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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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Characterization of the photoreceptor ribbon protein RIBEYE in wild-type and Bassoon mutant mice. (A) Immunoblot of mouse retinal homogenate probed with antibodies against RIBEYE A- and B-domain. Two protein bands (double arrow) of ∼110 kD and 120 kD, representing RIBEYE, are recognized by both antibodies. In addition, the anti-RIBEYE B-domain antibody labels the 50-kD CtBP2 protein band (arrow). Protein bands above and below the RIBEYE doublet are unspecific and appeared only occasionally. (B) Preincubation of the anti-GST-RIBEYE A-domain antiserum with an excess of the MBP-RIB179-448 fusion protein (+) completely prevents the immunodetection of RIBEYE. −, no RIBEYE antigen included. Incubation of the same blot with the anti-RIBEYE B-domain antibody shows the presence of RIBEYE in both lanes. (C) In a vertical cryostat section through mouse retina, the anti-RIBEYE A-domain antiserum stains the photoreceptor ribbons in the outer plexiform layer (OPL) and the bipolar cell ribbons in the inner plexiform layer (IPL). (D) Preadsorption of the anti-RIBEYE antiserum with the antigen results in a complete loss of RIBEYE staining. (E and F) EM and postembedding immunogold labeling shows that photoreceptor ribbons are decorated with gold particles for RIBEYE A- (E) and B-domain (F). Note the absence of gold particles at the base of the ribbons (arrowheads), the region of the arciform density. (G) Confocal laser-scanning micrograph of a region of the OPL double labeled for RIBEYE A- (green) and B-domain (red) demonstrate the colocalization of the two immunoreactivities at the photoreceptor ribbons. (H) An aggregate of free-floating ribbons in a cone photoreceptor terminal of Bassoon mutant retina decorated with RIBEYE immunoreactivity (preembedding labeling). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Bars: 10 μm (C and D); 0.1 μm (E and F); 5 μm (G); and 0.4 μm (H).
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fig1: Characterization of the photoreceptor ribbon protein RIBEYE in wild-type and Bassoon mutant mice. (A) Immunoblot of mouse retinal homogenate probed with antibodies against RIBEYE A- and B-domain. Two protein bands (double arrow) of ∼110 kD and 120 kD, representing RIBEYE, are recognized by both antibodies. In addition, the anti-RIBEYE B-domain antibody labels the 50-kD CtBP2 protein band (arrow). Protein bands above and below the RIBEYE doublet are unspecific and appeared only occasionally. (B) Preincubation of the anti-GST-RIBEYE A-domain antiserum with an excess of the MBP-RIB179-448 fusion protein (+) completely prevents the immunodetection of RIBEYE. −, no RIBEYE antigen included. Incubation of the same blot with the anti-RIBEYE B-domain antibody shows the presence of RIBEYE in both lanes. (C) In a vertical cryostat section through mouse retina, the anti-RIBEYE A-domain antiserum stains the photoreceptor ribbons in the outer plexiform layer (OPL) and the bipolar cell ribbons in the inner plexiform layer (IPL). (D) Preadsorption of the anti-RIBEYE antiserum with the antigen results in a complete loss of RIBEYE staining. (E and F) EM and postembedding immunogold labeling shows that photoreceptor ribbons are decorated with gold particles for RIBEYE A- (E) and B-domain (F). Note the absence of gold particles at the base of the ribbons (arrowheads), the region of the arciform density. (G) Confocal laser-scanning micrograph of a region of the OPL double labeled for RIBEYE A- (green) and B-domain (red) demonstrate the colocalization of the two immunoreactivities at the photoreceptor ribbons. (H) An aggregate of free-floating ribbons in a cone photoreceptor terminal of Bassoon mutant retina decorated with RIBEYE immunoreactivity (preembedding labeling). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Bars: 10 μm (C and D); 0.1 μm (E and F); 5 μm (G); and 0.4 μm (H).

Mentions: We generated an antiserum against the RIBEYE-specific A-domain. In retina homogenate this antiserum recognizes two protein bands of ∼110 and 120 kD that are also detected with an antibody against the B-domain (Fig. 1 A). In addition, the B-domain antibody recognizes the 50-kD CtBP2 band (Fig. 1 A). In preadsorbed controls, the immunodetection of RIBEYE in Western blots and in mouse retinal sections was abolished (Fig. 1, B and D).


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)

Characterization of the photoreceptor ribbon protein RIBEYE in wild-type and Bassoon mutant mice. (A) Immunoblot of mouse retinal homogenate probed with antibodies against RIBEYE A- and B-domain. Two protein bands (double arrow) of ∼110 kD and 120 kD, representing RIBEYE, are recognized by both antibodies. In addition, the anti-RIBEYE B-domain antibody labels the 50-kD CtBP2 protein band (arrow). Protein bands above and below the RIBEYE doublet are unspecific and appeared only occasionally. (B) Preincubation of the anti-GST-RIBEYE A-domain antiserum with an excess of the MBP-RIB179-448 fusion protein (+) completely prevents the immunodetection of RIBEYE. −, no RIBEYE antigen included. Incubation of the same blot with the anti-RIBEYE B-domain antibody shows the presence of RIBEYE in both lanes. (C) In a vertical cryostat section through mouse retina, the anti-RIBEYE A-domain antiserum stains the photoreceptor ribbons in the outer plexiform layer (OPL) and the bipolar cell ribbons in the inner plexiform layer (IPL). (D) Preadsorption of the anti-RIBEYE antiserum with the antigen results in a complete loss of RIBEYE staining. (E and F) EM and postembedding immunogold labeling shows that photoreceptor ribbons are decorated with gold particles for RIBEYE A- (E) and B-domain (F). Note the absence of gold particles at the base of the ribbons (arrowheads), the region of the arciform density. (G) Confocal laser-scanning micrograph of a region of the OPL double labeled for RIBEYE A- (green) and B-domain (red) demonstrate the colocalization of the two immunoreactivities at the photoreceptor ribbons. (H) An aggregate of free-floating ribbons in a cone photoreceptor terminal of Bassoon mutant retina decorated with RIBEYE immunoreactivity (preembedding labeling). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Bars: 10 μm (C and D); 0.1 μm (E and F); 5 μm (G); and 0.4 μm (H).
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Related In: Results  -  Collection

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

fig1: Characterization of the photoreceptor ribbon protein RIBEYE in wild-type and Bassoon mutant mice. (A) Immunoblot of mouse retinal homogenate probed with antibodies against RIBEYE A- and B-domain. Two protein bands (double arrow) of ∼110 kD and 120 kD, representing RIBEYE, are recognized by both antibodies. In addition, the anti-RIBEYE B-domain antibody labels the 50-kD CtBP2 protein band (arrow). Protein bands above and below the RIBEYE doublet are unspecific and appeared only occasionally. (B) Preincubation of the anti-GST-RIBEYE A-domain antiserum with an excess of the MBP-RIB179-448 fusion protein (+) completely prevents the immunodetection of RIBEYE. −, no RIBEYE antigen included. Incubation of the same blot with the anti-RIBEYE B-domain antibody shows the presence of RIBEYE in both lanes. (C) In a vertical cryostat section through mouse retina, the anti-RIBEYE A-domain antiserum stains the photoreceptor ribbons in the outer plexiform layer (OPL) and the bipolar cell ribbons in the inner plexiform layer (IPL). (D) Preadsorption of the anti-RIBEYE antiserum with the antigen results in a complete loss of RIBEYE staining. (E and F) EM and postembedding immunogold labeling shows that photoreceptor ribbons are decorated with gold particles for RIBEYE A- (E) and B-domain (F). Note the absence of gold particles at the base of the ribbons (arrowheads), the region of the arciform density. (G) Confocal laser-scanning micrograph of a region of the OPL double labeled for RIBEYE A- (green) and B-domain (red) demonstrate the colocalization of the two immunoreactivities at the photoreceptor ribbons. (H) An aggregate of free-floating ribbons in a cone photoreceptor terminal of Bassoon mutant retina decorated with RIBEYE immunoreactivity (preembedding labeling). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Bars: 10 μm (C and D); 0.1 μm (E and F); 5 μm (G); and 0.4 μm (H).
Mentions: We generated an antiserum against the RIBEYE-specific A-domain. In retina homogenate this antiserum recognizes two protein bands of ∼110 and 120 kD that are also detected with an antibody against the B-domain (Fig. 1 A). In addition, the B-domain antibody recognizes the 50-kD CtBP2 band (Fig. 1 A). In preadsorbed controls, the immunodetection of RIBEYE in Western blots and in mouse retinal sections was abolished (Fig. 1, B and D).

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