Abstract
Ribbons are presynaptic structures that mediate synaptic vesicle release in some sensory cells of the auditory and visual systems. Although composed predominately of the protein Ribeye, very little is known about the structural dynamics of ribbons. Here we describe the in vivo mobility and turnover of Ribeye at hair cell ribbon synapses by monitoring fluorescence recovery after photobleaching (FRAP) in transgenic zebrafish with GFP-tagged Ribeye. We show that Ribeye can exchange between halves of a ribbon within ~1 minute in a manner that is consistent with a simple diffusion mechanism. In contrast, exchange of Ribeye between other ribbons via the cell’s cytoplasm takes several hours.
Highlights
At the presynaptic active zone, ribbons tether and organize a pool of synaptic vesicles adjacent to clusters of L-type calcium channels
We investigate in vivo real-time structural dynamics at ribbons in zebrafish lateral line hair cells
A major challenge in imaging ribbon synapses arises from the ribbon’s size being comparable to or smaller than the diffraction-limited resolution of light microscopy (~250 nm). This limitation makes it challenging to resolve the dynamics within ribbons in live-imaging preparations. To bypass this limitation, we exploit the larger size of ribbons (~1 μm) in neuromast hair cells of the lateral line system of larval (4–6 day old) transgenic ribeye b-EGFP zebrafish[25]
Summary
At the presynaptic active zone, ribbons tether and organize a pool of synaptic vesicles adjacent to clusters of L-type calcium channels. Based on the close association between ribbons and vesicles, the ribbon may serve as a synaptic vesicle “conveyor belt” or as a scaffold for compound fusion of vesicles (“safety belt”)[7, 8], and may perform key steps in preparing synaptic vesicles for fusion (“priming”)[9, 10]. In addition to these proposed roles in organizing and preparing vesicles for fusion, evidence of correlations between ribbon ultrastructure and synaptic output properties has mounted. The real-time dynamics of the ribbon’s ultrastructure, and its interactions with tethered synaptic vesicles, remain largely unknown. Our findings suggest a relatively slow synaptic Ribeye turnover rate for whole ribbons (~6–7 hours), but an internal mobility of Ribeye within these large spherical ribbons on timescales of a few minutes
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