Figure 1.
The cytoplasmic side of Shisa9 interacts with multiple PDZ domain-containing proteins in a PDZ-ligand motif dependent manner.
A. Schematic representation of Shisa9 and the two Shisa9 cytoplasmic domains (cd) used within the yeast two-hybrid screen and direct two-hybrid assay (SP, signal peptide; TM, transmembrane domain; EVTV, C-terminal PDZ-ligand motif. B. Putative Shisa9 interactors (Gene symbol, recommended Uniprot name) selected for validation, as identified by yeast two-hybrid. The “clone count” represents the number of hits in the screen, the “start position” refers to the first amino acid of the protein's reference sequence (Protein Refseq) conserved within the direct two-hybrid clone, and the “PDZ domains” column lists the number of complete PDZ domains anticipated within that clone. C. Direct two-hybrid assay performed under stringent nutritional selection (–LTAH). The red coloration results from the cell's inability to activate the adenine reporter gene.
Figure 2.
Validation of interaction between Shisa9 and its putative interactors by means of co-immunoprecipitation from HEK293T cells.
A. Schematic view of Shisa9-constructs used in co-immunoprecipitations. SP, signal sequence; TM, transmembrane domain; HA, HA-tag; EVTV, PDZ-ligand motif. B. Co-immunoprecipitation of Shisa9-interactor complexes from HEK293T cells. HA-Shisa9WT and HA-Shisa9ΔEVTV were overexpressed in HEK293T cells in combination with interacting proteins (one at a time). Anti-HA-tag antibody was added to immunoprecipitate HA-Shisa9-interactor complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-V5 antibody against V5-tagged interactors. Shisa9WT co-immunoprecipitates with PSD95, PSD93, GRIP1, PICK1 and Lin7b proteins, whereas Shisa9ΔEVTV lost the possibility to establish the interaction with named proteins (left panel). The right panel shows the same membranes as in the left panel stained with the anti-HA antibody in order to visualize the presence of Shisa9 in the immunoprecipitated samples. The 50 kDa band is indicated.
Figure 3.
Validation of the interaction of Shisa9-PSD95 the brain.
Shisa9 forms a complex with PSD95 in the hippocampus and cortex. Anti-Shisa9 antibody was added to the mouse cortex and hippocampus lysates to immunoprecipitate native Shisa9 complexes. Obtained samples were resolved on SDS-PAGE, western blotted and immunostained with anti-PSD95 antibody. The 75 and 100 kDa bands are indicated.
Figure 4.
TAT-Shisa9 C-terminus mimetic peptide disrupts interaction between Shisa9 and recombinant PSD95.
A. The TAT-tagged Shisa9WT C-terminal mimetic peptide, but not the TAT-scrambled and TAT-Shisa9ΔEVTV peptide, competes off the interaction between recombinant PSD95 and the biotinylated Shisa9WT peptide. The 100 kDa band is indicated. B. Quantification of the PSD95 band in the presence of TAT-scrambled, TAT-Shisa9WT or TAT-Shisa9ΔEVTV peptide. PSD95 band intensities were normalized to the intensity of PSD95 band in TAT-scrambled peptide lane. All experiments were performed 3 times.
Figure 5.
Partial disruption of C-terminals Shisa9 interaction affect AMPAR mediated currents in denate granule cells.
A. Diagram showing the recording site and the electrically stimulated fibers of the lateral perforant path (dark blue; adapted from [38]. B. Example traces of AMPAR-mediated EPSCs after incubation with either the TAT-Shisa9WT (active) or the TAT-Shisa9ΔEVTV (control) peptide. Traces were aligned to the onset of the current. C, D. Bar graphs (mean±SEM) summarize the changes in rise- and decay kinetics vs. the kinetics of the control pulse. ** p<0.01 (Student's t-test). E. Representative recordings of a paired-pulse protocol at different stimulation intervals after incubation with either the TAT-Shisa9WT (purple) or the TAT- Shisa9ΔEVTV (blue) peptide. The dotted line indicates the amplitude of the first pulse. Note the decreased paired-pulse facilitation for the TAT-Shisa9WT peptide. F. Averages (±SEM) summarizing the differences in paired-pulse ratio facilitation at different inter-event-intervals. *** p<0.001, * p<0.05 (Post-hoc testing).
Figure 6.
Shisa9 increases the synchrony of DHPG-induced hippocampal oscillations via PDZ domain interactions.
A. Wavelet display of recorded field potentials of DHPG-induced oscillations under the 3 experimental conditions: Control (no peptide application, top trace), PDZ interacting peptide TAT-Shisa9WT (middle) and inactive form of the peptide TAT-Shisa9ΔEVTV (bottom). Warmer colors indicate higher oscillation amplitude (dimension-less units). B. Comparison of the power spectral density of the DHPG-induced oscillations in the 3 experimental conditions: control (light blue), TAT-Shisa9WT (purple), TAT-Shisa9ΔEVTV (dark blue). C. TAT-Shisa9WT peptide significantly increases the spectral amplitude of DHPG-induced hippocampal oscillations with respect to no peptide application, as well as with respect to the inactive peptide. D. Application of TAT-Shisa9WT peptide has no significant effect on the frequency. E. TAT-Shisa9WT peptide significantly narrows the spectral half-width with respect to control conditions. *p<0.05 (Student's t-test).