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First published online 20 March 2007
doi: 10.1242/jcs.003905

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Vertebrate-specific sequences in the gephyrin E-domain regulate cytosolic aggregation and postsynaptic clustering

Barbara Lardi-Studler¹, Birthe Smolinsky², Caroline M. Petitjean¹, Franziska Koenig³, Corinne Sidler¹, Jochen C. Meier⁴, Jean-Marc Fritschy^1,*, and Guenter Schwarz^2,*

¹ Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland
² Institute of Biochemistry, University of Cologne, D-50674 Köln, Germany
³ Institute of Plant Biology, Technical University Braunschweig, D-38023 Braunschweig, Germany
⁴ Max-Delbrück-Center for Molecular Medicine, D-13125 Berlin-Buch, Germany

View larger version (39K): [in this window] [in a new window]   Fig. 1. Structure of EGFP-gephyrin deletion constructs and mutant constructs. (A) Scheme of the five truncated gephyrin constructs and the construct with E-domain duplication. All constructs were coupled to EGFP at the N-terminus. First and last residues of the G and E domains as well as terminating residues are highlighted; L1 and L2 indicate the position of the two loops investigated in the mutant constructs (587-592 and 611-622, respectively). (B) Sequence of Loop 1 (L1) and Loop 2 (L2) mutant constructs, created by swapping residues from rat gephyrin (green boxes) with the corresponding E. coli MoeA residues (red boxes). L2A, L2B and L2C are three variants differing in the number of swapped residues: L2A (T611-F622), L2B (D613-I620), L2C (I614-K619). Boundary sequences in MoeA and gephyrin are indicated. Gray shading depicts the degree of conservation across species: black, 100% conservation; gray with black letters, at least 70% conservation; gray with white letters, type of amino acid conserved; white with black letters, variable. (C) Structure of gephyrin-E dimer shown in trace mode. The subdomains of one monomer are shown in shades of red and orange whereas the other monomer is shown in gray. Loops 1 and 2 are highlighted in light blue. For comparison, the active site of Moco synthesis is highlighted. (D) Zoom of subdomain 3 of panel C with the first and last residues that were exchanged in the corresponding L1 and L2 variants. Figures were generated with MOLSCRIPT (Esnouf, 1997) and rendered with POVRAY (www.povray.org).

View larger version (176K): [in this window] [in a new window]   Fig. 2. Expression of EGFP-tagged full-length gephyrin (EGFP-geph-GCE). Images from immunofluorescence staining with the markers indicated in each panel were visualized by confocal laser-scanning microscopy. (A) In HEK293 cells, EGFP-geph-GCE forms intracellular aggregates independently of the presence of GABAA receptors (stained for the 1 subunit in red) at the cell surface. (B,B') In 6+2 hippocampal neurons, a clustered staining for the GABAA receptor 2 subunit (red) was detected whereas only few endogenous (blue) and heterologous gephyrin clusters were present (B'); the gephyrin antibody stained both endogenous gephyrin and EGFP-geph-GCE. The latter formed cytosolic aggregates (arrows) and a few clusters co-localized with the 2 subunit in dendrites (open arrowheads). Open arrows point to non-specific gephyrin staining of a cell nucleus. (C) In 6+6 cells, the number of gephyrin and 2 subunit clusters increased markedly, although some 2 subunit clusters lacking gephyrin were still detectable. No endogenous gephyrin clusters devoid of EGFP were present, as shown at higher magnification in the inset (c1-c3; open arrowheads), suggesting that both proteins interact within individual clusters. (D-D'') In cells with a strong EGFP-geph-GCE expression, large aggregates devoid of 2 subunit staining were formed in dendrites (arrow). (E) In 10+2 cells, the 2 subunit (red) and gephyrin (blue) had the same appearance as at 6+6; EGFP-geph-GCE (green) was associated with 2 subunit immunoreactivity in clusters (white), but not in cytosolic aggregates (cyan), as shown at higher magnification in the inset (e1-e3). (F-F'') Labeling for synapsin 1 (red) demonstrates the selective postsynaptic localization of EGFP-geph-GCE clusters. Bars, 10 µm (A,B); 10 µm (C-E); 5 µm (F).

View larger version (139K): [in this window] [in a new window]   Fig. 3. Dominant-negative effect of EGFP-geph-GC and EGFP-geph-E. Both truncated constructs produced a diffuse staining (green) in HEK293 cells (A,E) and in neurons at all stages examined. (B,F) In 6+2 cultures, only a few endogenous gephyrin clusters (blue) were detected, whereas the 2 subunit staining (red) was already distinct. (C,D and G,H) In 6+6 cultures, a variable density of endogenous gephyrin clusters was observed in transfected neurons. C and G each illustrate a cell almost devoid of gephyrin clusters, as seen best in the enlarged insets (c1,g1); 2 subunit clusters were not affected in such cells (open arrows in c1 and g1) and gephyrin clusters were seen in non-transfected cells (arrowheads in c1 and g1). Panels D and H depict a transfected cell with numerous gephyrin clusters (open arrowheads in d1,h1). (I,J) Double staining for gephyrin (red) and MAP2 (blue) shows that many gephyrin clusters in transfected dendrites are localized at cross points with non-transfected dendrites (arrowheads in I'' and J''). (K-N) In 10+2 cultures, similar features were evident for both constructs; K and M illustrate a strong dominant-negative effect of each construct, and L and N a weak dominant-negative effect. Open arrowheads in L' and N' indicate postsynaptic gephyrin clusters containing the 2 subunit; open arrows in M' show the presence of 2 subunit clusters devoid of gephyrin. Bars, 10 µm (A,B,E-H); 2.5 µm (C-D); 5 µm (I-N).

View larger version (33K): [in this window] [in a new window]   Fig. 4. Reduction of endogenous gephyrin clusters after transfection of EGFP-gephyrin constructs. Frequency distribution of endogenous gephyrin clusters on dendrites of mock-transfected cells (A,D) and cells transfected with EGFP-geph-GC (B,E), EGFP-geph-E (C,F), EGFP-geph-GCEE (G) or EGFP-geph-L2B (H). Red, 6+6; green, 10+2 experiments. Dominant-negative effects are evidenced by a shift of the distribution towards lower cluster numbers per segment (see Table 1 for statistical analysis). Dendrites devoid of gephyrin clusters are highlighted in pale color. The strongest effect occurred after transfection of EGFP-geph-GC and EGFP-geph-E. In cells transfected with EGFP-geph-GCEE (G), dendrites devoid of endogenous gephyrin clusters were from cells with a strong transgene expression. The 10+2 experiments show that the reduction of gephyrin clusters also occurs after only 2 days of recombinant gephyrin expression, suggesting a rapid replacement of gephyrin in postsynaptic clusters. (I) Summary table indicating the proportion of dendrites with an almost complete loss of endogenous gephyrin clusters. Note that for EGFP-geph-E and EGFP-geph-GC, this fraction is approximately double after long-term expression (6+6) compared with short-term expression (10+2). Mock-transfected dendrites show no evidence from toxic effects of the transfection reagent after long-term treatment.

View larger version (145K): [in this window] [in a new window]   Fig. 5. Aggregation, but defective postsynaptic clustering, of EGFP-geph-GCEE in HEK293 cells and neurons. (A) Aggregates of EGFP-geph-GCEE (green) in HEK293 cells without colocalization with 1 subunit immunoreactivity (red) on the cell surface. (B) EGFP-geph-GCEE formed aggregates in 6+2 cells, located in the soma and in dendrites and lacking 2 subunit staining (arrowheads). (C) In 6+6 cells, only few EGFP-positive clusters colocalized with 2 subunit immunoreactivity were detected (open arrowheads). Cytosolic aggregates (arrowheads) were larger and more numerous. (D) In contrast to EGFP-geph-GCE, EGFP-geph-GCEE was not integrated in all gephyrin-immunoreactive clusters (open arrows); (E) Only a few EGFP-geph-GCEE-positive clusters associated with the 2 subunit and apposed to a synapsin-1-positive presynaptic terminal (blue) were observed (open arrowheads in E''); aggregates (green) were devoid of 2 subunit staining and were not postsynaptic (arrowheads in E''). (F-H) Three examples of cortical neurons transfected by nucleofection with EGFP-geph-GCEE, showing different aggregation and clustering in dependence of expression levels. In cells containing numerous aggregates (F; arrows), only few postsynaptic clusters were present (open arrowhead in f1); clusters of endogenous gephyrin were present in reduced numbers (arrowhead). When aggregates were few and small (G,g1; arrows), a larger number of postsynaptic clusters (g1, open arrowheads) was seen; finally, almost normal postsynaptic clustering was evident (H,h1; open arrowheads) when aggregates were nearly absent (arrow). Bars, 10 µm (A-C,F-H); 5 µm (D,E).

View larger version (40K): [in this window] [in a new window]   Fig. 6. Functional analysis of gephyrin constructs. (A) Moco activity of E. coli MoeA in cells expressing gephyrin mutant constructs (L1, L2B, L2C), a gephyrin variant unable to bind glycine receptors (P713E), gephyrin-GCEE, the isolated CE-domain, gephyrin (GCE) and as negative controls, the isolated G-domain (G) or empty vector (pQE30). Activities were determined with the nit-1 reconstitution assay in the absence of external molybdate (black bars). Different dilutions of crude protein extracts were used according to the linear range of the assay and units are defined as reconstituted enzyme activity (A540 over 20-minute reaction time per mg crude protein extract). Error bars (s.e.) are derived from at least three independent experiments. Enzymatic activity was normalized to the expression levels of gephyrin variants (white bars) measured by densitometry in western blots (B). (B) Western blot analysis of crude protein extracts that were used for Moco synthesis analysis (A) using monoclonal (mAB, upper blot) and polyclonal gephyrin antibodies (pAB, Puszta serum; lower blot); arrows indicate the relevant bands. The amount of protein loaded is stated for each lane. For comparison, different dilutions of gephyrin (GCE) are shown that were used for densitometric quantification of band intensities. The upper blot shows dotted lines for the molecular weight standards at 116 and 67 kDa.

View larger version (111K): [in this window] [in a new window]   Fig. 7. Differential aggregation of EGFP-gephyrin mutant constructs. (A) Postsynaptic clusters of EGFP-geph-L1 transfected in neurons, as seen at 6+6 by double staining with the 2 subunit (red) and gephyrin (blue). (B,C) In HEK293 cells, EGFP-geph-L2B (B) was strongly expressed but also produced a diffuse fluorescence excluding the nucleus, whereas EGFP-geph-L2C (C) formed intracellular aggregates. (D,E) In neurons, EGFP-geph-L2B also produced a diffuse fluorescence, but induced a variable reduction of endogenous gephyrin clusters (open arrowheads; compare D and E). Despite the reduction of gephyrin clusters, 2 subunit clusters still were detected on transfected dendrites (open arrows). In neighboring non-transfected dendrites, gephyrin clusters associated with the 2 subunit (arrows) were not affected. (F) EGFP-geph-L2C formed numerous clusters associated with the 2 subunit (yellow). Nearly all endogenous gephyrin clusters (blue) on transfected cells contained EGFP-geph-L2C; except intracellular aggregates, all EGFP-positive clusters were also colocalized with the 2 subunit (white; F'). (G) Co-staining for the 2 subunit (red) and synapsin-1 (blue) revealed that almost all EGFP-geph-L2C-positive clusters were postsynaptic. (H,I) PSD-95 staining (red) in combination with EGFP-geph-L2C (H) and EGFP-geph-GCE (I). As expected, no colocalization was apparent between the two markers. EGFP-geph-L2C transfected cells showed an increase in gephyrin clusters relative to control. (J-L) Reduced EGFP cluster size (J; cumulative distribution analysis) but increased density (K) in 6+6 cells transfected with EGFP-geph-L2C compared with EGFP-geph-GCE. (L) Opposite changes in EGFP-positive clusters versus PSD-95-positive clusters in EGFP-geph-GCE and EGFP-geph-L2C transfected neurons (mean ± s.d.). The significant increase of EGFP-geph-L2C cluster number was accompanied by a concomitant decrease of PSD95 clusters whereas the total number of postsynaptic sites remains unchanged. Bars, 10 µm (A-E); 5 µm (F-I).