The physical interaction partners Abp1 and ProSAPs/Shanks cooperate in controlling mushroom-type spine morphology. A–F, Images show enlarged heads of mushroom-shaped spines of primary hippocampal neurons at 14 DIV during overexpression of ProSAP1 (C) and ProSAP2 (D) compared with spine heads of mRFP-expressing cells (control, A) and Abp1-expressing cells (B). Coexpression of ProSAP1+Abp1 (E) and ProSAP2+Abp1 (F), respectively, led to an additional promotion of ProSAP-induced head extension and to a decrease in Abp1-induced spine neck elongation. Images represent the fluorescence signals of cell-filling molecules cotransfected to highlight the morphology. Bottom rows in C–F show the fluorescence signals of GFP–ProSAP1 and GFP–ProSAP2. G, H, Diagrams depict the means ± SEM for the length of mushroom spines (G) and the head width of mushroom spines (H) in percentage deviation from mRFP-transfected control cells for neurons expressing ProSAP1, ProSAP2, Abp1, or combinations thereof as indicated. *p < 0.05; **p < 0.01; ***p < 0.001. Scale bar, 5 μm.