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

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

Bassoon contains a 14-3-3 interaction interface. (A)

<p>Domain structure of Bassoon with the position of the fragments Bsn28 and Bsn11 (horizontal bars) containing serine-2845 (vertical line) critical for 14-3-3η interaction are depicted. (PBH 1-10: Bassoon/ Piccolo homology domains 1-10; Zn: Zinc finger domain; cc: coiled coil domain) The deletion of exon 4 and 5 in BsnΔEx4/5 mutant mice and positions of epitopes of antibodies used in the study are depicted. (<b>B</b>) Alignment of the amino acid sequences of the region containing the 14-3-3 binding motif from Bassoon orthologues of different species is shown. The RSM/LpSDP 14-3-3 binding motif is bold and the critical serine residue S2845 is boxed.</p

Analysis of molecular dynamics of GFP-Bsn and GFP-BsnS2845A by FRAP. (A)

<p>Primary hippocampal neurons were transfected with GFP-Bsn or GFP-Bsn^S2845A at DIV3 and analyzed at DIV 14-16. Synaptic targeting of both constructs was assessed by co-staining with synaptic markers homer and synaptophysin. The arrows highlight synapses of transfected cells stained for both markers. Scale bar, 20 µm. <b>(B)</b> Curves in the left panel show averaged fluorescence recovery of all analyzed puncta plotted as actual spot intensity relative to pre-bleaching intensity. The whiskers show SEM for each value. GFP-Bsn (wild-type) shows a higher recovery rate compared to GFP-Bsn^S2845A. Recovery was significantly lowered for GFP-Bsn^S2845A 300 s after photobleaching. Columns in right plot represent mean value normalized to GFP-Bsn recovery, whiskers SEM, * indicates P<0.05. The values were obtained from 4 independent imaging sessions. <b>(C)</b> Representative example image showing bleaching and recovery of a GFP-Bsn and GFP-Bsn^S2845A puncta. Scale bar is 1 µm.</p

Expression of CtBP1 and CtBP2 in hippocampus and cortex.

<p>Images showing the region of hippocampus (A–D) and visual cortex (E, F) taken from sagittal mouse brain slices stained with DAPI, antibodies against CtBP1 or 2 and Bassoon and corresponding fluorescent secondary antibodies. The images in A, B, E, F always show staining of the same slice. In C and D overlay of staining in all channels are shown. The bars are 600 µm in A and B, and 250 µm in E, F. Arrows in A show scattered cell bodies labeled with mCtBP1 antibody in corpus calosum, arrow in B depicts immunoreactivity of rbCtBP2 in the stratum lucidum. CA1–3 – CA1 to 3 regions of hippocampus, gDG – granular layer of dentate gyrus, lu- stratum lucidum, mDG – molecular layer of dentate gyrus, ori – stratum oriens, pDG – polymorph dentate gyrus, rad – stratum radiatum.</p

Localization of CtBP1 and CtBP2 in the molecular layer of the cerebellum.

<p>Electron micrographs showing immunoreactivity for CtBP1 (A–E) and CtBP2 (F–I) in presynaptic elements in the molecular layer of the cerebellum as detected by pre-embedding immunoperoxidase method. Arrowheads mark the postsynaptic density of clearly displayed asymmetric synapses and asterisks mark the postsynaptic side of symmetric synapses. (A) and (C) show examples of immunolabeled axonal varicosities (of presumed parallel fibers) contacting unstained postsynaptic elements (thorns of Purkinje dendrites). (B) Symmetric synaptic junction between a CtBP1-immunopositive presynaptic element (presumed basket cell axon) and a Purkinje cell body. The peroxidase reaction product is concentrated at the synaptic contact zone. (D, E) Axon varicosities synapse two dendritic thorns at the section plane. In D the densely packed vesicles co-localize with the peroxidase reaction product. In E the immunoreactivity is concentrated at region apposing PSD, however, an axonal profile without synaptic contact at the section plane shows also a strong labeling.(F–H) Immunopositive varicosities make asymmetric contacts to thorns and a dendrite (in the left of F). A gradient of increasing amount of peroxidase reaction product towards the synaptic contact zone is detectable. Postsynaptic structures are free of immunoreactivity. In H some axonal profiles are also labeled. (I) CtBP2-immunolocalization in a presumed basket cell axon making a symmetric contact on a Purkinje cell body. The peroxidase reaction product is accumulated in the presynaptic element. Scale bars correspond to 250 nm.</p

Overall expression pattern of CtBP1, Bassoon and CtBP2.

<p>The sagittal slices of adult mouse brain were stained with antibody from mouse against CtBP1 (A), from rabbit against Bassoon (Bsn) (B) and rabbit against CtBP2 (C) and corresponding fluorescently coupled secondary antibodies. A and B show staining with two antibodies in the same slice. Am – amygdala, Cb – cerebellum, cbp – cerebellar peduncle, cc – corpus callosum, cp – cerebral peduncle, CPu – caudate putamen, Cx – cortex, GP – globus pallidus, Hi – hippocampus, ic – internal capsula, Md – midbrain, MO – medula oblongata, NAc – nucleus accumbens, ob – olfactory bulb, oc – olfactory cortex, ot – olfactory tuberculus, Po – pons, SN – substantia nigra, Sth – subthalamus, sub – subiculum, Th – thalamus, VP – ventral palidium.</p

Cell-type specific expression of CtBP1 and CtBP2.

<p>Rat hippocampal neurons grown for 14 days in dissociated cultures were stained with rabbit antibodies against CtBP1 (A) or CtBP2 (B). Staining with GAD65-specific antibody was used to mark cell bodies of inhibitory neurons. Non-stained neurons were considered to be excitatory (marked by asterisk). The CtBP1 and CtBP2 immunoreactivity in nuclei (highlighted by staining with DAPI) was measured and quantified. The quantification revealed significantly lower nuclear expression of CtBP2 in GAD65-positive neurons compared with GAD65 negative ones, whereas CtBP1 expression was not different in these two cell types. Scalebar is 10 µm.</p

List of antibodies.

<p>List of antibodies.</p

Confocal images of CtBP1 and CtBP2 localization at synapses of hippocampus and cerebellum.

<p>Slices were stained with following antibodies: anti CtBP1 from mouse and anti Bsn from rabbit (A–F) and anti CtBP2 from rabbit and anti Bsn from mouse in G and H. Images from hippocampal region CA1 (A, B), and CA3 (C, D) and from cerebellum (E–H) are shown. B shows a high-resolution scan of transition between pyramidal cell layer and stratum radiatum, D transition between pyramidal cell layer and stratum lucidum and F and H transition between granular and molecular cell layer. Note significant overlap of staining for CtBPs and Bassoon in punctate pattern in neuropil in high-resolution scans. Scalebars are 50 µm in G and 10 µm in H. gl- granular layer, lu – stratum lucidum, ml – molecular layer, ori – stratum oriens, Py – pyramidal cell layer, rad – stratum radiatum, the cell body of a Purkinje cell is marked by asterisk.</p