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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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Reconstitution of Bassoon–RIBEYE protein complexes by cotransfection of the Bassoon and RIBEYE binding domains in COS cells. (A–C) Mito-RIBEYE B-domain constructs (A; anti-flag immunosignal) localize to mitochondria (B; MitoTracker Red CMXRos), best seen in the inset in the merge of the two stainings in C. (D–F) The GFP-RB46 (D) construct is distributed diffusely when expressed alone and shows no similarity in pattern with mitochondria (E and F). (G–J) In double-transfection experiments with the Mito-RIB-B-domain, the GFP-RB46 construct (I) adopts a pattern that overlaps with that of the Mito-RIB-B-domain (G) and the MitoTracker (H), as clearly seen when images are merged (J). (K–M) In contrast, the GFP-RB45 construct (K) remains diffusely distributed and shows no enrichment at mitochondria in cells that are cotransfected with the Mito-RIB-B-domain (L), as seen in the merge of the two stainings (M). Bars, 10 μm.
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fig5: Reconstitution of Bassoon–RIBEYE protein complexes by cotransfection of the Bassoon and RIBEYE binding domains in COS cells. (A–C) Mito-RIBEYE B-domain constructs (A; anti-flag immunosignal) localize to mitochondria (B; MitoTracker Red CMXRos), best seen in the inset in the merge of the two stainings in C. (D–F) The GFP-RB46 (D) construct is distributed diffusely when expressed alone and shows no similarity in pattern with mitochondria (E and F). (G–J) In double-transfection experiments with the Mito-RIB-B-domain, the GFP-RB46 construct (I) adopts a pattern that overlaps with that of the Mito-RIB-B-domain (G) and the MitoTracker (H), as clearly seen when images are merged (J). (K–M) In contrast, the GFP-RB45 construct (K) remains diffusely distributed and shows no enrichment at mitochondria in cells that are cotransfected with the Mito-RIB-B-domain (L), as seen in the merge of the two stainings (M). Bars, 10 μm.

Mentions: The RIBEYE B-domain was targeted to the outer mitochondrial membrane by generating a fusion construct with an appropriate mitochondrial targeting sequence and a flag epitope tag (Mito-RIB-B). The construct is successfully localized to mitochondria as indicated by costaining with anti-Flag antibodies and MitoTracker (Fig. 5, A–C). A clear ring-like structure of the anti-Flag immunostaining (green) encircling mitochondria (red) suggests that the RIBEYE construct is successfully targeted to mitochondria and inserted in a correct orientation into the outer mitochondrial membrane (Fig. 5 C, inset).


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)

Reconstitution of Bassoon–RIBEYE protein complexes by cotransfection of the Bassoon and RIBEYE binding domains in COS cells. (A–C) Mito-RIBEYE B-domain constructs (A; anti-flag immunosignal) localize to mitochondria (B; MitoTracker Red CMXRos), best seen in the inset in the merge of the two stainings in C. (D–F) The GFP-RB46 (D) construct is distributed diffusely when expressed alone and shows no similarity in pattern with mitochondria (E and F). (G–J) In double-transfection experiments with the Mito-RIB-B-domain, the GFP-RB46 construct (I) adopts a pattern that overlaps with that of the Mito-RIB-B-domain (G) and the MitoTracker (H), as clearly seen when images are merged (J). (K–M) In contrast, the GFP-RB45 construct (K) remains diffusely distributed and shows no enrichment at mitochondria in cells that are cotransfected with the Mito-RIB-B-domain (L), as seen in the merge of the two stainings (M). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171818&req=5

fig5: Reconstitution of Bassoon–RIBEYE protein complexes by cotransfection of the Bassoon and RIBEYE binding domains in COS cells. (A–C) Mito-RIBEYE B-domain constructs (A; anti-flag immunosignal) localize to mitochondria (B; MitoTracker Red CMXRos), best seen in the inset in the merge of the two stainings in C. (D–F) The GFP-RB46 (D) construct is distributed diffusely when expressed alone and shows no similarity in pattern with mitochondria (E and F). (G–J) In double-transfection experiments with the Mito-RIB-B-domain, the GFP-RB46 construct (I) adopts a pattern that overlaps with that of the Mito-RIB-B-domain (G) and the MitoTracker (H), as clearly seen when images are merged (J). (K–M) In contrast, the GFP-RB45 construct (K) remains diffusely distributed and shows no enrichment at mitochondria in cells that are cotransfected with the Mito-RIB-B-domain (L), as seen in the merge of the two stainings (M). Bars, 10 μm.
Mentions: The RIBEYE B-domain was targeted to the outer mitochondrial membrane by generating a fusion construct with an appropriate mitochondrial targeting sequence and a flag epitope tag (Mito-RIB-B). The construct is successfully localized to mitochondria as indicated by costaining with anti-Flag antibodies and MitoTracker (Fig. 5, A–C). A clear ring-like structure of the anti-Flag immunostaining (green) encircling mitochondria (red) suggests that the RIBEYE construct is successfully targeted to mitochondria and inserted in a correct orientation into the outer mitochondrial membrane (Fig. 5 C, inset).

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