The Journal of Neuroscience, March 1, 2006, ():
cAMP-Dependent Protein Kinase Postsynaptic Localization Regulated by NMDA Receptor Activation through Translocation of an A-Kinase Anchoring Protein Scaffold Protein
J. Neurosci. Smith et al.
26: 2391
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. No regulation of MAP2 dendritic localization by NMDA receptor activation in hippocampal neurons. (A) Cultured hippocampal neurons fixed and stained at 19-21 DIV for MAP2 (red) and synaptophysin (green) under control conditions. Small panels show high magnifications of the dendrites. (B) NMDA treatment (25μM NMDA for 3 min at 37°C) does not affect the localization of either MAP2 or synaptophysin immunostaining.
- supplemental material
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Supplemental Figure 2. NMDA treatment induces parallel redistribution of AKAP79 and PKA-RII away from dendritic spines in living hippocampal neurons. (A) Time-course 3D-imaging of chem-LTD regulation of AKAP79-YFP postsynaptic localization. Hippocampal neurons transfected with AKAP79-YFP (green) and PSD-95-CFP (blue) were treated with 25μM NMDA for 3 min at 33°C and then multi-plane Z image stacks were taken at 5 minute intervals after NMDA treatment. The images shown are 2D-projection images from these z-stacks showing PSD-95 puncta remain in dendritic spines while AKAP79 goes from membrane localized in dendrite shafts and spines to the cytoplasm in shafts. (B) AKAP79-CFP (blue) and PKA-RIIα-YFP (green) redistribute from dendritic spines and shaft membranes to the shaft cytoplasm over the same time-frame in response to NMDA treatment. Time-lapse imaging of hippocampal neurons treated with 50μM NMDA at 33°C and imaged at 5 minute intervals. Note some microtubular-like localization of PKA-RIIα-YFP fluorescence in the dendrite shafts at all time points.
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Supplemental Figure 3. Chem-LTD treatment induced redistribution of the AKAP79-PKA complex visualized using FRET microscopy. A, Cultured hippocampal neurons transfected with AKAP79-CFP (blue) and PKA-RIIα-YFP (green) imaged under control untreated conditions and after NMDA (25μM, 3 min) treatment (NMDA, 30 min.). The pseudocolor FRETc image shows that under control conditions, there is FRETc in dendritic spines as well as in cell soma membrane structures. After NMDA treatment, the FRET signal has moved away from the dendritic spine into the shaft and soma cytoplasm. B, Magnification of dendrites illustrating the redistribution of both CFP and YFP fluorescence and FRETc (arrows) from dendritic spines to the shaft 30 minutes after chem-LTD treatment. C, Quantitation of Spine/Shaft fluorescence ratio from neurons 15 and 30 minutes after chem-LTD treatment. The two time points were combined as there was no difference between them. FRETc spine/shaft ratio decreases from 1.37 ± 0.07 to 0.71 ± 0.04 after NMDA treatment. The AKAP79-CFP and PKA-RII-YFP fluorescence ratios change in the same manner (AKAP79-CFP: con 1.26 ± 0.04, NMDA 0.91 ± 0.04; PKA-RII-YFP: con 1.02 ± 0.07, NMDA 0.65 ± 0.05). ** p < 0.001, t test; control n = 10; NMDA n = 9 for all measurements.
