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Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex.

tom Dieck S, Altrock WD, Kessels MM, Qualmann B, Regus H, Brauner D, Fejtová A, Bracko O, Gundelfinger ED, Brandstätter JH - J. Cell Biol. (2005)

Bottom Line: Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1.A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex.Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

ABSTRACT
The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

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CtBP1, a RIBEYE homologue, is expressed at ribbon and conventional synapses. (A–C) Confocal laser-scanning micrographs of a vertical section through mouse retina double labeled for CtBP1 (A) and Piccolo (B), which marks ribbon and conventional synapses in the retina. As seen in the merge of the two stainings (C), CtBP1 and Piccolo immunoreactivities completely colocalize at the synapses in the outer plexiform layer (OPL) and inner plexiform layer (IPL) of the retina. (D and E) Electron micrographs of a photoreceptor (D) and an amacrine cell synapse (E) immunogold labeled for CtBP1. At the ribbon synapse the gold particles for CtBP1 decorate the ribbon (arrowheads), at the amacrine cell synapse they are located some distance from the active zone (arrowheads) at the edge of the electron-dense CAZ material. (F) Western blots of retina homogenate probed with antibodies against CtBP1 and the RIBEYE A- and CtBP2/RIBEYE B-domain. The antibody against CtBP1 recognizes the 50-kD CtBP1 protein and does not cross-react with RIBEYE. (G–I) Micrographs of cultured hippocampal neurons double labeled for CtBP1 (G) and Piccolo (H). In hippocampal neurons, like in retinal neurons, a fraction of CtBP1 immunoreactivity is localized at synapses where it colocalizes with Piccolo as seen in the merge of the two stainings (I). (J and K) CtBP2 (J) is not present at hippocampal synapses labeled with Piccolo (K). (L) Immunoblots showing that CtBP1 can be coimmunoprecipitated from brain synaptosomes with a monoclonal anti-Bassoon antibody. The boxes in G–K mark the regions that are shown at higher magnification. INL, inner nuclear layer; GCL, ganglion cell layer; Ext., brain synaptosomal extract; IP, immunoprecipitate; S, supernatant. Bars: 20 μm (A–C), 0.2 μm (D and E), and 10 μm (G–K).
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fig7: CtBP1, a RIBEYE homologue, is expressed at ribbon and conventional synapses. (A–C) Confocal laser-scanning micrographs of a vertical section through mouse retina double labeled for CtBP1 (A) and Piccolo (B), which marks ribbon and conventional synapses in the retina. As seen in the merge of the two stainings (C), CtBP1 and Piccolo immunoreactivities completely colocalize at the synapses in the outer plexiform layer (OPL) and inner plexiform layer (IPL) of the retina. (D and E) Electron micrographs of a photoreceptor (D) and an amacrine cell synapse (E) immunogold labeled for CtBP1. At the ribbon synapse the gold particles for CtBP1 decorate the ribbon (arrowheads), at the amacrine cell synapse they are located some distance from the active zone (arrowheads) at the edge of the electron-dense CAZ material. (F) Western blots of retina homogenate probed with antibodies against CtBP1 and the RIBEYE A- and CtBP2/RIBEYE B-domain. The antibody against CtBP1 recognizes the 50-kD CtBP1 protein and does not cross-react with RIBEYE. (G–I) Micrographs of cultured hippocampal neurons double labeled for CtBP1 (G) and Piccolo (H). In hippocampal neurons, like in retinal neurons, a fraction of CtBP1 immunoreactivity is localized at synapses where it colocalizes with Piccolo as seen in the merge of the two stainings (I). (J and K) CtBP2 (J) is not present at hippocampal synapses labeled with Piccolo (K). (L) Immunoblots showing that CtBP1 can be coimmunoprecipitated from brain synaptosomes with a monoclonal anti-Bassoon antibody. The boxes in G–K mark the regions that are shown at higher magnification. INL, inner nuclear layer; GCL, ganglion cell layer; Ext., brain synaptosomal extract; IP, immunoprecipitate; S, supernatant. Bars: 20 μm (A–C), 0.2 μm (D and E), and 10 μm (G–K).

Mentions: As the interaction of Bassoon was originally observed with CtBP1, a homologue of the RIBEYE B-domain, we monitored the distribution of this protein in vertical sections of wild-type mouse retina using a monoclonal mouse anti-CtBP1 antibody. Interestingly, in addition to the expected nuclear staining, strong CtBP1 labeling was observed in the two synaptic layers of the retina (Fig. 7 A). Double-labeling experiments with an antiserum against the CAZ protein Piccolo, which is present at ribbon and conventional synapses in the retina (Fig. 7 B; Dick et al., 2001), show a complete colocalization of CtBP1 and Piccolo in the two plexiform layers of the retina (Fig. 7 C). Postembedding immunogold EM further proved the presence of CtBP1 at ribbon and conventional synapses in the retina (Fig. 7, D and E). Immunoblot analysis revealed no cross-reactivity of the CtBP1 antibody with RIBEYE (Fig. 7 F), demonstrating that indeed the 50-kD CtBP1 protein is present synaptically. In the Bassoon mutant photoreceptor terminals with their free-floating ribbons, CtBP1 stays associated with RIBEYE (unpublished data), adding CtBP1 to the list of potential interactors with the photoreceptor ribbon.


Molecular dissection of the photoreceptor ribbon synapse: physical interaction of Bassoon and RIBEYE is essential for the assembly of the ribbon complex.

tom Dieck S, Altrock WD, Kessels MM, Qualmann B, Regus H, Brauner D, Fejtová A, Bracko O, Gundelfinger ED, Brandstätter JH - J. Cell Biol. (2005)

CtBP1, a RIBEYE homologue, is expressed at ribbon and conventional synapses. (A–C) Confocal laser-scanning micrographs of a vertical section through mouse retina double labeled for CtBP1 (A) and Piccolo (B), which marks ribbon and conventional synapses in the retina. As seen in the merge of the two stainings (C), CtBP1 and Piccolo immunoreactivities completely colocalize at the synapses in the outer plexiform layer (OPL) and inner plexiform layer (IPL) of the retina. (D and E) Electron micrographs of a photoreceptor (D) and an amacrine cell synapse (E) immunogold labeled for CtBP1. At the ribbon synapse the gold particles for CtBP1 decorate the ribbon (arrowheads), at the amacrine cell synapse they are located some distance from the active zone (arrowheads) at the edge of the electron-dense CAZ material. (F) Western blots of retina homogenate probed with antibodies against CtBP1 and the RIBEYE A- and CtBP2/RIBEYE B-domain. The antibody against CtBP1 recognizes the 50-kD CtBP1 protein and does not cross-react with RIBEYE. (G–I) Micrographs of cultured hippocampal neurons double labeled for CtBP1 (G) and Piccolo (H). In hippocampal neurons, like in retinal neurons, a fraction of CtBP1 immunoreactivity is localized at synapses where it colocalizes with Piccolo as seen in the merge of the two stainings (I). (J and K) CtBP2 (J) is not present at hippocampal synapses labeled with Piccolo (K). (L) Immunoblots showing that CtBP1 can be coimmunoprecipitated from brain synaptosomes with a monoclonal anti-Bassoon antibody. The boxes in G–K mark the regions that are shown at higher magnification. INL, inner nuclear layer; GCL, ganglion cell layer; Ext., brain synaptosomal extract; IP, immunoprecipitate; S, supernatant. Bars: 20 μm (A–C), 0.2 μm (D and E), and 10 μm (G–K).
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fig7: CtBP1, a RIBEYE homologue, is expressed at ribbon and conventional synapses. (A–C) Confocal laser-scanning micrographs of a vertical section through mouse retina double labeled for CtBP1 (A) and Piccolo (B), which marks ribbon and conventional synapses in the retina. As seen in the merge of the two stainings (C), CtBP1 and Piccolo immunoreactivities completely colocalize at the synapses in the outer plexiform layer (OPL) and inner plexiform layer (IPL) of the retina. (D and E) Electron micrographs of a photoreceptor (D) and an amacrine cell synapse (E) immunogold labeled for CtBP1. At the ribbon synapse the gold particles for CtBP1 decorate the ribbon (arrowheads), at the amacrine cell synapse they are located some distance from the active zone (arrowheads) at the edge of the electron-dense CAZ material. (F) Western blots of retina homogenate probed with antibodies against CtBP1 and the RIBEYE A- and CtBP2/RIBEYE B-domain. The antibody against CtBP1 recognizes the 50-kD CtBP1 protein and does not cross-react with RIBEYE. (G–I) Micrographs of cultured hippocampal neurons double labeled for CtBP1 (G) and Piccolo (H). In hippocampal neurons, like in retinal neurons, a fraction of CtBP1 immunoreactivity is localized at synapses where it colocalizes with Piccolo as seen in the merge of the two stainings (I). (J and K) CtBP2 (J) is not present at hippocampal synapses labeled with Piccolo (K). (L) Immunoblots showing that CtBP1 can be coimmunoprecipitated from brain synaptosomes with a monoclonal anti-Bassoon antibody. The boxes in G–K mark the regions that are shown at higher magnification. INL, inner nuclear layer; GCL, ganglion cell layer; Ext., brain synaptosomal extract; IP, immunoprecipitate; S, supernatant. Bars: 20 μm (A–C), 0.2 μm (D and E), and 10 μm (G–K).
Mentions: As the interaction of Bassoon was originally observed with CtBP1, a homologue of the RIBEYE B-domain, we monitored the distribution of this protein in vertical sections of wild-type mouse retina using a monoclonal mouse anti-CtBP1 antibody. Interestingly, in addition to the expected nuclear staining, strong CtBP1 labeling was observed in the two synaptic layers of the retina (Fig. 7 A). Double-labeling experiments with an antiserum against the CAZ protein Piccolo, which is present at ribbon and conventional synapses in the retina (Fig. 7 B; Dick et al., 2001), show a complete colocalization of CtBP1 and Piccolo in the two plexiform layers of the retina (Fig. 7 C). Postembedding immunogold EM further proved the presence of CtBP1 at ribbon and conventional synapses in the retina (Fig. 7, D and E). Immunoblot analysis revealed no cross-reactivity of the CtBP1 antibody with RIBEYE (Fig. 7 F), demonstrating that indeed the 50-kD CtBP1 protein is present synaptically. In the Bassoon mutant photoreceptor terminals with their free-floating ribbons, CtBP1 stays associated with RIBEYE (unpublished data), adding CtBP1 to the list of potential interactors with the photoreceptor ribbon.

Bottom Line: Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1.A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex.Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroanatomy, Max Planck Institute for Brain Research, D-60528 Frankfurt/Main, Germany.

ABSTRACT
The ribbon complex of retinal photoreceptor synapses represents a specialization of the cytomatrix at the active zone (CAZ) present at conventional synapses. In mice deficient for the CAZ protein Bassoon, ribbons are not anchored to the presynaptic membrane but float freely in the cytoplasm. Exploiting this phenotype, we dissected the molecular structure of the photoreceptor ribbon complex. Identifiable CAZ proteins segregate into two compartments at the ribbon: a ribbon-associated compartment including Piccolo, RIBEYE, CtBP1/BARS, RIM1, and the motor protein KIF3A, and an active zone compartment including RIM2, Munc13-1, a Ca2+ channel alpha1 subunit, and ERC2/CAST1. A direct interaction between the ribbon-specific protein RIBEYE and Bassoon seems to link the two compartments and is responsible for the physical integrity of the photoreceptor ribbon complex. Finally, we found the RIBEYE homologue CtBP1 at ribbon and conventional synapses, suggesting a novel role for the CtBP/BARS family in the molecular assembly and function of central nervous system synapses.

Show MeSH
Related in: MedlinePlus