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

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 α1 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

Epilepsy-induced abnormal striatal plasticity in Bassoon mutant mice

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Differential Spatial Expression and Subcellular Localization of CtBP Family Members in Rodent Brain

C-terminal binding proteins (CtBPs) are well-characterized nuclear transcriptional co-regulators. In addition, cytoplasmic functions were discovered for these ubiquitously expressed proteins. These include the involvement of the isoform CtBP1-S/BARS50 in cellular membrane-trafficking processes and a role of the isoform RIBEYE as molecular scaffolds in ribbons, the presynaptic specializations of sensory synapses. CtBPs were suggested to regulate neuronal differentiation and they were implied in the control of gene expression during epileptogenesis. However, the expression patterns of CtBP family members in specific brain areas and their subcellular localizations in neurons in situ are largely unknown. Here, we performed comprehensive assessment of the expression of CtBP1 and CtBP2 in mouse brain at the microscopic and the ultra-structural levels using specific antibodies. We quantified and compared expression levels of both CtBPs in biochemically isolated brain fractions containing cellular nuclei or synaptic compartment. Our study demonstrates differential regional and subcellular expression patterns for the two CtBP family members in brain and reveals a previously unknown synaptic localization for CtBP2 in particular brain regions. Finally, we propose a mechanism of differential synapto-nuclear targeting of its splice variants CtBP2-S and CtBP2-L in neurons

Regulation of presynaptic anchoring of the scaffold protein Bassoon by phosphorylation-dependent interaction with 14-3-3 adaptor proteins.

The proper organization of the presynaptic cytomatrix at the active zone is essential for reliable neurotransmitter release from neurons. Despite of the virtual stability of this tightly interconnected proteinaceous network it becomes increasingly clear that regulated dynamic changes of its composition play an important role in the processes of synaptic plasticity. Bassoon, a core component of the presynaptic cytomatrix, is a key player in structural organization and functional regulation of presynaptic release sites. It is one of the most highly phosphorylated synaptic proteins. Nevertheless, to date our knowledge about functions mediated by any one of the identified phosphorylation sites of Bassoon is sparse. In this study, we have identified an interaction of Bassoon with the small adaptor protein 14-3-3, which depends on phosphorylation of the 14-3-3 binding motif of Bassoon. In vitro phosphorylation assays indicate that phosphorylation of the critical Ser-2845 residue of Bassoon can be mediated by a member of the 90-kDa ribosomal S6 protein kinase family. Elimination of Ser-2845 from the 14-3-3 binding motif results in a significant decrease of Bassoon's molecular exchange rates at synapses of living rat neurons. We propose that the phosphorylation-induced 14-3-3 binding to Bassoon modulates its anchoring to the presynaptic cytomatrix. This regulation mechanism might participate in molecular and structural presynaptic remodeling during synaptic plasticity

Quantitative analysis of nuclear and synaptosomal expression of CtBP1 and CtBP2.

<p>Synaptosomal (A) and nuclear (B) fraction from whole rat brain were analysed with antibodies against CtBP1, CtBP2, nuclear marker NeuN and synaptic markers synaptophysin (sph), Bassoon (Bsn) and Piccolo (Pclo). Bars and numbers indicate position and size (in kDa) of the molecular weight markers. The arrow shows specific double-band corresponding to NeuN, the band migrating above 70 kDa marker is not specific. Note enrichment of nuclear marker NeuN in nuclear fraction and its absence in synaptosomes. Sph, Bsn and Pclo are enriched in synaptosomes. Expression levels of CtBP1 and CtBP2 in nuclear and synaptosomal fraction prepared from whole brain (C), cortex (D) or cerebellum (E) were detected using specific antibodies from mouse and rabbit. The enrichment of signal in the synaptosomal or nuclear fraction was expressed in percentages relative to signal measured in homogenates. Note higher relative expression in synaptosomes than in nuclei for CtBP1 in contrast to higher relative expression of CtBP2 in nuclei compared to synaptosomes. The plot in F shows the results of quantification; bars represent the mean values, whiskers the SEMs.</p

List of antibodies.

<p>List of antibodies.</p

Specificity test of antibodies against CtBP1 and CtBP2.

<p>Schematic representation of domain structure of members of CtBP protein family is shown in A. The region in grey represents the high homology region shared by all family members. The red, yellow and blue marked regions depict unique N-terminal sequence expressed in CtBP1-L, CtBP2 and RIBEYE respectively. The positions of antigens used for generating antibodies used in this study are depicted as bars above the corresponding sequence. To test specificity of available antibodies, the indicated samples were tested by Western blot analysis using mouse monoclonal or rabbit polyclonal antibodies against CtBP1 or CtBP2 (B, C, E, F), rabbit polyclonal antibody against GFP (D) and RIBEYE specific antibody from rat (G). Bars and numbers indicate position and size (in kDa) of the molecular weight markers.</p