Vesicle sub‐pool organization at inner hair cell ribbon synapses

Abstract

The afferent inner hair cell synapse harbors the synaptic ribbon, which ensures a constant vesicle supply. Synaptic vesicles (SVs) are arranged in morphologically discernable pools, linked via filaments to the ribbon or the presynaptic membrane. We propose that filaments play a major role in SV resupply and exocytosis at the ribbon. Using advanced electron microscopy, we demonstrate that SVs are organized in sub-pools defined by the filament number per vesicle and its connections. Upon stimulation, SVs increasingly linked to other vesicles and to the ribbon, whereas single-tethered SVs dominated at the membrane. Mutant mice for the hair cell protein otoferlin (, ) are profoundly deaf with reduced sustained release, serving as a model to investigate the SV replenishment at IHCs. Upon stimulation, multiple-tethered and docked vesicles (rarely observed in wild-type) accumulated at active zones due to an impairment downstream of docking. Conclusively, vesicles are organized in sub-pools at ribbon-type active zones by filaments to support vesicle supply, transport, and finally release.

Functions of Vertebrate Ferlins

“…Following the fusion of SVs with PM, the clearance of the active zone including the efficient transport of the material away from the places of exocytosis and endocytosis per se are dependent on otoferlin and, likely, on its interaction with endocytic adaptor proteins AP-2µ and endophilin-A1, motor protein myosin VI, and GTPase Rab8b [149][150][151][152][153][154][155] (reviewed in [3]). It was shown, that SVs on average have larger diameters in a temperature-sensitive (I515T) and pachanga mutants, indicating a defect in the reformation/maturation of SVs and thus, a role of otoferlin in this process [156,157]. The functions of otoferlin in the IHCs could be regulated by phosphorylation, e.g., changing Ca 2+ -sensitivity of C2C and C2F domains [158].…”

Balancing presynaptic release and endocytic membrane retrieval at hair cell ribbon synapses

“…Morphologically, AZ‐tethered SVs are usually classified into two main populations, the membrane‐proximal and the ribbon‐associated pool of SVs . Functionally, patch‐clamp recordings of depolarization‐evoked changes in IHC membrane capacitance similarly suggest multiple pools of SVs.…”

Nanomachinery Organizing Release at Neuronal and Ribbon Synapses

“…Application of 3D structural methods like serial-section 3D reconstruction, electron tomography (ET), conical and scanning transmission EM (STEM) tomography after conventional aldehyde fixation (CAF), have made it possible to analyze the vesicle pool dynamics in a broad range of neuronal but also ribbon synapses. For example ribbon synapses of frog saccular hair cells [114,115,116,117], murine IHCs [16,81,89,90,98,118,119,120,121,122,123,124], retinal bipolar cells [125], and photoreceptors [126,127,128] have been studied extensively.…”

“…Moreover, applying different methods for EM sample preparation such as freeze-etching, CAF or rapid freezing, exposed that SVs are associated with the AZ via delicate filamentous structures that appear at vertebrate NMJ, central neuronal and ribbon-type synapses [16,19,38,90,116,118,120,121,122,125,129,130,131,132,133,134,135,136,137,138,139] (Figure 1C,D). Notably, we will use ‘filaments’ as a generic term in this review for these structures.…”

“…Notably, we will use ‘filaments’ as a generic term in this review for these structures. Two categories of presynaptic filaments are described across synapses and species: (i) Filaments that link SVs within the presynaptic terminal lumen to form a network, coined as interconnectors from now on (Figure 2A’,A’’) and (ii) filaments, commonly termed as tethers, which connect SVs to the AZ-membrane (Figure 2C–E) [16,90,116,118,120,121,122,125,129,130,131,132,133,134,135,136]. Notably, we will use ‘filaments’ as a generic term.…”