How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release.
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ABSTRACT:
SUBMITTER: Maxeiner S
PROVIDER: S-EPMC4868958 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Munc119 (also denoted as RG4) is a mammalian ortholog of the Caenorhabditis elegans protein unc119 and is essential for vision and synaptic transmission at photoreceptor ribbon synapses by unknown molecular mechanisms. Munc119/RG4 is related to the prenyl-binding protein PrBP/delta and expressed at high levels in photoreceptor ribbon synapses. Synaptic ribbons are presynaptic specializations in the active zone of these tonically active synapses and contain RIBEYE as a unique and major component. In the present study, we identified Munc119 as a RIBEYE-interacting protein at photoreceptor ribbon synapses using five independent approaches. The PrBP/delta homology domain of Munc119 is essential for the interaction with the NADH binding region of RIBEYE(B) domain. But RIBEYE-Munc119 interaction does not depend on NADH binding. A RIBEYE point mutant (RE(B)E844Q) that no longer interacted with Munc119 still bound NADH, arguing that binding of Munc119 and NADH to RIBEYE are independent from each other. Our data indicate that Munc119 is a synaptic ribbon-associated component. We show that Munc119 can be recruited to synaptic ribbons via its interaction with RIBEYE. Our data suggest that the RIBEYE-Munc119 interaction is essential for synaptic transmission at the photoreceptor ribbon synapse.
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Project description:Synaptic ribbons are structures made largely of the protein Ribeye that hold synaptic vesicles near release sites in non-spiking cells in some sensory systems. Here, we introduce frameshift mutations in the two zebrafish genes encoding for Ribeye and thus remove Ribeye protein from neuromast hair cells. Despite Ribeye depletion, vesicles collect around ribbon-like structures that lack electron density, which we term "ghost ribbons." Ghost ribbons are smaller in size but possess a similar number of smaller vesicles and are poorly localized to synapses and calcium channels. These hair cells exhibit enhanced exocytosis, as measured by capacitance, and recordings from afferent neurons post-synaptic to hair cells show no significant difference in spike rates. Our results suggest that Ribeye makes up most of the synaptic ribbon density in neuromast hair cells and is necessary for proper localization of calcium channels and synaptic ribbons.
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Project description: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.
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Project description:Active zones at chemical synapses are highly specialized sites for the regulated release of neurotransmitters. Despite a high degree of active zone protein conservation in vertebrates, every type of chemical synapse expresses a given set of protein isoforms and splice variants adapted to the demands on neurotransmitter release. So far, we know little about how specific active zone proteins contribute to the structural and functional diversity of active zones. In this study, we explored the nanodomain organization of ribbon-type active zones by addressing the significance of Piccolino, the ribbon synapse-specific splice variant of Piccolo, for shaping the ribbon structure. We followed up on previous results, which indicated that rod photoreceptor synaptic ribbons lose their structural integrity in a knockdown of Piccolino. Here, we demonstrate an interaction between Piccolino and the major ribbon component RIBEYE that supports plate-shaped synaptic ribbons in retinal neurons. In a detailed ultrastructural analysis of three different types of retinal ribbon synapses in Piccolo/Piccolino-deficient male and female rats, we show that the absence of Piccolino destabilizes the superstructure of plate-shaped synaptic ribbons, although with variable manifestation in the cell types examined. Our analysis illustrates how the expression of a specific active zone protein splice variant (e.g., Piccolino) contributes to structural diversity of vertebrate active zones.SIGNIFICANCE STATEMENT Retinal ribbon synapses are a specialized type of chemical synapse adapted for the regulated fast and tonic release of neurotransmitter. The hallmark of retinal ribbon synapses is the plate-shaped synaptic ribbon, which extends from the release site into the terminals' cytoplasm and tethers hundreds of synaptic vesicles. Here, we show that Piccolino, the synaptic ribbon specific splice variant of Piccolo, interacts with RIBEYE, the main component of synaptic ribbons. This interaction occurs via several PxDLS-like motifs located at the C terminus of Piccolino, which can connect multiple RIBEYE molecules. Loss of Piccolino disrupts the characteristic plate-shaped structure of synaptic ribbons, indicating a role of Piccolino in synaptic ribbon assembly.
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Project description:Non-spiking sensory hair cells of the auditory and vestibular systems encode a dynamic range of graded signals with high fidelity by vesicle exocytosis at ribbon synapses. Ribeye, the most abundant protein in the synaptic ribbon, is composed of a unique A domain specific for ribbons and a B-domain nearly identical to the transcriptional corepressor CtBP2. CTBP2 and the B-domain of Ribeye contain a surface cleft that binds to proteins harboring a PXDLS/T peptide motif. Little is known about the importance of this binding site in synaptic function. Piccolo has a well-conserved PVDLT motif and we find that overexpressed Ribeye exhibits striking co-localization with Piccolo in INS-cells, while two separate mutants containing mutations in PXDLS/T-binding region, fail to co-localize with Piccolo. Similarly, co-transfected Ribeye and a piccolo fragment containing the PVDLT region co-localize in HEK cells. Expression of wild-type Ribeye-YFP in zebrafish neuromast hair cells returns electron densities to ribbon structures and mostly rescued normal synaptic transmission and morphological phenotypes in a mutant zebrafish lacking most Ribeye. By contrast, Ribeye-YFP harboring a mutation in the PXDLS/T-binding cleft resulted in ectopic electron dense aggregates that did not collect vesicles and the persistence of ribbons lacking electron densities. Furthermore, overexpression failed to return capacitance responses to normal levels. These results point toward a role for the PXDLS/T-binding cleft in the recruitment of Ribeye to ribbons and in normal synaptic function.
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Project description:Ribbon synapses of tonically releasing sensory neurons must provide a large pool of releasable vesicles for sustained release, while minimizing spontaneous release in the absence of stimulation. Complexins are presynaptic proteins that may accomplish this dual task at conventional synapses by interacting with the molecular machinery of synaptic vesicle fusion at the active zone to retard spontaneous vesicle exocytosis yet facilitate release evoked by depolarization. However, ribbon synapses of photoreceptor cells and bipolar neurons in the retina express distinct complexin subtypes, perhaps reflecting the special requirements of these synapses for tonic release. To investigate the role of ribbon-specific complexins in transmitter release, we combined presynaptic voltage clamp, fluorescence imaging, electron microscopy, and behavioral assays of photoreceptive function in zebrafish. Acute interference with complexin function using a peptide derived from the SNARE-binding domain increased spontaneous synaptic vesicle fusion at ribbon synapses of retinal bipolar neurons without affecting release triggered by depolarization. Knockdown of complexin by injection of an antisense morpholino into zebrafish embryos prevented photoreceptor-driven migration of pigment in skin melanophores and caused the pigment distribution to remain in the dark-adapted state even when embryos were exposed to light. This suggests that loss of complexin function elevated spontaneous release in illuminated photoreceptors sufficiently to mimic the higher release rate normally associated with darkness, thus interfering with visual signaling. We conclude that visual system-specific complexins are required for proper illumination-dependent modulation of the rate of neurotransmitter release at visual system ribbon synapses.
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Project description:In the mammalian retina, bipolar cells and ganglion cells which stratify in sublamina a of the inner plexiform layer (IPL) show OFF responses to light stimuli while those that stratify in sublamina b show ON responses. This functional relationship between anatomy and physiology is a key principle of retinal organization. However, there are at least three types of retinal neurons, including intrinsically photosensitive retinal ganglion cells (ipRGCs) and dopaminergic amacrine cells, which violate this principle. These cell types have light-driven ON responses, but their dendrites mainly stratify in sublamina a of the IPL, the OFF sublayer. Recent anatomical studies suggested that certain ON cone bipolar cells make axonal or ectopic synapses as they descend through sublamina a, thus providing ON input to cells which stratify in the OFF sublayer. Using immunoelectron microscopy with 3-dimensional reconstruction, we have identified axonal synapses of ON cone bipolar cells in the rabbit retina. Ten calbindin ON cone bipolar axons made en passant ribbon synapses onto amacrine or ganglion dendrites in sublamina a of the IPL. Compared to the ribbon synapses made by bipolar terminals, these axonal ribbon synapses were characterized by a broad postsynaptic element that appeared as a monad and by the presence of multiple short synaptic ribbons. These findings confirm that certain ON cone bipolar cells can provide ON input to amacrine and ganglion cells whose dendrites stratify in the OFF sublayer via axonal synapses. The monadic synapse with multiple ribbons may be a diagnostic feature of the ON cone bipolar axonal synapse in sublamina a. The presence of multiple ribbons and a broad postsynaptic density suggest these structures may be very efficient synapses. We also identified axonal inputs to ipRGCs with the architecture described above.
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Project description:Synaptic ribbons are presynaptic structures formed by the self-association of RIBEYE-the main structural component of ribbon synapses. RIBEYE consists of two domains: a unique N-terminal A-domain and a C-terminal B-domain that is identical to the transcription co-repressor C-terminal binding protein 2 (CtBP2). Previous studies in cell lines have shown that RIBEYE A-domain alone is sufficient to form ribbon-like aggregates and that both A- and B- domains form homo-and heterotypic interactions. As these interactions are likely the basis for synaptic-ribbon assembly and structural plasticity, we wanted to examine how zebrafish Ribeye A- and B- domains interact with synaptic ribbons in vivo. To that end, we characterized the localization of exogenously expressed Ribeye A- and B- domains and the closely related protein, CtBP1, in the hair cells of transgenic zebrafish larvae. Unexpectedly, exogenously expressed Ribeye A-domain showed variable patterns of localization in hair cells; one zebrafish paralog of A-domain failed to self-associate or localize to synaptic ribbons, while the other self-assembled but sometimes failed to localize to synaptic ribbons. By contrast, Ribeye B-domain/CtBP2 was robustly localized to synaptic ribbons. Moreover, both exogenously expressed B-domain/CtBP2 and CtBP1 were preferentially localized to the basal end of ribbons adjacent to the postsynaptic density. Overexpression of B-domain/CtBP2 also appeared to affect synaptic-ribbon composition; endogenous levels of ribbon-localized Ribeye were significantly reduced as hair cells matured in B-domain/CtBP2 transgenic larvae compared to wild-type. These results reveal how exogenously expressed Ribeye domains interact with synaptic ribbons, and suggest a potential organization of elements within the ribbon body.
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