RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons

Shankhwar, Soni; Schwarz, Karin; Katiyar, Rashmi; Jung, Martin; Maxeiner, Stephan; Südhof, Thomas C.; Schmitz, Frank

doi:10.3389/fnmol.2022.838311

Cited by 6 publications

(22 citation statements)

References 68 publications

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“…In Ribeye KI mice, the enzymatic B-domain was replaced with GCamP3 and flanked with loxP sites for Cre-dependent deletion. As reported recently [ 34 ], we discovered that substituting GCamP3 for the RIBEYE B-domain eliminated ribbons throughout the retina. From OMR assays, we found that eliminating retinal ribbons reduced temporal resolution and contrast sensitivity.…”

Section: Introductionsupporting

confidence: 75%

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment

Mesnard

Barta

Sladek

et al. 2022

IJMS

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Glutamate release from rod and cone photoreceptor cells involves presynaptic ribbons composed largely of the protein RIBEYE. To examine roles of ribbons in rods and cones, we studied mice in which GCamP3 replaced the B-domain of RIBEYE. We discovered that ribbons were absent from rods and cones of both knock-in mice possessing GCamP3 and conditional RIBEYE knockout mice. The mice lacking ribbons showed reduced temporal resolution and contrast sensitivity assessed with optomotor reflexes. ERG recordings showed 50% reduction in scotopic and photopic b-waves. The readily releasable pool (RRP) of vesicles in rods and cones measured using glutamate transporter anion currents (IA(glu)) was also halved. We also studied the release from cones by stimulating them optogenetically with ChannelRhodopsin2 (ChR2) while recording postsynaptic currents in horizontal cells. Recovery of the release from paired pulse depression was twofold slower in the rods and cones lacking ribbons. The release from rods at −40 mV in darkness involves regularly spaced multivesicular fusion events. While the regular pattern of release remained in the rods lacking ribbons, the number of vesicles comprising each multivesicular event was halved. Our results support conclusions that synaptic ribbons in rods and cones expand the RRP, speed up vesicle replenishment, and augment some forms of multivesicular release. Slower replenishment and a smaller RRP in photoreceptors lacking ribbons may contribute to diminished temporal frequency responses and weaker contrast sensitivity.

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Section: Introductionsupporting

confidence: 75%

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment

Mesnard

Barta

Sladek

et al. 2022

IJMS

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“…Secondary antibody dilutions and references are given in Table 2. Following several washes with PBS, sections were embedded in NPG antifade, as previously described [12,17,38,75,76]. Immunolabeled sections were analyzed by confocal microscopy with an A1R confocal microscope, see below.…”

Section: Immunohistochemistry Of 10 µM Thick Cryostat Sections Of Mou...mentioning

confidence: 99%

“…The RIBEYE protein is the major component of synaptic ribbons [12][13][14][15][16][17]. RIBEYE consists of two main domains, an amino-terminal A domain that is unique to RIBEYE, and a carboxyterminal B domain that is identical, except for the first 20 amino-terminal amino acids, with the transcriptional co-repressor CtBP2 [12].…”

Section: Introductionmentioning

confidence: 99%

A Novel Cre Recombinase Mouse Strain for Cell-Specific Deletion of Floxed Genes in Ribbon Synapse-Forming Retinal Neurons

Suiwal,

Wartenberg,

Boehm

et al. 2024

IJMS

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We generated a novel Cre mouse strain for cell-specific deletion of floxed genes in ribbon synapse-forming retinal neurons. Previous studies have shown that the RIBEYE promotor targets the expression of recombinant proteins such as fluorescently tagged RIBEYE to photoreceptors and retinal bipolar cells and generates fluorescent synaptic ribbons in situ in these neurons. Here, we used the same promotor to generate a novel transgenic mouse strain in which the RIBEYE promotor controls the expression of a Cre-ER(T2) recombinase (RIBEYE-Cre). To visualize Cre expression, the RIBEYE-Cre animals were crossed with ROSA26 tau-GFP (R26-τGFP) reporter mice. In the resulting RIBEYE-Cre/R26 τGFP animals, Cre-mediated removal of a transcriptional STOP cassette results in the expression of green fluorescent tau protein (tau-GFP) that binds to cellular microtubules. We detected robust tau-GFP expression in retinal bipolar cells. Surprisingly, we did not find fluorescent tau-GFP expression in mouse photoreceptors. The lack of tau-GFP reporter protein in these cells could be based on the previously reported absence of tau protein in mouse photoreceptors which could lead to the degradation of the recombinant tau protein. Consistent with this, we detected Cre and tau-GFP mRNA in mouse photoreceptor slices by RT-PCR. The transgenic RIBEYE-Cre mouse strain provides a new tool to study the deletion of floxed genes in ribbon synapse-forming neurons of the retina and will also allow for analyzing gene deletions that are lethal if globally deleted in neurons.

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“…Visualization of cone synapses with PNA-Alexa568 and immunolabeling of PNAstained cone terminals was performed exactly as previously described [83]. For the immunolabeling, the rabbit polyclonal Arl13b (Proteintech) was used in a 1:50 dilution in blocking solution, containing 0.5% BSA in PBS.…”

Section: Immunostaining Of Pna-stained Cone Synapses On Cryostat Sect...mentioning

confidence: 99%

“…For the immunolabeling, the rabbit polyclonal Arl13b (Proteintech) was used in a 1:50 dilution in blocking solution, containing 0.5% BSA in PBS. PNA-Alexa568 was diluted 1:200 in blocking buffer, exactly as described [83].…”

Section: Immunostaining Of Pna-stained Cone Synapses On Cryostat Sect...mentioning

confidence: 99%

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses

Suiwal

Dembla

Schwarz

et al. 2022

IJMS

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The Unc119 protein mediates transport of myristoylated proteins to the photoreceptor outer segment, a specialized primary cilium. This transport activity is regulated by the GTPase Arl3 as well as by Arl13b and Rp2 that control Arl3 activation/inactivation. Interestingly, Unc119 is also enriched in photoreceptor synapses and can bind to RIBEYE, the main component of synaptic ribbons. In the present study, we analyzed whether the known regulatory proteins, that control the Unc119-dependent myristoylated protein transport at the primary cilium, are also present at the photoreceptor synaptic ribbon complex by using high-resolution immunofluorescence and immunogold electron microscopy. We found Arl3 and Arl13b to be enriched at the synaptic ribbon whereas Rp2 was predominantly found on vesicles distributed within the entire terminal. These findings indicate that the synaptic ribbon could be involved in the discharge of Unc119-bound lipid-modified proteins. In agreement with this hypothesis, we found Nphp3 (Nephrocystin-3), a myristoylated, Unc119-dependent cargo protein enriched at the basal portion of the ribbon in close vicinity to the active zone. Mutations in Nphp3 are known to be associated with Senior–Løken Syndrome 3 (SLS3). Visual impairment and blindness in SLS3 might thus not only result from ciliary dysfunctions but also from malfunctions of the photoreceptor synapse.

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RIBEYE B-Domain Is Essential for RIBEYE A-Domain Stability and Assembly of Synaptic Ribbons

Cited by 6 publications

References 68 publications

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment

Eliminating Synaptic Ribbons from Rods and Cones Halves the Releasable Vesicle Pool and Slows Down Replenishment

A Novel Cre Recombinase Mouse Strain for Cell-Specific Deletion of Floxed Genes in Ribbon Synapse-Forming Retinal Neurons

Ciliary Proteins Repurposed by the Synaptic Ribbon: Trafficking Myristoylated Proteins at Rod Photoreceptor Synapses

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