Otoferlin acts as a Ca2+ sensor for vesicle fusion and vesicle pool replenishment at auditory hair cell ribbon synapses
- Institut Pasteur, France
- UMRS 1120, Institut National de la Santé et de la Recherche Médicale, France
- Sorbonne Universités, UPMC Université Paris 06, Complexité du Vivant, France
- Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina
- Yale University School of Medicine, United States
- Université de Bordeaux, France
- University Paris Descartes, Sorbonne Paris Cité, France
- Ultrapole, Institut Pasteur, France
- Centre National de la Recherche Scientifique, France
- Texas Tech University Health Sciences Center, United States
- Université Clermont Auvergne, France
- UMR 1107, Institut National de la Santé et de la Recherche Médicale, France
- Centre Jean Perrin, France
- Institute of Neurology, University College London, United Kingdom
- Institut de la Vision, France
- Collège de France, France
Figures
Figure 1
Homology model of the otoferlin C2C domain.
(A) Predicted domain structure of the otoferlin protein. Otoferlin is a transmembrane vesicular protein (1997 amino acids in the mouse) consisting of six C2 domains (C2A-F), one Fer1 domain, and one …
Figure 2 with 1 supplement
Smaller amplitude of ABR wave-I in Otof C2C/C2C mice.
(A) ABR thresholds of one-month-old Otof C2C/+ (black) and Otof C2C/C2C (blue) mice, for pure tone frequencies between 5 kHz and 32 kHz. (B) Mean ABR trace recorded in Otof C2C/+ (black line, n = 7 …
Figure 2—figure supplement 1
Progressive hearing loss in Otof C2C/C2C mice after one month of age.
(A) ABR thresholds in dB SPL (sound pressure level), for five different pure tone frequencies (5, 10, 15, 20, and 32 kHz), in Otof +/+ (brown) and Otof C2C/+ (black) mice at the age of one month. (B)…
Figure 3 with 2 supplements
Normal expression and subcellular distribution of otoferlin in Otof C2C/C2C IHCs.
(A) Confocal microscopy images of IHCs from whole-mount preparations of the organ of Corti from P15 Otof C2C/+ and Otof C2C/C2C mice triple-labeled for otoferlin (blue), ribeye (red), and …
Figure 3—figure supplement 1
Normal number of ribbon synapses in Otof C2C/C2C IHCs.
Confocal microscopy images of IHCs from whole-mount preparations of the P15 organ of Corti, triple-labeled for the synaptic ribbon protein ribeye (red), presynaptic Cav1.3 L-type Ca2+ channels …
Figure 3—figure supplement 2
Normal colocalization of otoferlin and Vglut3 in Otof C2C/C2C IHCs.
Analysis of two representative STED images of individual IHCs from whole-mount preparations of organs of Corti from a P15 Otof C2C/+ mouse (left panels) and a P15 Otof C2C/C2C mouse (right panels) …
Figure 4 with 2 supplements
Ultrastructural analysis of the different vesicle pools in Otof C2C/C2C IHC ribbon synapses.
(A) Representative transmission electron micrographs of ribbon synapses from an Otof C2C/+ IHC (upper panels) and an Otof C2C/C2C IHC (lower panels). Side views of the electron tomographic …
Figure 4—video 1
Example of a ribbon synapse from an Otof C2C/+ mouse reconstructed by electron tomography
https://doi.org/10.7554/eLife.31013.009
Figure 4—video 2
Example of a ribbon synapse from an Otof C2C/C2C mouse reconstructed by electron tomography
https://doi.org/10.7554/eLife.31013.010
Figure 5 with 1 supplement
Ca2+-dependence of the RRP vesicle fusion defect in Otof C2C/C2C IHCs.
(A1) Protocol used to depolarize IHCs from −95 mV to potentials between −65 to +35 mV (top). Examples of Ca2+ currents (ICa) (middle) and corresponding Cm traces (bottom) for P15-P18 Otof C2C/+ and O…
Figure 5—figure supplement 1
Ca2+-dependence of RRP vesicle fusion is normal in Otof C2C/+ IHCs.
(A) Example of Ca2+ current (ICa) and corresponding Cm, membrane conductance (Gm), and series conductance (Gs) traces recorded in a P15 Otof C2C/+ IHC after 20 ms of depolarization to −10 mV (same …
Figure 6
Abnormal kinetics of exocytosis evoked by intracellular Ca2+ uncaging in Otof C2C/C2C IHCs.
(A) Left: Mean ΔCm response curves in P14-P16 Otof C2C/+ and Otof C2C/C2C IHCs after a single UV flash (purple arrowhead) uncaging intracellular Ca2+. Right: Bar graph showing mean and individual …
Figure 7
Reduced Ca2+-sensitivity of exocytosis in Otof C2C/C2C IHCs.
(A) Mean ΔCm response curves (top) and normalized exocytosis rates (dCm/dt) (bottom), with mean intracellular fluorescent Ca2 signals of OGB-5N (green traces) recorded simultaneously after Ca2+ …
Figure 8 with 1 supplement
Reduced sustained release in Otof C2C/C2C IHCs.
(A) Paired-pulse stimulation protocol, showing two consecutive 20 ms depolarizations from a holding potential of −95 mV to −10 mV (top), and example Cm traces from Otof C2C/+ and Otof C2C/C2C IHCs …
Figure 8—figure supplement 1
Normal Ca2+ entry, assessed by high-speed two-dimensional Ca2+ imaging, in Otof C2C/C2C IHCs.
(A0) Left: transverse section of an IHC from a P15 wild-type mouse transduced on P3 with an AAV2/8 viral vector expressing mCherry-conjugated ribeye-A to render the synaptic ribbons visible (red …
Figure 9 with 1 supplement
Normal endocytosis, assessed by prolonged Cm measurements, in Otof C2C/C2C IHCs.
(A) Left: Examples of Cm traces, recorded over a period of 15 s, in response to a 100 ms depolarization to −10 mV from a holding potential of −95 mV, in P14-P17 Otof C2C/+ and Otof C2C/C2C IHCs. …
Figure 9—figure supplement 1
Example of a protocol used to probe endocytosis in an Otof C2C/+ IHC and corresponding recorded traces.
Example of Ca2+ current (ICa) and corresponding Cm, Gm, and Gs traces recorded in an Otof C2C/+ P15 IHC over a period of 15 s, in response to a 100 ms long depolarization to −10 mV from a holding …
Figure 10
Lower EPSC rates in Otof C2C/C2C IHCs.
(A) Superimposed example EPSC traces (IEPSC) recorded at Otof +/+ and Otof C2C/C2C IHC afferent boutons on P8-P11, after extracellular application of 25 mM KCl (top), and histograms of EPSC …
Figure 11
A mass action model of synaptic release reproduces Ca2+ sensitivity defects in Otof C2C/C2C IHCs.
(A) Diagram of the synaptic vesicle pools described by the model: the RRP (red), the recycling pool (violet), the reserve pool (green), and the distant pool (yellow). The process of synaptic …
Author response image 1
SDS-PAGE analysis showing the poor quality of the Otoferlin C2C proteins purified.
Tables
Table 1
Values of the parameters used in the mass action model of synaptic release
https://doi.org/10.7554/eLife.31013.021| Parameter | Otof C2C/+ | Otof C2C/C2C |
|---|---|---|
| VM1 | 616 ± 105 vesicles | 570 ± 81 vesicles |
| VM2 | 1 602 ± 248 vesicles | 1 877 ± 487 vesicles |
| VM3 | 16772 ± 8792 vesicles | 11 312 ± 3835 vesicles |
| VM4 | 11 491 ± 2015 vesicles | 14 171 ± 3562 vesicles |
| k1 | 1062 ± 583 C−1.s−1 | 349 ± 169 C−1.s−1 |
| k2 | 6.4 ± 3.9 C−1.s−1 | 0.8 ± 0.4 C−1.s−1 |
| k3 | 4.5 ± 9.9 C−1.s−1 | 0.95 ± 0.86 C−1.s−1 |
| k4 | (6.6 ± 5.2) x 10−3 C−1.s−1 | (8.5 ± 3.6) x 10−3 C−1.s−1 |
| [Ca2+]1 | (1.7 ± 0.8) x 10−14 C | (2.1 ± 0.1) x 10−14 C |
| [Ca2+]2 | (8.3 ± 5.9) x 10−13 C | (6.7 ± 2.7) x 10−13 C |
| [Ca2+]3 | (8. 7 ± 2. 6) x 10−12 C | (10.2 ± 1.4) x 10−12 C |
| [Ca2+]4 | (9.7 ± 3.4) x 10−12 C | (9.7 ± 1.6) x 10−12 C |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.31013.022
