Cupidin-Drebrin interaction and colocalization in the spines of hippocampal neurons, and its inhibition by expressing a mutant with a deletion of the Cupidin Cdc42-binding domain. (A) Mouse Drebrin contains an actin depolymerization factor/cofilin-like domain (ADF) in the N-terminal region and two putative PPxxF Homer-binding motifs (592-PPPP ATF-596 and 674-PP PVF-678) in the C-terminal region. Both Drebrin-A and Drebrin-E were co-immunoprecipitated with anti-Homer1, 2 and 3 antibodies (Homer-mix) and an anti-pan Homer antibody that recognizes all three Homer family members (right panel). Immunoprecipitates with control rabbit IgG showed no Drebrin bands. (B) The GST-fused mouse Drebrin A C-terminal region (Drebrin-C WT, aa 579-706 containing two Homer ligand motifs 592-PP ATF-596 and 674-PP PVF-678) and its double mutant (Drebrin-C mutant with P593A and P675A) were bacterially expressed, electrophoresed and transferred to a nitrocellulose membrane followed by an overlay assay with GST-CPD protein extracts (see Methods). Drebrin-Cupidin binding was proven by Western blot analysis using anti-Cupidin (CPD C) antibody. Top panel, CCB stain; bottom panel, Western blot. (C) Immuno-fluorescent staining of endogenous Cupidin (green) and Drebrin (red) proteins colocalized in the dendritic spines of primary cultured rat hippocampal neurons. Bar: 5 μm. (D) GFP fluorescence (green) and Drebrin-immuno-fluorescence (red) images in the dendrites of hippocampal neurons overexpressing GFP, GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) by recombinant adenovirus infection. Bar: 5 μm. (E) The relative puncta number (top graph) and size (bottom graph) of Drebrin-positive puncta along dendrites overexpressing either GFP-CPD or GFP-CPDΔ191–230 (= GFP-Δ191–230) were quantified and normalized to those of dendrites overexpressing GFP alone, which was set to 1.0. *, p < 0.01.