Figure 4.

AF-6 PDZ domain mediates interaction with kalirin-7 in neurons. a, In cortical neurons, exogenous kalirin-7 is targeted to punctate structures along dendrites, coexpression of AF-6 enhanced kalirin-7 clustering into puncta; coexpression with myc-AF-6-PDZ* reduced kalirin-7 clustering. b, Quantification of a: kalirin-7 integrated intensity per cluster (**p < 0.01 compared with control). c, Functional PDZ domain in AF-6 is necessary for N-cadherin activation-dependent recruitment of kalirin-7. Neurons transfected with myc-AF6-PDZ* were treated with clust FcNcad or untreated (control) and immunostained for kalirin-7 (green) and myc (purple). Insets show endogenous kalirin-7 puncta in dendrites of a control neuron and in clust FcNcad-treated neurons expressing myc-AF6-PDZ* (#) or nontransfected (##). The insets are from magnified regions denoted with dashed white lines. d, Integrated intensities of kalirin-7 clusters in c; quantification done for individual clusters and for all clusters within a segment of dendrite normalized to dendritic length; myc-AF-6-PDZ* prevents spine kalirin-7 recruitment by N-cadherin adhesion (***p < 0.001 compared with control; **p < 0.01 compared with clust FcNcad). e, Differential association of AF-6, kalirin-7, and PSD-95 with the synaptic cytoskeleton. P2 synaptosomal fraction was solubilized in buffers with detergents of different strengths, and soluble (S) or insoluble, particulate (P) fractions were separated by centrifugation. Presence in the S fraction indicates solubility in that detergent, whereas presence in P indicates association with the insoluble proteins. Error bars indicate SEM. Scale bars: a, c, insets, 10 μm; c, 20 μm.