The Journal of Neuroscience, July 19, 2006, ():
Differential Control of Postsynaptic Density Scaffolds via Actin-Dependent and -Independent Mechanisms
J. Neurosci. Kuriu et al.
26: 7693
Supplemental data
Files in this Data Supplement:
- supplemental material
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Figure 1 Relationship between GluR1 and GluR2 immunoreactivity and the extent of overexpression of EGFP-tagged scaffolding proteins.
Neurons infected with recombinant adenoviruses of four EGFP-tagged scaffolding proteins were fixed and stained with polyclonal anti-GluR1 (Chemicon) and monoclonal anti-GluR2 (Chemicon) antibodies. After reaction with secondary antibodies conjugated with Cy3 and Alexa633, GFP fluorescence and GluR1/GluR2 immunoreactivity were quantitated in single synapses by using confocal microscopy. GFP-positive clusters (20-30 clusters per cell) were randomly selected and the average fluorescence intensity of individual cells was plotted for 10 neurons. The plots indicate that there is no strong positive correlation between GFP intensity and the amount of GluR1 and GluR2 in single synapses except for PSD-95 (r=0.77, p=0.0089 for GluR1/PSD-95, r=0.41, p=0.23 for GluR2/PSD-95, r=0.19, p=0.59 for GluR1/GKAP, r=-0.16, p=0.65 for GluR2/GKAP, r=-0.17, p=0.63 for GluR1/Shank, r=-0.69, p=0.027 for GluR2/Shank, r=0.19, p=0.59 for GluR1/Homer, r=-0.095, p=0.79 for GluR2/Homer). In the case of PSD-95, we estimate that moderate overexpression of PSD-95 in our imaging experiments (corresponding to GFP intensity of 400-500 in this experimental condition) can potentially induce 9% increase of GluR1 and 6% increase of GluR2.
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Figure 2 Relationship between cluster dynamics and cluster intensity.
For individual clusters, we calculated CV of cluster intensities at different time points from t = 0 to 5.5 hr and the maximal fluorescence intensity of the clusters during the same period. The plots indicate that there is no strong correlation between the maximal cluster intensities and the dynamic property (r=-8.0X10-4, p=0.99 for PSD-95, r=0.12, p=0.62 for GKAP, r=-0.17. p=0.47 for Shank, r=-0.27, p=0.25 for PSD-Zip45, n=20 for each). The plots were based on time-lapse imaging of neurons maintained 16 days in culture.
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Figure 3 Effect of NMDA receptor inhibition on FRAP kinetics of four scaffolding proteins.
Neurons expressing EGFP-tagged scaffolding proteins were maintained in culture medium and FRAP analysis was performed on 10 neurons for each condition with their locations recorded by using XY stage controller. Cells were incubated with 100 μM of APV for 1 hr and the second FRAP experiments on different PSD clusters in the same set of neurons were performed in the presence of APV. No substantial difference of FRAP kinetics could be detected.
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Figure 4 Redistribution of GKAP L694A mutant by bicuculline/4AP treatment and suppression of its dynamics. A. Time-lapse images showing enhanced GKAP L694A clustering after application of bicuculline/4AP. Bar, 5 μm. B. Quantitation of fluorescence change after bicuculline/4AP treatment. C. FRAP analysis of neurons expressing wild type GKAP or GKAP L694A mutant with or without bicuculline/4AP treatment.Bicuculline/4AP induced significant suppression of FRAP kinetics in GKAP L694A expressing neurons. The extent of suppression was comparable to wild type GKAP.
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Figure 5 Unaltered localization of scaffolding proteins in neurons lacking NMDARs or mGluR5. A. Immunocytochemistry of NR1-/- neurons indicating absence of NR1, NR2A and NR2B clusters without disruption of PSD-95 clusters in the same dendrites. B. Immunocytochemistry of mGluR5-/- neurons. Clustering of PSD-Zip45, PSD-95, and Shank was not altered. In addition, colocalization of PSD scaffolding proteins was also unaffected. Bars, 5 μm.
