Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses
- University of Göttingen, Germany
- Max Planck Institute for Multidisciplinary Sciences, Germany
- University Medical Center Göttingen, Germany
- South Westphalia University of Applied Sciences, Germany
- University of Göttingen Medical Center, Germany
Abstract
Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and transmitter release. Ultrastructurally, the number of docked SVs increased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.
Data availability
All research materials and biological reagents used in this paper are reported in the Materials and Method section. The custom routines and scripts used in the manuscript are provided as Source Codes: Source Code 1: IMARIS custom plug-ins for the analysis of Figure 1D; Source code 2: Igor Pro custom-written analysis (OptoEPSCs) of light-evoked EPSCs related to Figure 3C-F; Source code 3: MATLAB scripts (HPMacquire) for the computer interface to control the light pulse for Opto-HPF. Related to Figure 4A; Source code 4: MATLAB script (Intensityprofilecalculator) for the analysis of the irradiance in Figure 4E; Source code 5: MATLAB scripts (HPManalyse) for the alignment of the data obtained from the Opto-HPF sensors. Related to Figure 5C. The raw data files, including the numerical data associated with the figures, are available on the Open Science Framework DOI 10.17605/OSF.IO/WFJVE .
Article and author information
Author details
Lina María Jaime Tobón
Funding
Deutsche Forschungsgemeinschaft (CRC 889,Project A02)
- Tobias Moser
Multiscale Bioimaging is a Cluster of Excellence of the University of Göttingen, Germany (EXC 2067/1- 390729940)
- Tobias Moser
MPI-NAT (Erwin Neher Fellowship)
- Lina María Jaime Tobón
Deutsche Forschungsgemeinschaft (CRC 889,Project A07)
- Carolin Wichmann
Deutsche Forschungsgemeinschaft (CRC 1286,Project A04)
- Carolin Wichmann
Deutsche Forschungsgemeinschaft (CRC 1286,Project B05)
- Tobias Moser
Deutsche Forschungsgemeinschaft (CRC 1286,Project Z04)
- Felipe Opazo
Leibniz Program (Leibniz Prize)
- Tobias Moser
Niedersächsisches Ministerium für Wissenschaft und Kultur (Niedersächsisches Vorab)
- Tobias Moser
Erasmus Mundus (Neurasmus Scholarship)
- Lina María Jaime Tobón
Fondation Pour l'Audition (FPA RD-2020-10)
- Tobias Moser
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal handling and all experimental procedures were in accordance with the national animal care guidelines issued by the animal welfare committees of the University of Göttingen and the Animal Welfare Office of the State of Lower Saxony (AZ 509.42502/01-27.03).
Copyright
© 2022, Chakrabarti et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses
eLife 11:e79494.
https://doi.org/10.7554/eLife.79494
Further reading
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- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
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- Cell Biology
Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
