Dai Z , Peng HB .

NS-23583 NINDS NIH HHS , R01 NS023583 NINDS NIH HHS

	Figure 2. Dispersal of AChR hot spots as a function of distance to its nearest bead-induced new cluster. (A) Two hot spots at positions a and b underwent dispersal in response to bead-induced new cluster formation. The arrows point to two bead-induced clusters which were nearest to these two spots. Hot spot a is closer to a developing bead-induced cluster (top arrow) than hot spot b (bottom arrow). As the time sequence shows, a is dispersed at a much faster rate than b. The image at 24 h was contrast-enhanced to illustrate the bead-induced cluster (arrows). The same is true of the last time point in C and D. (B) Quantification of hot spot dispersal. Two hot spots on the same cell, one at a distance of 20 μm (squares) and the other at 30 μm (circles) away from its nearest bead-induced cluster, were recorded throughout the dispersal process. Triangles were measurements of a hot spot from a control cell not treated with beads. Points of each dispersal process can be well fitted with a simple exponential decay curve: I(t) = I0e−t/τ, where I(t) and I0 are intensities at time t and 0, respectively, and τ is the time constant. This allows the calculation of the dispersal time constant. (C) A hot spot (a) immediately underneath a bead. It underwent dispersal quickly while new clusters formed under beads (arrows). (D) A hot spot (b) at a long distance (190 μm) from the closest bead. It showed very slow decrease in R-BTX fluorescence. Its intensity at 25 h was not much different from a control cluster (a). (E) Rate of dispersal of the two hot spots depicted in C (D = 0 mm) and D (D = 190 mm). The intermediate hot spot is from another cell. (F) Time constants of hot spot dispersal as a function of distance to nearest bead-induced AChR cluster. Each dot represents a single hot spot. Data were collected from 12 cells. A linear regression line is drawn through the data points. The correlation is highly significant with a correlation coefficient of 0.87 (P < 0.0001, Pearson product moment correlation test).
	Figure 3. Innervation-induced hot spot dispersal. (A) Top panel: phase contrast; second panel: time zero of observation period (after overnight nerve–muscle coculture); third panel: 24 h after observation started; fourth panel: the same 24-h image visualized with a 10-fold increase in excitation light intensity. On this innervated muscle cell (bottom cell in the top panel), the motor nerve (arrow) induced the formation of an AChR cluster at nerve–muscle contact (arrowheads in the third panel, 24 h) and a preexisting hot spot (arrows in 2nd to 4th panels) was undergoing dispersal. After 24 h, features of the dispersing hot spot were still conserved as pointed out by arrows. (B) Fine structure of a hot spot undergoing nerve-induced dispersal. Well-preserved structural features are pointed out by arrows. The 24 h image was taken with 10× excitation. (C) Change in hot spot intensity as a function of its distance to nerve–muscle contact. Fa and Fb, fluorescence intensity of hot spots (after background subtraction) at 0 and 24 h of observation period. A linear regression line is drawn through the data points. The correlation is significant with a correlation coefficient of 0.84 (P < 0.05, Pearson product moment correlation test).
	Figure 4. Effect of PTPase inhibitors on AChR cluster dispersal and formation. (A) PV at 50 μM prevented the dispersal of hot spots (two-tailed arrowheads) on a bead-stimulated muscle cell. This image was taken 24 h after bead treatment. Both bead-induced clusters (arrows) and hot spots coexisted. (B and C) Effect of PV on the formation of bead-induced clusters. AChRs, as well as phosphotyrosine labeling, were discretely concentrated at the site of bead stimulation in the control (B). PV (50 μM) caused AChR and phosphotyrosine cluster to assume a more scattered appearance (C). Small punctate aggregates were seen over a broad area around the bead. (D and E) PV effect on NMJ formation. AChR clusters form discretely at nerve–muscle contacts in the control (D). Clusters appeared more scattered in the presence of PV at 50 μM (E). Nerve–muscle contacts are shown in phase contrast on the left of each example. (F) Dose dependence of the PV effect on bead-induced cluster formation and dispersal. At 50 μM, PV nearly abolished the dispersal without significantly affecting the cluster formation. At higher concentrations, discrete clusters failed to form in the presence of pervanadate. The equation for fitting the hot spot data is described in the text. The data on bead-induced clustering were fitted with the equation: N = Nmax/(1 + exp[0.06{[PV] − 90}]), where Nmax and N are the percentage of beads with AChR clusters at 0 and a given PV concentration, respectively, and [PV] is the PV concentration. The curves are normalized to the control. (G) Effect of PAO (10 nM) on preserving hot spots. Both new clusters induced by beads (arrows) and preexisting hot spots (two-tailed arrowheads) coexist. Bead-induced clusters also assume a diffuse appearance under PAO treatment. (H) Dose-response of PAO effects on AChR formation and dispersal. The equation for fitting the hot spot data is the same as that for PV (F). The data on bead-induced clustering were fitted with the equation: N = Nmax/(1 + exp[0.56{[PAO] − 8.395}]).
	Figure 5. Effect of PTPase injection on cluster dispersal. (A) PTPase and FITC-dextran injection. The cell in the middle was injected with Yersinia PTPase together with FITC-dextran. Before injection, all cells in the field had AChR hot spots (arrows and two-tailed arrowhead in the top R-BTX panel). 24 h after PTPase injection (bottom R-BTX panel), the hot spot disappeared on the PTPase- injected cell, while they persisted on uninjected cells (arrows). (B) Control FITC-dextran injection. The hot spot on dextran-injected cell was unaffected 24 h after injection. (C) PTPase and FITC-dextran injection in the presence of PV (75 μM). The hot spot on the injected cell remained intact (two-tailed arrowheads). (D) Time course of hot spot dispersal resulting from direct PTPase injection compared with control cells. (E) Ratio of mean fluorescence intensity of AChR hot spots at 24 h after injection over its original state. Control (FITC-dextran), n (number of cells) = 11; PTPase, n = 11; PV+PTPase, n = 3. The difference between control and PTPase is highly statistically significant (P < 0.001).
	Figure 6. A model for PTPase in AChR cluster formation and dispersal. (A) In the absence of innervation, AChRs form clusters spontaneously. These sites are usually associated with phosphotyrosine concentration. The left side shows the level of either kinase activity (blue) or PTPase activity (red). The right side shows a lateral profile of the muscle cell. (B) Innervation or growth factor–coated bead locally activates receptor tyrosine kinases at the site of stimulation and also raises the PTPase activity in a diffuse manner. This results in AChR clustering discretely at the site of stimulation and the dispersal of AChR hot spots distally. (C) Inhibition of PTPase activity by pervanadate causes the diffusion of the kinase signal, resulting in more diffuse clustering process at the site of stimulation, and the retention of AChR hot spots. (D) Injection of exogenous PTPase raises the activity of this enzyme throughout the muscle cell and causes hot spot dispersal in the absence of synaptogenic stimulus.

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