrast-stained with 4% uranyl acetate in dHthworks)  to reducthworks) .Analyses of the ribbon synapses, including segmentation, 3D reconstruction, and rendering, were carried out with AMIRA software  and with custom Matlab functions (Mathworks). The contours of the ribbon, the presynaptic density of the afferent dendrite, and nearby organelles, such as mitochondria, coated pits, and tubular structures, were drawn on every section. Spheres of constant diameter were used to mark synaptic vesicles. The ribbon was defined as the center of the active zone. For each ribbon, we counted the number of synaptic vesicles within 80 nm of the ribbon surface. These vesicles were considered to constitute the ribbon-attached vesicle pool (RAP), which is thought to correspond to the functional recycling and reserve pools . A subseAuditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were recorded, as previously described , in micef1 and f2 were applied simultaneously, with f2 between 5 and 20 kHz, and the f2 /f1 ratio kept constant at 1.2. The cubic difference tone at 2f1 – f2, the most prominent distortion product tone produced by the mammalian ear, was measured for primary tone frequencies of equal levels, ranging from 30 to 75 dB SPL.DPOAEs were collected with a miniature microphone positioned at the entry of the ear canal. Two primary pure-tone stimuli of frequencies Otof+/+, OtofC2C/+, and OtofC2C/C2C mice, as previously described . The patch pipette electrodes were made of borosilicate glass (World Precision Instruments).Electrophysiological recordings were carried out on excised cochlear apical coils of either prehearing (P8-P12) or hearing (P15-P18) escribed . The dis2+ current and ΔCm were recorded with an EPC-9 patch-clamp amplifier and Patchmaster software . We used a single 30 mV amplitude sine wave from a holding potential of −95 mV. Except for endocytosis recordings, the acquisition frequency was 50 kHz, low-pass filtered at 6–10 kHz, and the frequency of the sine wave was 1 kHz. In the case of endocytosis recordings, the acquisition frequency was set to 10 kHz, and the frequency of the sine wave to 800 Hz. The resulting maximal depolarization to ∼−65 mV was sufficiently small to avoid activation of the Ca2+ current. Ca2+ current recordings were corrected for the linear leak conductance measured near −95 mV. Liquid junction potential (~−15.5 mV) was corrected off-line for Cs-gluconate-based intracellular solutions. The extracellular recording solution consisted of 111.5 mM NaCl, 6 mM KCl, 10 mM CaCl2, 1 mM MgCl2, 27 mM TEA-Cl, 2 mM sodium pyruvate, 5 mM glucose, 10 mM Na-HEPES, pH 7.4 (adjusted with NaOH). Tetrodotoxin (1 μM) and apamin (1 μM) were added to the extracellular solution. The intracellular pipette solution for cell membrane capacitance recordings contained 140 mM Cs-gluconate, 20 mM TEA-Cl, 0.5 mM EGTA, 5 mM creatine phosphate, 4 mM Mg-ATP, 0.3 mM Na2-GTP, 10 mM HEPES, pH 7.2 (adjusted with CsOH). We used 10 mM extracellular Ca2+ and 0.5 mM intracellular EGTA unless otherwise specified. The ΔCm evoked by membrane depolarization was measured as ΔCm = Cm (response) − Cm (baseline), and was used as a measure of synaptic vesicle exocytosis in IHCs. Cm (baseline) was obtained by averaging capacitance data points before the depolarizing pulse, and Cm (response) was obtained by averaging capacitance data points after the transient current following the depolarizing pulse .For cell capacitance experiments on IHCs, the pipette resistance in the external solution was 2–3 MΩ. Only cells with a series resistance below 10 MΩ (uncompensated) were included in the study. Cang pulse . With de2+ imaging experiments were carried out by adding 200 µM Oregon Green Bapta 5N dye  to the intracellular patch pipette solution. Images were acquired at a rate of 500 Hz, with a high-speed camera (Photometrics) mounted on a swept-field confocal microscope . Imaging protocols began two minutes after cell opening, to allow the OGB-5N dye to diffuse. Ca2+ entry was assessed by selecting regions of interest (a circle with a 6-pixel radius), in which the standard deviation of the fluorescent Ca2+ signal was maximal during IHC depolarization , using a single flash from a high-power UV LED light source . The UV LED TTL triggered through the EPC10 patch clamp amplifier, was directly connected to the epi-illumination port at the rear of an upright Nikon FN1 microscope, and illumination was focused through the 60 x objective . Hair cells were loaded with 145 mM CsCl, 5 mM HEPES, 20 mM TEA, 10 mM DM-nitrophen and 10 mM CaCl2. After patch rupture, we systematically kept the cells at a holding potential of −70 mV (without leak correction) for 2 min, for cell loading and equilibration.We triggered a rapid rise in the intracellular Ca2+ concentration was measured by adding 50 µM OGB-5N to the intracellular solution, as previously described  coupled to the FN1 Nikon upright microscope. The dye was excited with a 488 nm solid-state laser  and emission was recorded at 500–530 nm. OGB-5N-emission fluorescence was continuously monitored before and after the UV flash, by fast line scan analysis at 1 kHz spanning the middle part of the IHC. The Ca2+-dependent fluorescent signal of OGB-5N was calibrated in situ, in the hair cell cytoplasm, as previously described (2+]free), from 1 to 100 µM. The patch pipettes were filled with a Cs-based solution  and various concentrations of Ca2+ and EGTA. The Ca-EGTA Maxchelator freeware  was used to determine [Ca2+]free. After 2 min of cell loading and equilibrium, the fluorescence signal was measured at each [Ca2+]free in different cells. Fluorescence emission values were plotted as F/Fmax against [Ca2+]free. Data points were best fitted by a sigmoidal function with a DK of 23.30 ± 0.71 µM. This DK value is very close to the value reported in vitro . We quantified Ca2+ responses in each hair cell during the Ca2+ uncaging experiments, by calculating the intracellular Ca2+ concentration ([Ca2+]i) as follows ([2+]i = DK (F-Fmin)/(Fmax-F), where DK is the estimated constant of dissociation, F the fluorescence at time t, Fmin the minimum fluorescence, and Fmax the maximal fluorescence.In some experiments, intracellular Caescribed . Changesescribed . Hair cefollows , and the pipette solution for intracellular recording contained 135 mM KCl, 3.5 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, 5 mM HEPES, 2.5 mM Na2ATP, pH 7.2 (adjusted with KOH). EPSCs were induced by increasing extracellular K+ concentration from 5.8 mM to 25.8 mM, by replacing 20 mM NaCl with 20 mM KCl in the external solution. EPSC recordings were performed with a MultiClamp 700B amplifier (Molecular Devices), and a National Instruments digitizer (NI-PCIe 6351). They were low-pass filtered at 6 kHz, and digitized at 50 kHz.Postsynaptic bouton recordings of EPSCs were performed as previously described . PipetteData were analyzed as previously described . In the t tests with Welch’s correction, which does not assume equal variances, were used, unless otherwise stated. The other tests performed included two-way ANOVA with Bonferroni post hoc tests to assess the interaction between two independent variables, Kolmogorov-Smirnov tests to compare distributions, Fisher’s exact tests to compare proportions, and nonparametric Mann-Whitney tests when the data could not be assumed to be normally distributed, precluding use of the parametric Student’s t test. The normality of data distribution was assessed with the D’Agostino and Pearson normality test. Data are expressed as the mean ± standard error of the mean (SEM) unless otherwise stated. Numbers (n) in the figures and text indicate the number of biological replicates derived from independent experiments. Asterisks on bar graphs denote the statistical significance of the differences indicated in brackets , whereas ns indicates ‘not significant’ (p>0.05).The data were analyzed with Igor Pro  and Prism  softwares. Values of p<0.05 were considered to indicate that the differences observed between groups were statistically significant. Two-tailed unpaired Student's OtofC2C/C2C and OtofC2C/+ IHCs were carried out with a modified version of a published mass action model of synaptic vesicle fusion in IHCs (K1), and for vesicle trafficking from the recycling pool to the RRP (K2), from the reserve pool to the recycling pool (K3), and from the distant pool to the reserve pool (K4) (see 2+ concentration ([Ca2+]i) (here expressed in units of charge (C), which corresponds to taking the effective volume in which Ca2+ diffusion occurs as a unit volume) as follows:Simulations of synaptic vesicle fusion in  in IHCs   see , were se1k, 2k, 3,k and 4k are Ca2+-independent rate constants associated with the various pools, [Ca2+]1, …, [Ca2+]4 represent the minimum Ca2+ concentrations triggering RRP fusion, and vesicle transitions from the recycling pool, the reserve pool, and the distant pool, respectively  and vesicle number V(t) in each of the four vesicle pools:where ectively . MinimumInitial conditions were set as follows:2+]1, [Ca2+]2, [Ca2+]3, [Ca2+]4, 1k, 2k, 3k, 4k, VM1, VM2, VM3, and VM4 were obtained by least-squares fitting to the experimental data obtained for OtofC2C/+ and OtofC2C/C2C IHCs. In detail, the sum-of-squares error on the Cm curve was defined as the sum of squared differences between the averaged Cm values measured as a function of some experimental parameter  and the corresponding Cm values predicted by the model for a given set of fitting parameters. This error was minimized using the Matlab built-in minimization function (fminsearch function), which is a general-purpose nonlinear minimization function based on the simplex algorithm  and in Matlab. The parameters 2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses" for consideration by eLife. Your article has been evaluated by a Senior Editor and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your work entitled "Otoferlin acts as a CaeLife in its current form.Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered for publication in The reviewers note that this paper addresses important outstanding questions about the putative role of otoferlin as a calcium sensor for vesicle fusion in auditory hair cell ribbon synapses, an area of some controversy. While the reviewers generally found your paper impressive and liked it overall, they had substantial concerns, as you can see from their comments.The following points will likely require more experiments:2+ sensitivity? Data about changes in affinity and/or cooperatively, for example using biochemistry and caged Ca2+ experiments combined with electrophysiology, would strengthen the authors' claims and make the observed phenotypes easier to interpret.1) What is the nature of the shift in Ca2) The capacitance experiments related to the endocytosis phenotype are somewhat preliminary and would benefit from more controls .3) The unaltered EPSC frequency is surprising given the other phenotypes; this needs to be addressed, possibly with additional experiments. In addition, the reviewers thought there may be a need for control experiments to show that putative changes in expression levels of the mutant do not contribute to the phenotype.Reviewer #1:In this paper, the authors have developed KI mice having mutations in the Ca binding sites of Otoferin, a possible Ca sensor of exocytosis from inner hair cells. As expected, the apparent Ca sensitivity of exocytosis has been changed, and vesicle replenishment has been affected. Overall, the results are convincing, but there are several points which need authors attention.1) It is a bit unclear how much the Ca sensitivity of mutations is altered. Some evidence from biochemistry may be helpful. Are mutations shifting the Ca sensitivity without changing the slope or has the slope (cooperativity) been changed?2) In 3) For Ca uncaging experiments, it is a pity that the authors did not perform experiments with changing intracellular Ca concentrations. As it is, it is unclear if the maximal rate of secretion has been changed (which is nothing to do with Ca sensitivity) or else if Ca sensitivity is shifted. The authors may need additional experiments or need some comments or clarification. Also, the authors should cite the works by Heidelberger/Matthews and Beutner/Moser, both of whom have developed Ca uncaging at ribbon synapses.4) In Figure 7 , I am not convinced if synaptic vesicle replenishment is Ca dependent and otoferin is the sensor. The authors need some comments.5) Figure 8 is difficult to understand. Why the EPSC frequency is not reduced in mutant mice? This is due to compensation or better conditions have to be found (higher or lower K concentrations)?6) I do not feel that the modeling part is really required, because it is not that informative.Reviewer #2:The paper by Michalski et al. examines the molecular identity and functional properties of the calcium / release sensor at auditory hair cell ribbon synapses. To address this question, the authors combine genetic approaches (mutant mice), immunohistochemistry, electron tomography, recording of evoked potentials, presynaptic capacitance measurements, calcium imaging, calcium uncaging, and modeling. The main findings are:- Point mutations in the C2C domain of otoferlin reduce the amplitude of auditory evoked brainstem potentials.- Otoferlin mutations change the calcium sensitivity of exocytosis, as probed with capacitance measurements from hair cells.- Otoferlin mutations also change release during long-lasting repetitive stimulation, suggesting a role for the protein in vesicle replenishment.- Modeling data further suggest that the difference in the sensitivity of the calcium sensor can explain the synaptic phenotype.Based on these findings, the authors conclude that otoferlin functions as a calcium sensor of exocytosis and vesicle replenishment in hair cell ribbon synapses. Overall, I found this a quite impressive paper. The results are interesting, because the manuscript for the first time uses the approach of Fernandez et al., 2001 (pioneered for synaptotagmin) to a different calcium-sensing protein. Furthermore, the experiments are technically well done , and the manuscript is concisely and accurately written. One caveat is that the role of otoferlin in exocytosis from hair cells was surrounded by controversy in the past, but I feel the present manuscript provides definitive answers to many of the outstanding questions.1) Although otoferlin is clearly expressed in the mutant (as shown by immunocytochemistry), the exact expression levels are unknown. The authors should address this point by quantitative analysis, or at the very least put caveat sentences into the paper. If expression levels were different between wild-type and mutant, this would affect the main conclusions.2+ sensor mediating exocytosis? The authors might have the high-resolution imaging data to address this question.2) Is otoferlin present on synaptic vesicles, as required for a Ca3) The authors show that their capacitance versus calcium curves were fitted power functions with exponents less than one, suggesting the absence of cooperativity. If I read the early literature on hair cell exocytosis correctly, data were well fit by a model with five binding sites for calcium and cooperativity in the unbinding rates . How the present data relate to these earlier findings is presently unclear. This point should be better discussed.4) The authors generally use the heterozygous rather than wild-type mice as controls. This should be better justified in the paper. The argument that there are only minimal differences between wild-type and heterozygous animals in the auditory evoked brainstem potentials is not very compelling, because this assay may not have the sensitivity to detect small differences.5) A design flaw in the uncaging experiments is that the absolute calcium concentration is not simultaneously monitored, as often done by the ratiometric calcium the indicator fura 2. Furthermore, the authors mention absolute calcium concentrations several times. However, it is unclear how these concentration estimates were obtained with the non-ratiometric indicator OGB5-N.6) In the capacitance measurements, the authors only illustrate Cm traces. The authors should at least once show the corresponding gm and ga traces to convince the readers of the reliability of the measurements.7) The endocytosis data are preliminary. First, longer traces should be shown to illustrate whether capacitance decays back to baseline. Second, the authors should analyze the decay more quantitatively, for example by exponential fitting. Finally, it is surprising that the mutant apparently does not affect endocytosis, despite its clear effects on replenishment rate. This point should be better discussed.8) The EPSC recordings from postsynaptic boutons are a little preliminary. It is unclear how the authors determine the frequency of events and how they measure amplitude and decay time constant in complex waveforms. Finally, it is unexpected that there is no significant difference in frequency between control and mutant. Increasing the number of experiments may resolve this issue. [Editors’ note: the author responses to the first round of peer review follow.]Reviewer #1:In this paper, the authors have developed KI mice having mutations in the Ca binding sites of Otoferin, a possible Ca sensor of exocytosis from inner hair cells. As expected, the apparent Ca sensitivity of exocytosis has been changed, and vesicle replenishment has been affected. Overall, the results are convincing, but there are several points which need authors attention.1) It is a bit unclear how much the Ca sensitivity of mutations is altered. Some evidence from biochemistry may be helpful. Are mutations shifting the Ca sensitivity without changing the slope or has the slope (cooperativity) been changed?2C domains for biochemical assays. Three independent laboratories made several attempts with a view to probing the effects of the C2C domain mutations on Ca2+‐binding in vitro .Unfortunately, we were unable to produce and purify high‐quality C2+ levels near release sites during Ca2+ channel activation, we had reported that the C2C domain mutations almost halved the Ca2+ sensitivity of the RRP sensor  loaded with the low‐affinity fluorescent Ca2+ indicator, OGB‐5N. This approach made it possible to show ex vivo that the Ca2+ sensitivity of release is reduced in OtofC2C/C2CIHCs. Our results indicate that C2C domain mutations strongly decrease the Ca2+ affinity of otoferlin, by a factor of about 4, but not the cooperativity of release.By contrast, uncaging experiments were more successful. In the first version of the manuscript, through the various experimental protocols used to manipulate intracellular Caf fusion . In the data new . We simu2+ binding mutant (D519A/D521A) versions of the otoferlin C2C domain for biochemical validation of the importance of the Ca2+‐binding site in the otoferlin C2C domain. The chosen domain boundaries of otoferlin C2C were based on the known structural properties of C2 domains: prediction of eight β‐strands, the periodicity of alternating hydrophobic residues in betastrands consistent with known C2 domain structures, identification of betabulge residues to ascertain C2 domain topology, geometric arrangement of consensus Ca2+ binding residues, location of β‐strand 3 with consensus residues conserved in all C2 domains, and overall length of the domain. Once these criteria were met, a 3D model was generated with Modeller, by aligning the selected primary sequence with a template appropriate for C2 domain topology. For confirmation that the hydrophobic core and β‐sheet assignments of the model domain were stable, the model was simulated for 100 ns with NAMD. This led us to test two different C2C domain boundaries (residues 403‐554 and residues 449‐600). We used a bacterial system to test various expression and growth parameters, the two different protein boundaries , additives and affinity tags . Despite this comprehensive analysis, most of the clones resulted in little or no protein production. The only exception was Clone #1, with the C‐terminal His tag. However, the proteins produced from this clone were of very poor quality, giving rise to multiple bands on protein gels. This was confirmed by mass spectrometry and Edman N‐terminal sequencing analysis, which also showed additional peaks and sequence variability, respectively. Attempts to purify the otoferlin C2C domain further, by removing contaminants through size‐exclusion and affinity chromatography, were unsuccessful. We nevertheless performed the liposome aggregation, membrane binding, and liposome fusion assays described in a previous study , to check the effect of Ca2+‐binding, but we observed no effect of Ca2+ addition on the WT or mutated C2C domains, probably because of the suboptimal quality of the proteins used.Biochemistry: We attempted to produce and purify wild‐type and Ca2) In m decline (in the range of 2‐10 fF/s) upon short depolarizations , and a fast phase with an exponential decline (with a time constant in the 2‐4 s range) upon longer depolarizations  . In the former and current m, Gm, and Gs traces shortly after the end of the depolarization, to make sure that these transients are not included in our ΔCm estimate. As suggested, to avoid confusion, we now provide example Cm, Gm, and Gs traces and illustrate how we evaluate ΔCm  For Ca uncaging experiments, it is a pity that the authors did not perform experiments with changing intracellular Ca concentrations. As it is, it is unclear if the maximal rate of secretion has been changed (which is nothing to do with Ca sensitivity) or else if Ca sensitivity is shifted. The authors may need additional experiments or need some comments or clarification. Also, the authors should cite the works by Heidelberger/Matthews and Beutner/Moser, both of whom have developed Ca uncaging at ribbon synapses.2+ uncaging experiments (new 2+ concentration in IHCs loaded with OGB‐5N. Our results indicate that C2C domain mutations strongly reduce Ca2+ affinity (by a factor of about four), without changing apparent cooperativity.As mentioned above (see response to point 1), we now provide a new set of Caents new , in whic2+ uncaging Results section.Heidelberger/Matthews and Beutner/Moser are now cited at the start of the Ca4) In Figure 7 , I am not convinced if synaptic vesicle replenishment is Ca dependent and otoferin is the sensor. The authors need some comments.2+‐dependent and we refer to the original article  in the manuscript. The grounds for our conclusion concerning the role of otoferlin as a Ca2+ sensor in synaptic vesicle replenishment are as follows:Synaptic vesicle replenishment has been shown to be CaOtofC2C/C2C IHCs. In accordance with the reviewers’ suggestions, we now provide a more detailed description of endocytosis (see response to point 2). The fast and slow modes of membrane retrieval were similar in OtofC2C/+ and OtofC2C/C2C IHCs. Accordingly, vesicle pool numbers at the ribbon synapses were also normal. Moreover, rapid and global increases in intracellular Ca2+concentration in response to UV‐flash photolysis led to similar total synaptic release levels in OtofC2C/+ and OtofC2C/C2C IHCs, strongly suggesting that endocytosis and vesicle reformation are not affected in OtofC2C/C2C IHCs. This particular point is now discussed as follows: “Finally, the RRP replenishment defect in OtofC2C/C2C IHCs was partially rescued, in terms of total synaptic release, by making Ca2+ available at high concentrations throughout the cytoplasm, as in Ca2+ uncaging experiments, suggesting that synaptic vesicle reformation is not affected in OtofC2C/C2C IHCs.” Moreover, this result indicates that the loss of Ca2+ sensitivity does not prevent the total synaptic pool of the IHCs to be depleted by high calcium loads. In previous studies of other otoferlin mutant mice, Ca2+ uncaging failed to elicit normal total synaptic release .i) Endocytosis is normal in OtofC2C/C2C IHCs than in OtofC2C/+ IHCs, as shown by long depolarizations or trains of 50 successive short depolarizations, suggesting an effect of C2C mutations on Ca2+‐dependent vesicle replenishment. Moreover, paired pulse ratio experiments, which probe synaptic vesicle pool replenishment efficiency independently of fusion efficiency, showed that peak vesicle pool replenishment efficiency in OtofC2C/C2C IHCs was only half that in OtofC2C/+ IHCs, excluding the possibility that the synaptic vesicle pool replenishment defect was merely a consequence of the impairment of RRP fusion.ii) The rate of synaptic vesicle replenishment was lower in OtofC2C/C2C IHCs with low intracellular Ca2+‐buffering conditions, the exocytotic response was rapidly saturated for depolarizations lasting 15‐20 ms . This saturation is apparent for depolarizations of up to 100 ms . In OtofC2C/C2C IHCs, release begins to increase again for depolarizations lasting more than 200 ms , and was insensitive to 5 mM intracellular EGTA, suggesting that vesicle pool replenishment at the release sites was also impaired in these cells .” This result provides evidence that the replenishment deficit is not only due to impaired synaptic fusion, but also a direct consequence of the C2C domain mutation.iii) In 0 ms see . This re2+ sensor for vesicle replenishment.Based on all these findings, we conclude that otoferlin functions as a Ca5) Figure 8  is difficult to understand. Why the EPSC frequency is not reduced in mutant mice? This is due to compensation or better conditions have to be found (higher or lower K concentrations)?n = 8 recordings for each genotype. Secondly, in the previous version of the paper, we calculated EPSC activation frequency throughout the length of each recording, regardless of its length. This led to a non‐uniform comparison of EPSC activation frequencies. Finally, the application of high levels of K+ typically induces a rapid increase in activation frequency within a few seconds, followed by a slowing over tens of seconds or minutes. This first phase is greatly influenced by IHC release capacity. For this reason, we now evaluate EPSC activation frequency in the first 10 seconds after the application of 25mM K+. It is now clear that the mutation has a strong effect on EPSC rate  I do not feel that the modeling part is really required, because it is not that informative.K1) and the rate of transition from the recycling pool to the RRP (2K), but that there is no change in Ca2+ thresholds for the recruitment of each vesicle pool or in vesicle number in each pool.We think that the first part of the modeling results is noteworthy new  because We have removed the second part of the modeling results, in which the same model settings were used to reproduce our experimental results for 100 ms depolarizations.Reviewer #2:[…] 1) Although otoferlin is clearly expressed in the mutant (as shown by immunocytochemistry), the exact expression levels are unknown. The authors should address this point by quantitative analysis, or at the very least put caveat sentences into the paper. If expression levels were different between wild-type and mutant, this would affect the main conclusions.OtofC2C/+ and OtofC2C/C2C inner hair cells (IHCs), based on comparisons of otoferlin immunofluorescence, as previously described for other otoferlin mutants . We found no difference at the apex, middle, or base of OtofC2C/+ and OtofC2C/C2C IHCs  Is otoferlin present on synaptic vesicles, as required for a CaOtofC2C/+ and OtofC2C/C2C IHCs, and significantly smaller than the mean distance between a random Vglut3 spot and its nearest neighboring Vglut3 spot (the expected distribution of nearest‐neighbor distances for randomly distributed points). The distributions of distances between a given otoferlin immunostaining spot and the closest Vglut3 immunostaining spot were also similar in OtofC2C/+ and OtofC2C/C2C IHCs, suggesting that the C2C mutations did not affect the association of otoferlin with synaptic vesicles.We now provide super‐resolution images obtained by stimulated emission depletion imaging (STED) microscopy, of IHCs stained for otoferlin and a marker of IHC vesicles (Vglut3) see new . Using n3) The authors show that their capacitance versus calcium curves were fitted power functions with exponents less than one, suggesting the absence of cooperativity. If I read the early literature on hair cell exocytosis correctly, data were well fit by a model with five binding sites for calcium and cooperativity in the unbinding rates . How the present data relate to these earlier findings is presently unclear. This point should be better discussed.m measurements in response to IHC depolarization .These two sets of experiments cannot be directly compared because of their very different experimental conditions. In the case of ΔCrization , Ca2+ enm measurements in response to IHC depolarization ). [It should be noted that this relationship is not linear in immature IHCs]. In this experimental setting, we plotted ΔCm as a function of ICa at the end of the stimulus, so we had no access to the early kinetics of release during depolarization. In the Ca2+ uncaging experiments reported by Beutner et al. , the initial release rate (dCm/dt) is plotted. In these uncaging experiments, if the final ΔCm were plotted as a function of ICa, a flat relationship would probably have been observed, as suggested by the examples shown in figure 2 of the same paper , because the same maximal Cm is reached whatever the original intracellular calcium concentration, suggesting that release is saturated after a certain time.For ΔCrization , we obta2+ uncaging experiments in OtofC2C/+ and OtofC2C/C2C IHCs. In control cells, we obtained cooperativity values similar to those reported by Beutner et al. , between 3 and 4.As described below (see reply to point 5), we provide new data for Ca4) The authors generally use the heterozygous rather than wild-type mice as controls. This should be better justified in the paper. The argument that there are only minimal differences between wild-type and heterozygous animals in the auditory evoked brainstem potentials is not very compelling, because this assay may not have the sensitivity to detect small differences.m recordings for Otof+/+ IHCs, further demonstrating that synaptic release is similar in OtofC2C/+ and Otof+/+ IHCs. We measured Ca2+ currents (ICa) and the corresponding ΔCm in response to depolarizations of various amplitudes  and 20 ms duration in Otof+/+ IHCs, and compared these results with those of OtofC2C/+ IHCs (see Otof+/+ IHCs to periodic stimulation with a train of 50 short (5 ms long) depolarizations to ‐10 mV, separated by 10 ms intervals. We found that this protocol elicited superlinear release in around half the recorded Otof+/+ IHCs, as for OtofC2C/+ IHCs.We now provide new ΔCIHCs see . We also5) A design flaw in the uncaging experiments is that the absolute calcium concentration is not simultaneously monitored, as often done by the ratiometric calcium the indicator fura 2. Furthermore, the authors mention absolute calcium concentrations several times. However, it is unclear how these concentration estimates were obtained with the non-ratiometric indicator OGB5-N.2+and membrane capacitance upon UV‐flash photolysis of DM‐nitrophen for each cell (see new 2+ dye Oregon Green Bapta‐5N (OGB‐5N) as an intracellular calcium probe because it allows continuous rapid monitoring of the Ca2+ signal under confocal microscopy (line scan at 1 kHz), before and after UV‐flash Ca2+ uncaging. OGB‐5N is a dye displaying excitation at a long wavelength (488 nm), a visible wavelength at which there is no photolysis of DM‐nitrophen, by contrast to UV epifluorescence experiments using the ratiometric dye Fura‐2 (excitation at 340 and 380 nm). Another advantage of OGB‐5N is its Ca2+ affinity, which is lower (DK= 20 µM) than that of Fura‐2 (DK = 140 nM), making it possible to obtain more precise measurements of variations of Ca2+concentration, in the μM range.We have run additional experiments for simultaneous quantification of the increase in intracellular Ca see new . We used2+‐dependent fluorescent signal of OGB‐5N was first calibrated in situ, in the hair cell cytoplasm, as previously described . Hair cells were voltage‐clamped at ‐70 mV in the whole‐cell configuration with various solutions differing in their free calcium concentrations ([Ca2+]free), which ranged from 1 to 100 µM. The patch pipettes were filled with a Cs+‐based solution  and various concentrations of Ca2+ and EGTA. Ca‐EGTA Maxchelator freeware  was used to determine [Ca2+]free. After 2 min of cell loading and equilibration, the fluorescence signal was measured at each [Ca2+]free, in different cells. Fluorescence emission values were plotted as F/Fmax against [Ca2+]free. Data points were best fitted by a sigmoidal function with a DK of 23.30 ± 0.71 µM. This DK value is very close to the value reported in vitro . We quantified the Ca2+ responses during the Ca2+ uncaging experiments in each hair cell, by calculating intracellular [Ca2+] as follows , chapter 42): [Ca2+]i= DK (F Fmin)/(Fmax‐F) where DK is the estimated constant of dissociation, F the fluorescence at time t, Fmin the minimum fluorescence and Fmax the maximal fluorescence.As now described in the Materials and methods section, the Ca6) In the capacitance measurements, the authors only illustrate Cm traces. The authors should at least once show the corresponding gm and ga traces to convince the readers of the reliability of the measurements.m, Gm, and Gs traces and illustrate how we typically evaluate ΔCm(see We now provide an example of Ce ΔCmsee . We havee ΔCmsee .7) The endocytosis data are preliminary. First, longer traces should be shown to illustrate whether capacitance decays back to baseline. Second, the authors should analyze the decay more quantitatively, for example by exponential fitting. Finally, it is surprising that the mutant apparently does not affect endocytosis, despite its clear effects on replenishment rate. This point should be better discussed.m trace returned to baseline). We studied the two modes of endocytosis with different kinetics that have been described: the slow phase, with a linear Cm decline, upon short depolarizations , and the fast phase, with an exponential decline upon longer depolarizations  . The kinetics of the slow and fast components of endocytosis were similar in OtofC2C/+ and OtofC2C/C2C IHCs . Vesicle reformation is now discussed as follows: “Finally, the RRP replenishment defect in OtofC2C/C2C IHCs was partially rescued, in terms of total synaptic release, by making Ca2+ available at high concentrations throughout the cytoplasm, as in Ca2+ uncaging experiments, suggesting that synaptic vesicle reformation is not affected in OtofC2C/C2C IHCs.”The normal endocytosis observed is consistent with the normal otoferlin levels, normal vesicle numbers at ribbon synapses, and the similar total synaptic release in 8) The EPSC recordings from postsynaptic boutons are a little preliminary. It is unclear how the authors determine the frequency of events and how they measure amplitude and decay time constant in complex waveforms. Finally, it is unexpected that there is no significant difference in frequency between control and mutant. Increasing the number of experiments may resolve this issue.n = 8 recordings for each genotype. Secondly, in the previous version of the paper, we calculated EPSC activation frequency throughout the length of each recording, regardless of its length. This led to a non‐uniform comparison of EPSC activation frequencies. Finally, the application of high levels of K+ typically induces a rapid increase in activation frequency within a few seconds, followed by a slowing over tens of seconds or minutes. This first phase is greatly influenced by IHC release capacity. For this reason, we now evaluate EPSC activation frequency in the first 10 seconds after the application of large amounts of K+. It is now clear that the mutation has a strong effect on EPSC rate , and a National Instruments digitizer (NI‐PCIe 6351). […] Decay time constants were calculated with monophasic recorded EPSCs only.”


Our data show that phosphorylation of Grb7 protein on the Ser194-Pro motif by c-Jun N-terminal kinase facilitates its binding with the WW domain of Pin1. Subsequently, Grb7 is degraded by the ubiquitin- and proteasome-dependent proteolytic pathway. Indeed, we found that Pin1 exerts its peptidyl-prolyl cis/trans isomerase activity in the modulation of Grb7 protein stability in regulation of cell cycle progression at the G2-M phase. This study illustrates a novel regulatory mechanism in modulating Grb7-mediated signaling, which may take part in pathophysiological consequences.Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions. The molecular mechanisms underlying the regulation of Grb7 remain unclear. Here, we revealed a novel negative post-translational regulation of Grb7 by the peptidyl-prolyl  Growth factor receptor bound protein 7 (Grb7) is a member of the Grb7 adaptor protein family that includes Grb10 and Grb14 proteins. The entire Grb7 family proteins are composed of five major protein-binding modules, including an N-terminal proline-rich region, a putative RA (Ras-associating) domain, a central PH (pleckstrin homology) domain, a BPS motif (between PH and SH2 domains), and a C-terminal SH2 domain . AlthougSeveral studies have suggested that the tyrosine phosphorylation state of Grb7 is crucial for its regulation and functionality. Various stimuli, such as epidermal growth factor , ephrin cis/trans isomerase, Pin1, is an essential regulator for multiple post-translational modifications by catalyzing the conversion of phospho-Ser/Thr–Pro motifs between two distinct cis and trans isomers of a protein [cis/trans isomerization of peptidyl-prolyl peptide bonds [The peptidyl-prolyl  protein . Pin1 code bonds . Pin1 isde bonds , 15, prode bonds , proteinde bonds , phosphode bonds , and thede bonds . As a rede bonds , 20–22. In the present study, we first identified a serine phosphorylation site preceding a proline residue, Ser194, on Grb7 protein. This phosphorylation was catalyzed by JNK, which enables interaction with Pin1 via its WW domain. Then, the interaction between Grb7 and Pin1 then subjects Grb7 ubiquitination and subsequent degradation through proteasome-mediated proteolysis in a Pin1 isomerase activity-dependent manner. Consequently, we revealed Pin1 involved in Grb7-mediated cell cycle progression.Glutathione-agarose beads, protein A-sepharose 4B beads, human plasma fibronectin, poly-L-lysine, EGF, G-418 disulfate salt, 5’-bromo-2-deoxyuridine (BrdU), puromycin, cycloheximide, LY294002, and SB431542 were purchased from Sigma-Aldrich . His-Tag binding agarose was purchased from Bioman Scientific . SB203580, PD98059, JNK inhibitor II and MG-132 were obtained from Calbiochem . CGP74514A, SB-415286, and TDZD-8 were gifts form Dr. Pei-Jung Lu . Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM), RPMI, DMEM/F-12 (1:1), and Opti-MEM media were purchased from Invitrogen . The mouse monoclonal anti-Cyclin D1  antibody was purchased from Enzo Life Science . The mouse monoclonal anti-Akt-1 , anti-HA , and anti-GFP  antibodies were obtained from Santa Cruz Biotechnology . The rabbit polyclonal anti-Grb7 , anti-Grb7 , anti-JNK , anti-ERK 1 , anti-p38α , anti-HA-probe , and anti-Pin1  antibodies were obtained from Santa Cruz Biotechnology . The rabbit polyclonal anti-phospho-cdc2 , the rabbit polyclonal anti-phospho-Erk1/2 , the mouse monoclonal anti-phospho-SAPK/JNK , the rabbit monoclonal anti-GSK-3β , and the rabbit monoclonal anti-phospho-GSK-3α/β  antibodies were purchased from Cell Signaling . The rabbit monoclonal anti-phospho-p38α , the mouse monoclonal anti-phospho-Ser/Thr motif  and anti-actin  were obtained from Millipore . The mouse monoclonal anti-BrdU , anti-Flag , and anti-GST  antibodies were purchased from Sigma-Aldrich . The secondary horseradish peroxidase-conjugated goat anti-rabbit antibodies and goat anti-mouse antibodies were obtained from Jackson ImmunoResearch Laboratories .Saccharomyces cerevisiae strain Y187), pGBTK7 (GAL4 DNA-binding domain vector), pGADT7 (GAL4 activation domain vector), and Saccharomyces cerevisiae strain AH109 were obtained from Clontech Laboratories . The full length of Grb7 was used as a bait protein. A total of 1.4 million independent clones from the human brain cDNA library constructed in yeast strain Y187 were screened with yeast strain AH109 transformed with pGBTK7-Grb7. The combination of 1 ml of library strain Y187 and 5 ml of bait strain AH109 pGBTK7-Grb7 was cultured in a sterile 2-liter Erlenmeyer flask with 45 ml of 2 × YPDA (containing kanamycin) at 30°C for 20–24 hr. The mating culture was washed with 0.5 × YPDA (containing kanamycin) and resuspended in 0.5 × YPDA (containing kanamycin). Serial dilutions of yeast culture were spread on SD/-Leu, SD/-Trp, and SD/-Leu/-Trp plates and incubated at 30°C for 3–5 days to determine the mating efficiency. We streaked the remaining culture first on SD/-His/-Leu/-Trp plates at 30°C for one week, and then, we transferred all the colonies to SD/-Ade/-His/-Leu/-Trp plates by replica plating and incubated the plates at 30°C for one week. The library proteins that bind with pGBTK7 were confirmed by retreating on SD/-Ade/His/-Leu/-Trp agar plates, as well as by X-gal assay. All the positive clones were transformed into Escherichia coli strain DH5α to obtain the library plasmids, which were then sequenced. After sequencing, the sequences were subjected to a BLAST search in the National Center for Biotechnology Information database to find all the matching genes.The human brain Matchmaker cDNA library (cultured in +/+) and knockout (Pin1-/-) mouse embryonic fibroblasts were gifts form Dr. Pei-Jung Lu  and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum . All cells were incubated in a 37°C humidified 5% CO2 incubator. Cells were transfected with mammalian expression plasmids, as indicated, using Lipofectamine 2000TM transfection reagent  according to the manufacturer’s instructions. Experiments were conducted 24–48 hr after transfection. Transfected cells were selected by G418 .A431 human epithelial carcinoma and 293T human epithelial kidney cell lines were gifts from Dr. Jun-Lin Guan  and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum . Pin1 wild-type , were used as a template; individual point mutation was generated by site-directed mutagenesis, as described previously , using tTM RNA extraction reagent . The cDNA was synthesized from RNA using M-MLV reverse transcriptase according to the manufacturer’s recommendations . The primers were designed as follows: 5′-GAATTCGCCCCCATGTAGTAAAG-3′ (sense) and 5′-GAATTCGTTTTTCCGGAAGACGAA-3′ (antisense) for Grb7; 5′-GGAGCTGATCAACGGCTACA-3′ (sense) and 5′-TCAGTGCGGAGGATGATGT-3′ (antisense) for Pin1. The housekeeping gene GAPDH was used as a reference for normalization. Quantitation of PCR products was estimated by SYBR Green reagent  using the ABI StepOnePlus Real-Time PCR system, and the data were analyzed with ABI StepOnePlus Real-Time PCR software according to the manufacturer’s instructions. qPCRs were performed in triplicate, and copy number alterations were scored as validated if the 2ΔCt was ≥ 2 (gain) or ≤ 0.5 (loss) with CV ≤ 15% of 2ΔCt. The Grb7 and Pin1 gene expression levels were normalized by GAPDH, which was used as a reverse transcription control.RNA was extracted from cells using MaestroZolLentiviruses encoding GRB7, PIN1, or LUCIFERASE small-hairpin RNA (shRNA) were obtained from the TRC lentiviral shRNA library in the National RNAi Core Facility of Academia, Taiwan. The targeting sequencings of various shRNAs were as follows: GRB7 shRNA (clone ID: TRCN0000061387) 5′-CCAGGGCTTTGTCCTCTCTTT-3′; PIN1 shRNA (clone ID: TRCN0000010577) 5′-GCCATTTGAAGACGCCTCGTT-3′; PIN1 shRNA (clone ID: TRCN0000001034) 5′-CCAGAAGATCAAGTCGGGAGA-3′; MAPK8 shRNA (clone ID: TRCN0000196850) 5’-GTGTCTTCAATGTCAACAGAT-3’. Production and infection of lentiviruses were processed according to the guidelines of the National RNAi Core Facility of Academia Sinica .3VO4, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, and 20 mg/ml leupeptin) on ice for at least 20 min. Next, lysates were collected and clarified by centrifugation at maximum speed for 30 min at 4°C. Immunoprecipitations were carried out by incubating cell lysates with antibodies, as indicated, for 4 hr at 4°C, followed by incubation for an additional 3 hr with protein A-sepharose 4B beads. Then, after washing three times with lysis buffer, immune complexes were resolved using SDS-PAGE. Finally, Western blotting was performed using horseradish peroxidase-conjugated IgG as a secondary antibody and the Western Lightning-ECL system  for detection. All experiments were conducted at least three independent times.Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then lysed with 1% Nonidet P-40 lysis buffer  in DMEM as the chemoattractant or DMEM only as a control in the lower wells for 7 hr in a 37°C humidified 5% CO2 incubator. At the end of the experiments, cells on the upper side of the polycarbonate membrane were removed, and the bottom-side cells were fixed in methanol for 10 min and stained with crystal violet . The migrated cells were counted in four randomly selected fields of each well under a light microscope  using a 20× objective lens.Cell migration assays were carried out by a Neuro Probe 48-well chemotaxis Boyden chamber  as described previously [Sixteen hours after transfection, cells were first serum starved for 24 hr in DMEM with 0.2% FBS. Cells were then washed with DMEM and incubated for 20 hr in DMEM with 10% FBS and 100 μM BrdU . Thereafter, cells were fixed in 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.5% Triton X-100, treated with DNase I , and then processed for immunofluorescence staining with anti-BrdU  antibody, as described previously  with modCells were washed with cold PBS and then fixed in 4% paraformaldehyde at 4°C for 1 hr. Thereafter, cells were washed in cold PBS and treated with propidium iodide staining solution  in the dark for 30 min. Cells were analyzed by flow cytometry .p < 0.05 was considered significant differences among the experimental groups.Student’s t-test was used for statistical analyses. The data in this study were represented as mean ± SD of at least three independent experiments. *, E. coli was capable of efficiently pulling down Grb7  exhibited high homology to the sequence of Pin1 . This reown Grb7 . Collect194-Pro motif, rather than with the phospho-Ser361-Pro motif of Grb7  rather than the N-terminus of Grb7 . Given t of Grb7 . Similarin vitro pull down assay utilizing the GST-tagged full-length, WW domain (amino acids 1–39), or PPIase domain (amino acids 45–163) of Pin1 recombinant proteins, we found that the WW domain of Pin1 was sufficient to interact with Grb7 , GSK-3 (SB-415286), GSK-3β (TDZD-8), AKT (LY294002), ERK (PD98059), JNK (JNK-in) and p38 MAPK (SB203580) inhibitors, were employed to examine if the corresponding kinase is involved in the phosphorylation of the Ser194-Pro motif of Grb7. Among the inhibitors used, the JNK inhibitor exhibited an apparent interference of the interaction between Pin1 and Grb7  or K63A  mutantin Pin1 knockdown cells  or Pin1 Furthermore, we sought to elucidate the underlying mechanism by which Pin1 negatively modulates Grb7 protein stability. Several studies have demonstrated that Pin1 enables control of protein stability via ubiquitin-mediated proteolysis, for example, NF-kB, interferon-regulatory factor 3 , 27. As Moreover, the promotion of ubiquitinated Grb7 protein was impeded upon co-transfection with W34A (the pSer/Thr-Pro motif binding deficient mutant) or K63A (the PPIase deficient mutant) mutant of Pin1 compared to the wild-type Pin1 . This reGiven the essential role of Grb7 in cell cycle regulation and cell migration, we then explored the effect of Pin1 interaction with Grb7 on these cellular functions. First, the A431 cells stably expressing wild-type, S194A or S361A mutant Grb7  were sub194-Pro motif of Grb7 enables the physical interaction with Pin1  was found to contain minimal phosphoaccepter Ser-Pro (SP) motif for phosphorylation by JNK [We found that JNK, but not p38, ERK, CDK1 or GSK3, enables binding to the Ser-Pro motif and further inducing phosphorylation at the Ser protein . Consistn by JNK , 29. Ourn by JNK . In linen by JNK –32.194-Pro motif by Pin1 might facilitate the accessibility to E3 ubiquitin ligase, thereby contributing to the ubiquitination of Grb7 (194 residue (S194A) of Grb7 abolished Pin1-binding ability and isomerization of Grb7 by Pin1 , JNK-in (25 μM), SB203580 (10 μM), LY294002 (10 nM), CGP74514A (5 μM), TDZD-8 (50 μM), or SB-415286 (20 μM) for 1 hr. Cell lysates were collected and subjected to Western blot analysis to analyze the phosphorylation and expression of indicated signal molecules.(PDF)Click here for additional data file.