The Journal of Neuroscience, June 28, 2006, ():
Lateral Diffusion Drives Constitutive Exchange of AMPA Receptors at Dendritic Spines and Is Regulated by Spine Morphology
J. Neurosci. Ashby et al.
26: 7046
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure S1. Intracellular acidification during low pH wash. (A) Image shows distribution of free SEP throughout the cell interior. Graph shows pooled and averaged (meanąs.e.m.) changes in fluorescence of SEP (and hence cytoplasmic pH) when the cell is exposed the low pH wash. Inset show the averaged fluorescence changes in paired spines and neighboring shaft areas. (B) Typical traces from different cells expressing either SEP-GluR2 or free SEP allowing direct comparison of the effects of low pH wash on fluorescence in a spine head and in the neighboring dendritic shaft. Note that the effect on the fluorescence of intracellular free SEP is minimal compared to the instantaneous effect on SEP-GluR2.
- supplemental material
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Supplemental Movie S2. Three-dimensional projected images of SEP-GluR2 in spiny dendritic region showing the rapid loss of surface fluorescence caused by wash with acidic buffer (pH 6.0). Note the rapid loss and recovery of fluorescence in spine heads when the buffer is added and then removed. There are 10 seconds between each frame.
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Supplemental Movie S3. Three-dimensional projected images (shown in glow scale) from experimental time-course showing that photobleached SEP-GluR2 AMPARs on the surface of spine heads (in white circle) are constitutively exchanged for fluorescent AMPARs. There are 30 seconds between each frame and bleaching occurs at between 3rd and 4th frame.
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Supplemental Figure S4. Recovery of SEP-GluR2 following bleaching is blocked by cell fixation. (A) Images from FRAP experiment on SEP-GluR2 in spiny dendrite of a neuron that had been fixed with 4% paraformaldehyde for 15 minutes. There is little or no detectable fluorescence recovery in the bleached spines (white circles) (B) Pooled and averaged (meanąs.e.m.) FRAP recovery curve for spine head regions from fixed cells. There is little fluorescence recovery after bleaching indicating that FRAP is not due to recovery of fluorescence of individual bleached molecules.
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Supplemental Figure S5. Bleaching causes no detectable photodamage. (A) Time-course of SEP-GluR2 fluorescence from a region exposed to three consecutive bleaches. Note the similarly-shaped recovery after each event. (B) SEP-GluR2 mobile fractions from individual experiments with multiple bleach events. (C) Pooled data (meanąs.e.m.) of mobile fractions relative to the first of consecutive bleaches. There are no differences in mobile fraction caused by repetitive bleaching, indicating that there is little or no photodamage.
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Supplemental Movie S6. Experimental sequence of SEPGluR2 fluorescence (shown in green) from single confocal plane at edge of cell body (shown in Figure2A). Bleaching of SEPGluR2 fluorescence (in red circle) is followed by rapid recovery of fluorescence along the membrane from the edges of bleach region towards the centre. There is a 5 second interval between images and bleaching occurs between 3rd and 4th frame. Total length of movie is 4 minutes.
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Supplemental Figure S7. Histogram showing the mean ąs.e.m. of the area of stubby and mushroom spine heads measured using fluourescence from SEP-GluR2 and pmGFP. There are no measurable differences between the sizes of the heads of either group of spines.
