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First published online 18 October 2006
doi: 10.1242/dev.02619

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The dynamics of recycled acetylcholine receptors at the neuromuscular junction in vivo

Department of Molecular, Cellular and Developmental Biology and Neuroscience Program, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA.

View larger version (39K): [in a new window]   Fig. 1. Recycled and pre-existing AChRs are removed at different rates from the same synapse. The sternomastoid muscle was labeled first with BTX-biotin/strept-488, then 3-4 days later with strept-594 to selectively label recycled AChRs. (A) High resolution image of a NMJ branch showing that recycled (red and yellow overlay) and pre-existing (retaining their strept-488 after initial labeling; green and yellow overlay) AChRs are intermingled in the postsynaptic membrane. (B) Preexisting and recycled AChRs labeled with different fluorophores were assayed for fluorescence intensities immediately after strept-594 labeling of recycled AChRs (d0), and 2 days later (d2). The total fluorescence intensity of each AChR pool was normalized to 100% at initial imaging. Pseudo-color images provide a linear representation of the density of AChRs. (C) Graph summarizing the results obtained from all junctions by the approach shown in B. Each data point represents the mean percentage of fluorescence intensity ±s.d. Note that recycled receptors are removed significantly faster than pre-existing receptors. Scale bars, 20 µm.

View larger version (31K): [in a new window]   Fig. 2. Insertion of newly recycled AChRs matches the removal of recycled receptors. (A) The sternomastoid muscle was labeled with BTX-biotin followed by a saturating dose of strept-488. Three to 4 days later, the animal was anaesthetized and the sternomastoid muscle bathed with a saturating dose of strept-594 to specifically label the recycled receptors; superficial synapses were imaged immediately, and again 2 days later, and the loss of recycled receptors determined. The sternomastoid muscle was then bathed with fresh strept-594, and the synapses re-imaged to determine the insertion of newly recycled AChRs. Scale bar, 20 µm. (B) Bar graph summarizing the recycled AChR loss and insertion results (±s.d.) obtained by the approach shown in A. Note that nearly all of the AChRs from the recycled pool lost over the 2 days were replaced by newly recycled receptors re-inserted into the postsynaptic membrane of the NMJ.

View larger version (117K): [in a new window]   Fig. 3. Fluorescent tags from recycled receptors are endocytosed and targeted to vesicles containing strept-488 from pre-existing AChRs. The sternomastoid muscle was labeled first with BTX-biotin/strept-488, then 3 days later with strept-594 to label recycled receptors. (A) Recycled and pre-existing AChRs imaged 2 days after recycled receptor labeling. Note that all the vesicles containing red fluorescence from recycled AChRs also have green signals from preexisting AChRs (arrows), whereas some of the green spots lack red signal (arrowheads). (B) A NMJ was labeled as in A and then imaged periodically over the next 28 hours. Note that a number of the vesicles containing strept-488 from the pre-existing AChRs at time 0 are stable over the next 28 hours (arrows), whereas others are transient. The strept-594 labels from recycled receptors are selectively targeted to the stable vesicles containing fluorescent label from pre-existing receptors. Scale bars, 20 µm.

View larger version (90K): [in a new window]   Fig. 4. Confocal images of a triply labeled NMJ. The sternomastoid muscle of a live animal was first labeled with BTX-biotin/strept-488 (green), then 3 days later with strept-594 (red) to label recycled receptors. Two days after recycled receptor labeling, the animal was perfused with 2% PFA, the sternomastoid muscle sectioned and immunostained with anti-AChR. Green arrows indicate accumulation of green fluorescent streptavidin that has been removed from pre-existing AChRs after internalization, which may correspond to degradative vesicles. Yellow arrows indicate accumulation of green and red streptavidin tags removed from pre-existing and recycled receptors after internalization, which probably also correspond to degradative vesicles. Blue arrows indicate vesicles containing AChRs, but lacking any streptavidin-Alexa tags; these vesicles may correspond to internalized AChRs in later stages of recycling and/or degradation, or newly synthesized receptors in the process of insertion. White arrows indicate vesicles containing receptors and their streptavidin tags after internalization. Colors in the overlay have been adjusted in Photoshop (Adobe) to maximize contrast. Boxed areas (above) are enlarged below. The results suggest that the vesicles containing fluorescent products (yellow arrows) may be stable, whereas those corresponding to cycling AChRs (white arrows) may be more dynamic. Scale bar, 20 µm.

View larger version (45K): [in a new window]   Fig. 5. Muscle denervation affects the contribution of recycled AChRs. (A) A denervated NMJ was labeled with BTX-biotin/strept-594 and imaged immediately. Three days later the synapse was re-imaged to measure the loss of fluorescence, then incubated with strept-594 to selectively label recycled AChRs. Note that 60% of the original fluorescence was lost after 3 days, whereas very little fluorescence was gained after strept-594 was added (6% of the original fluorescence), indicating that recycling of AChR is significantly decreased in denervated muscle. All three panels are displayed on the same intensity scale. (B) Bar graph summarizing the mean percentage of fluorescence intensity ±s.d. obtained from many denervated junctions. (C) An innervated synapse treated as in A, showing that a significant number of recycled AChRs return back to the postsynaptic membrane. (D) Bar graph summarizing measurements obtained from many innervated synapses.

View larger version (24K): [in a new window]   Fig. 6. Denervation increases the removal rates of recycled and pre-existing AChRs at the NMJ. (A) AChRs at denervated NMJs were first labeled with BTX-biotin/strept-488, and 3 days later recycled AChRs were labeled with strept-594. Fluorescence signals from both receptor pools were then imaged, and re-imaged 1 day later. (B) Bar graph showing the lifetime of recycled and pre-existing AChRs from the same synapses as assessed by the change in fluorescence over the 1-day period. In innervated synapses, recycled AChRs had an average half-life of 28 hours, whereas pre-existing AChRs had an average half-life of 102 hours. In denervated synapses, recycled AChR half-life dropped to 15 hours, and pre-existing AChR half-life decreased by the same proportion to 48 hours. Scale bar: 20 µm.

View larger version (20K): [in a new window]   Fig. 7. Muscle stimulation prevents loss of recycled AChRs from denervated NMJs. Three days after labeling with BTX-biotin/strept-488, recycled AChRs on denervated muscle were labeled with strept-594 and imaged immediately, and then again after 8 hours in the absence (A) and presence (B) of stimulation. Scale bar, 20 µm. (C) Graph summarizing the fluorescence measurements obtained from many synapses. After 8 hours, 66% of fluorescence from recycled receptors remained in denervated synapses; however, when denervated muscle was chronically stimulated, this loss of fluorescence was largely abolished.

View larger version (80K): [in a new window]   Fig. 8. Tyrosine phosphatase inhibition causes the accumulation of recycled receptors in the peri-synaptic membrane. (A) Live adult mouse NMJ previously saturated with BTX-biotin/strept-488 (green) at time 0 and viewed at high detector gain 9 hours later. The muscle was continuously bathed in the tyrosine phosphatase inhibitor PAO for 9 hours and then imaged. (B) The junction was then incubated with strept-594 (red) to determine if PAO affects AChR recycling. Perisynaptic areas were labeled (arrows), indicating that some of the recycled receptors had become mistargeted. (C) Overlay of pre-existing and recycled receptors (overlay images were adjusted for brightness and contrast using Photoshop). (D-F) Similar results were obtained using an alternative tyrosine phosphatase inhibitor, pervanadate. Note that whereas recycled receptors (red) appear in the peri-synaptic area, pre-existing receptors (green) remain restricted to the synapse. (G-I) When the serine/threonine phosphatase inhibitor okadaic acid was used instead, recycled receptors (red) were properly targeted to the synapse. Scale bars: 20 µm.