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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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Bassoon–RIBEYE interaction at the base of the photoreceptor ribbon. (A–C) Confocal laser-scanning micrographs of photoreceptor ribbon synapses double labeled for RIBEYE (A) and Bassoon (B). In the merge of the two stainings (C), the horseshoe-like appearance of the photoreceptor ribbon colabeled for RIBEYE and Bassoon is clearly visible. Bassoon labeling is surrounded by the RIBEYE labeling. (D) Electron micrographs of photoreceptor ribbons, en face and cross-section view (inset), postembedding immunogold double labeled for Bassoon (large gold particles) and RIBEYE (small gold particles). The gold particles for Bassoon are located closest to the active zone (arrowheads). (E) Immunoblots of 25 μg total protein per lane from wild-type (+/+) and Bassoon mutant (−/−) retina homogenates show a 50% reduction of RIBEYE immunoreactivity in the −/− retina. Data are normalized to the mean value (100%) obtained from wild-type samples. Statistical analysis by the unpaired t test shows a significant difference in the RIBEYE immunoreactivity between the two genotypes (asterisk, P < 0,01). Results are expressed as mean ± SD; n = 4 for both genotypes. (F) Immunoprecipitation experiments from wild-type (+/+) and mutant (−/−) retinal extracts with the rabbit antiserum BSN1.6 directed against the NH2-terminal part of wild-type and mutant Bassoon. The arrows on the left indicate wild-type Bassoon (420 and 320 kD), the arrowhead on the right indicates the 180-kD mutant protein. Note that the unspecific band (asterisk) is not precipitated. RIBEYE is coimmunoprecipitated from wild-type but not from Bassoon mutant retina. The same is true for the higher molecular weight band of Piccolo. In contrast, ERC2/CAST1 is found in both precipitates. Kinesin KIF3A and synaptojanin are not detected in either precipitate. Control experiments were performed with preimmune serum (preIS). IP, immunoprecipitation. Bars: 2 μm (A–C), 0.2 μm (D).
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fig6: Bassoon–RIBEYE interaction at the base of the photoreceptor ribbon. (A–C) Confocal laser-scanning micrographs of photoreceptor ribbon synapses double labeled for RIBEYE (A) and Bassoon (B). In the merge of the two stainings (C), the horseshoe-like appearance of the photoreceptor ribbon colabeled for RIBEYE and Bassoon is clearly visible. Bassoon labeling is surrounded by the RIBEYE labeling. (D) Electron micrographs of photoreceptor ribbons, en face and cross-section view (inset), postembedding immunogold double labeled for Bassoon (large gold particles) and RIBEYE (small gold particles). The gold particles for Bassoon are located closest to the active zone (arrowheads). (E) Immunoblots of 25 μg total protein per lane from wild-type (+/+) and Bassoon mutant (−/−) retina homogenates show a 50% reduction of RIBEYE immunoreactivity in the −/− retina. Data are normalized to the mean value (100%) obtained from wild-type samples. Statistical analysis by the unpaired t test shows a significant difference in the RIBEYE immunoreactivity between the two genotypes (asterisk, P < 0,01). Results are expressed as mean ± SD; n = 4 for both genotypes. (F) Immunoprecipitation experiments from wild-type (+/+) and mutant (−/−) retinal extracts with the rabbit antiserum BSN1.6 directed against the NH2-terminal part of wild-type and mutant Bassoon. The arrows on the left indicate wild-type Bassoon (420 and 320 kD), the arrowhead on the right indicates the 180-kD mutant protein. Note that the unspecific band (asterisk) is not precipitated. RIBEYE is coimmunoprecipitated from wild-type but not from Bassoon mutant retina. The same is true for the higher molecular weight band of Piccolo. In contrast, ERC2/CAST1 is found in both precipitates. Kinesin KIF3A and synaptojanin are not detected in either precipitate. Control experiments were performed with preimmune serum (preIS). IP, immunoprecipitation. Bars: 2 μm (A–C), 0.2 μm (D).

Mentions: With immunocytochemistry and confocal laser-scanning microscopy, we screened in wild-type photoreceptor terminals a number of presynaptic proteins for their localization as compared with RIBEYE and thus for their potential to interact with the photoreceptor ribbon. The proteins synaptotagmin, VGLUT1 (vesicular glutamate transporter 1), SNAP-25 (synaptosomal-associated protein of 25 kD), syntaxin 3, and synaptophysin were present throughout the photoreceptor terminals; they did not specifically colocalize with RIBEYE, and thus were excluded from further analysis (unpublished data). Proteins that colocalized with RIBEYE in wild-type photoreceptor terminals were Piccolo, the kinesin motor protein KIF3A, RIM1, RIM2, Munc13-1, a Ca2+ channel α1 subunit, ERC2/CAST1 (Fig. 2), and Bassoon (see Fig. 6).


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)

Bassoon–RIBEYE interaction at the base of the photoreceptor ribbon. (A–C) Confocal laser-scanning micrographs of photoreceptor ribbon synapses double labeled for RIBEYE (A) and Bassoon (B). In the merge of the two stainings (C), the horseshoe-like appearance of the photoreceptor ribbon colabeled for RIBEYE and Bassoon is clearly visible. Bassoon labeling is surrounded by the RIBEYE labeling. (D) Electron micrographs of photoreceptor ribbons, en face and cross-section view (inset), postembedding immunogold double labeled for Bassoon (large gold particles) and RIBEYE (small gold particles). The gold particles for Bassoon are located closest to the active zone (arrowheads). (E) Immunoblots of 25 μg total protein per lane from wild-type (+/+) and Bassoon mutant (−/−) retina homogenates show a 50% reduction of RIBEYE immunoreactivity in the −/− retina. Data are normalized to the mean value (100%) obtained from wild-type samples. Statistical analysis by the unpaired t test shows a significant difference in the RIBEYE immunoreactivity between the two genotypes (asterisk, P < 0,01). Results are expressed as mean ± SD; n = 4 for both genotypes. (F) Immunoprecipitation experiments from wild-type (+/+) and mutant (−/−) retinal extracts with the rabbit antiserum BSN1.6 directed against the NH2-terminal part of wild-type and mutant Bassoon. The arrows on the left indicate wild-type Bassoon (420 and 320 kD), the arrowhead on the right indicates the 180-kD mutant protein. Note that the unspecific band (asterisk) is not precipitated. RIBEYE is coimmunoprecipitated from wild-type but not from Bassoon mutant retina. The same is true for the higher molecular weight band of Piccolo. In contrast, ERC2/CAST1 is found in both precipitates. Kinesin KIF3A and synaptojanin are not detected in either precipitate. Control experiments were performed with preimmune serum (preIS). IP, immunoprecipitation. Bars: 2 μm (A–C), 0.2 μm (D).
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Related In: Results  -  Collection

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fig6: Bassoon–RIBEYE interaction at the base of the photoreceptor ribbon. (A–C) Confocal laser-scanning micrographs of photoreceptor ribbon synapses double labeled for RIBEYE (A) and Bassoon (B). In the merge of the two stainings (C), the horseshoe-like appearance of the photoreceptor ribbon colabeled for RIBEYE and Bassoon is clearly visible. Bassoon labeling is surrounded by the RIBEYE labeling. (D) Electron micrographs of photoreceptor ribbons, en face and cross-section view (inset), postembedding immunogold double labeled for Bassoon (large gold particles) and RIBEYE (small gold particles). The gold particles for Bassoon are located closest to the active zone (arrowheads). (E) Immunoblots of 25 μg total protein per lane from wild-type (+/+) and Bassoon mutant (−/−) retina homogenates show a 50% reduction of RIBEYE immunoreactivity in the −/− retina. Data are normalized to the mean value (100%) obtained from wild-type samples. Statistical analysis by the unpaired t test shows a significant difference in the RIBEYE immunoreactivity between the two genotypes (asterisk, P < 0,01). Results are expressed as mean ± SD; n = 4 for both genotypes. (F) Immunoprecipitation experiments from wild-type (+/+) and mutant (−/−) retinal extracts with the rabbit antiserum BSN1.6 directed against the NH2-terminal part of wild-type and mutant Bassoon. The arrows on the left indicate wild-type Bassoon (420 and 320 kD), the arrowhead on the right indicates the 180-kD mutant protein. Note that the unspecific band (asterisk) is not precipitated. RIBEYE is coimmunoprecipitated from wild-type but not from Bassoon mutant retina. The same is true for the higher molecular weight band of Piccolo. In contrast, ERC2/CAST1 is found in both precipitates. Kinesin KIF3A and synaptojanin are not detected in either precipitate. Control experiments were performed with preimmune serum (preIS). IP, immunoprecipitation. Bars: 2 μm (A–C), 0.2 μm (D).
Mentions: With immunocytochemistry and confocal laser-scanning microscopy, we screened in wild-type photoreceptor terminals a number of presynaptic proteins for their localization as compared with RIBEYE and thus for their potential to interact with the photoreceptor ribbon. The proteins synaptotagmin, VGLUT1 (vesicular glutamate transporter 1), SNAP-25 (synaptosomal-associated protein of 25 kD), syntaxin 3, and synaptophysin were present throughout the photoreceptor terminals; they did not specifically colocalize with RIBEYE, and thus were excluded from further analysis (unpublished data). Proteins that colocalized with RIBEYE in wild-type photoreceptor terminals were Piccolo, the kinesin motor protein KIF3A, RIM1, RIM2, Munc13-1, a Ca2+ channel α1 subunit, ERC2/CAST1 (Fig. 2), and Bassoon (see Fig. 6).

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