The Journal of Neuroscience, August 2, 2006, ():
The Role of Protein Interaction Motifs in Regulating the Polarity and Clustering of the Metabotropic Glutamate Receptor mGluR1a
J. Neurosci. Das and Banker
26: 8115
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplementary Figure S1. The level of expression of mGluR1a following transfection. On average, the level of receptor expression in transfected neurons was similar to that in the small population of interneurons that endogenously express mGluR1a. A few transfected cells expressed at extraordinarily high levels (more than 10 times the endogenous level). Such cells were excluded from analysis and are not shown on this plot. Cultures were transfected after 7-9 days in vitro, and then stained for mGluR1a protein 5-7 days later. The average staining intensity in endogenously expressing cells was normalized to 1.0. The plots show the median value (the horizontal bar within the box), the two central quartiles (the box) and the range (the vertical line).
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Supplementary Figure S2. mGluR1a(wild-type) is clustered along dendritic shafts and at the tips of spines. Receptor localization on the cell surface (a) was compared with fluorescence due to soluble GFP (b), which fills shafts and spines. An overlay of the two images in shown in c. Cells were transfected after 8 days in culture, then stained for cell surface receptor 7 days later.
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Supplementary Figure S3. There was no significant difference in the expression levels of the wild-type or mutant mGluR1a constructs. As a measure of the efficiency of receptor expression on the cell surface, we quantified the intensity of cell surface staining and normalized this to the level of CFP fluorescence (which also includes all intracellular pools of the receptor). The value of this ratio for cells expressing wild-type receptor was taken as 1.0. Hippocampal neurons were co-transfected with CFP-tagged mGluR1a constructs and soluble YFP. Cells were transfected between 7-9 days in vitro, then stained for cell surface receptor 5-7 days later. The vertical bars represent the standard errors of the mean. The absolute values of CFP-protein expression and of cell surface expression were also similar among the various constructs (data not shown).
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Supplementary Figure S4. The algorithm used for identifying receptor clusters. The left panels illustrate sample images of receptor clusters from cells transfected with either mGluR1a(wild-type) or mGluR1a(ΔCT). The orange spots in the middle panel (“thresholded image”) show the pixels identified by the algorithm as having more than twice the average fluorescence intensity of the dendrite as a whole. Two parameters were used as a measure of receptor clustering: the area occupied by clusters (as a percentage of the total dendritic area) and cluster intensity (average intensity within clusters divided by the average intensity over the remainder of the dendrite). The values of these parameters for the specific images illustrated are given in the panels on the right.
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Supplementary Figure S5. Use of correlation analysis to quantify the co-localization of receptors with a postsynaptic marker. The upper panels show sample images of receptor and Homer staining from cells transfected with either mGluR1a(wild-type) or mGluR1a(ΔCT). The plots below show the intensity of mGluR1a staining (x-axis) and of Homer immunostaining (y-axis) for every pixel in the image that lies within the dendrites, along with the correlation coefficient (r-value) for each plot. For the cell expressing mGluR1a wild-type, pixels that are bright in the image of receptor staining tend to be bright in the image stained for Homer, and vice versa, so that the points fall along a straight line. For the cell transfected with mGluR1a(ΔCT) there is little correlation between brightness in the two images. The correlation plots were prepared and r-values calculated using Metamorph software.
