The Journal of Neuroscience, October 26, 2005, ():
Identification of Nicotinic Acetylcholine Receptor Recycling and Its Role in Maintaining Receptor Density at the Neuromuscular Junction In Vivo
J. Neurosci. Bruneau et al.
25: 9949
Supplemantal data
Files in this Data Supplement:
- supplemental material
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Supplemental Figure 1. There was no significant difference between the rate of loss of AChR labeling assessed with fluorescent BTX or with fluorescent Streptavidin/biotin-BTX. (A) C2C12 myotubes were saturated with bungarotoxin-Alexa 594 (top panels) and individual clusters were imaged. Eight hours later the same clusters were re-imaged and fluorescence was quantified. In the bottom panels, C2C12 myotubes were saturated with bungarotoxin-biotin followed immediately by streptavidin-Alexa 594. Individual clusters were imaged after labeling and at 8 hours and their fluorescence was assayed. (B) Graph represents the fluorescence remaining at BTX-Alexa (38% ±12.0 SD, n= 11 clusters/3 cultures) or BTX-biotin-streptavidin-Alexa (33% ±10.0 SD, n= 14 clusters/3 cultures). The amount of fluorescence remaining detected with the two labeling schemes was not significantly different (p > 0.2). The data show the mean ± SEM.
- supplemental material
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Supplemental Figure 2. Streptavidin/biotin BTX dissociation does not occur on stimulated denervated muscle. (A) Denervated muscle was labeled with bungarotoxin-biotin/streptavidin-Alexa 594 to saturation, and the fluorescence at an individual synapse was imaged and quantified (first panel). After initial imaging the muscle was stimulated for the duration of the experiment. After 8 hours the same synapse was located and imaged and the fluorescence was again quantified (second panel). To identify any receptors that had been recycled during the period of stimulation, new streptavidin-Alexa 594 was added to the muscle and the synapse was again imaged and fluorescence was quantified (third panel). (B) Control denervated muscle not stimulated but labeled and imaged exactly as described in A. (C) Summary of the data obtained from many synapses as in (A) and (B). Note that stimulation of the sternomastoid muscle prevented fluorescence loss and that there was no evidence for recycling AChR, while the fluorescence in unstimulated muscle NMJs lost ~20% of their original fluorescence over 8 hours, and the addition of new streptavidin-Alexa 594 resulted in a significant increase in fluorescence intensity. Values represent mean ±SEM.
- supplemental material
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Supplemental Figure 3. A proposed model suggesting the way activity affects the insertion of recycled and new AChRs at the neuromuscular junction. (A) During normal synaptic transmission, the recycled (4) and new receptor (1) pathways contribute equally to the synaptic density. (B) In the absence of postsynaptic activity, the recycled and new receptor pools decrease. After internalization (2) the receptors presumably shift from the recycling pathway to the degradative (3) pathway.
