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. 2017 Dec 1;293(5):1551–1567. doi: 10.1074/jbc.M117.813808

Figure 7.

Figure 7.

CaMKII activity and AKAP150 lipidation state cooperate to mediate AKAP spine removal and structural plasticity during NMDA-induced LTD. A–E, CaMKII activity is required for NMDA-elicited AKAP150 depalmitoylation. DIV 14 hippocampal cultures treated with 50 μm NMDA showed reduced AKAP150 palmitoylation as assayed by ABE and BMCC palmitoylation assays. However, this decrease was blocked by tatCN21, a small peptide inhibitor of CaMKII activity (fold change relative to vehicle (Veh) control); ABE (A and B): NMDA, 0.27 ± 0.09, p < 0.01, one-sample t test; tatCN21, 1.14 ± 0.12, p > 0.05; NMDA + tatCN21, 0.72 ± 0.09, p > 0.05; IN, input; PD, pulldown after biotin-switch assay of palmitoylation; NS, not significant; **, p < 0.01. GluA1 palmitoylation was unaffected by NMDA treatment (C). Note: this assay involves labeling all palmitoylated proteins with biotin followed by pulldown with streptavidin beads, elution, SDS-PAGE, and finally Western blotting for proteins of interest; BMCC (D and E): NMDA, 0.31 ± 0.07, p < 0.001, one-sample t test; tatCN21, 1.34 ± 0.34, p > 0.05; NMDA + tatCN21, 1.47 ± 0.31, p > 0.05; IN, input; IP, immunoprecipitation for detection of palmitoylation. Note: this assay involves immunoprecipitating the protein of interest, followed by labeling the palmitoylated fraction of the protein with biotin, processing via SDS-PAGE, and Western blotting first with streptavidin-conjugated HRP and then antibody toward the protein of interest. Irreversible lipidation of AKAP79 blocks its NMDA-induced spine-to-shaft translocation and spine shrinkage. F, 12 DIV hippocampal neurons transfected with AKAP79WT-GFP or myr-AKAP79-GFP along with an mCh cell fill were imaged before and 10 min following 30 μm NMDA application. G, NMDA treatment reduced AKAP79WT spine-to-shaft ratio but had no effect on myr-AKAP79 (AKAP79WT, 0.81 ± 0.04; myr-AKAP79, 1.02 ± 0.04, p < 0.001 by unpaired t test). ***, p < 0.001. H, NMDA application resulted in a signification reduction in mean spine cross-sectional area in AKAP79WT-expressing cells but not in those expressing myr-AKAP79 (fold change relative to pretreatment; AKAP79WT, 0.83 ± 0.03; myr-AKAP79, 1.08 ± 0.09, p < 0.01 by unpaired t test). *, p < 0.05. I, there was no significant NMDA-induced change in the spine-to-shaft ratio of the mCh cell fill in either condition. Scale bar, 5 μm. NS, not significant.