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The formation of acetylcholine receptor (AChR) cluster is a key event during the development of the neuromuscular junction. It is induced through the activation of muscle-specific kinase (MuSK) by the heparan-sulfate proteoglycan agrin released from the motor axon. On the other hand, DC electric field, a non-neuronal stimulus, is also highly effective in causing AChRs to cluster along the cathode-facing edge of muscle cells.To understand its molecular mechanism, quantum dots (QDs) were used to follow the movement of AChRs as they became clustered under the influence of electric field. From analyses of trajectories of AChR movement in the membrane, it was concluded that diffuse receptors underwent Brownian motion until they were immobilized at sites of cluster formation. This supports the diffusion-mediated trapping model in explaining AChR clustering under the influence of this stimulus. Disrupting F-actin cytoskeleton assembly and interfering with rapsyn-AChR interaction suppressed this phenomenon, suggesting that these are integral components of the trapping mechanism induced by the electric field. Consistent with the idea that signaling pathways are activated by this stimulus, the localization of tyrosine-phosphorylated forms of AChR β-subunit and Src was observed at cathodal AChR clusters. Furthermore, disrupting MuSK activity through the expression of a kinase-dead form of this enzyme abolished electric field-induced AChR clustering.These results suggest that DC electric field as a physical stimulus elicits molecular reactions in muscle cells in the form of cathodal MuSK activation in a ligand-free manner to trigger a signaling pathway that leads to cytoskeletal assembly and AChR clustering.
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???displayArticle.pmcLink???PMC3201969 ???displayArticle.link???PLoS One
Figure 1. The effect of DC electric field on AChR clustering along the cathodal edge of muscle cells.AChR clusters formed spontaneously in non-innervated muscle cells. The position of these receptor âhotspotsâ could not be predicted as in control cultures (A, B). DC electric field (direction indicated by arrow in C from anode + to cathode -) caused the formation of AChR clusters along the cathode-facing edge of the cell (arrowheads in D). A and C are phase images and B and D corresponding R-BTX images. (E) The time and electric field-strength-dependence of AChR clustering. Increasing field strength accelerated the clustering process with half-maximum response reached after 2, 1.5 and 1 hr at field strength of 2.5, 5 and 7.5 V/cm, respectively. In control cells not exposed to electric field, no polarized appearance of AChR clusters was seen.
Figure 2. QD-AChR single-particle tracking and trajectory analysis.Single QD-labeled AChRs appeared as bright dots on the surface of muscle cells (A). An example of a 2-D trajectory of AChR single-particle movement under electric field exposure (direction indicated in A) is shown in B, with its projection to the x-axis parallel to the field in C. (D) The plot of MSD and time in logarithmic scale. This plot leads to the calculation of Hurst exponent and diffusion coefficient from the slope and the intercept of the regression line that best-fitted data points as shown. The calculated value of Hurst index is 0.5, indicating that the particle movement conforms to Brownian motion.
Figure 3. Clustering of QD-labeled AChRs along the cathodal edge of the cell with time.An obvious asymmetry in AChR density as reflected by QD fluorescence intensity was observed after 20 min exposure (arrowheads in C). This asymmetry increased with time.
Figure 4. QD-AChR diffusion coefficients.(A) Overall diffusion coefficients. (B) The trajectories were broken down into two components, parallel and perpendicular to the electric field direction and the ratios of respective 1D movement were calculated. (C) Trajectories of QD-AChRs located within or beyond 15 µm of the cathodal edge where receptors were clustered were used in the calculation. (D) Again, trajectories broken down into components parallel and perpendicular to the field direction were used to calculate the ratios. For each bar, more than 50 trajectories were analyzed. Error bars are SEM. No difference in diffusion coefficients under different field strengths, proximity to the cathodal edge or trajectory orientation with respect to the electric field was observed. (p>0.1)
Figure 5. Hurst exponents of QD-AChRs.(A) Overall values calculated from QD-AChR trajectories. (B) Trajectories were broken down into components parallel and perpendicular to the electric field. (C-D) Analyses of trajectories close to or away from cathodal clustering edge. Despite differences in field strength, proximity to the clustering edge and trajectory orientation, the mean Hurst exponent values were uniformly at 0.5, indicating the motion of QD-AChRs is truly Brownian in nature under all conditions. Analyses of each data bar were based on at least 50 trajectories. Indicating lack of differences, the p-values are greater than 0.1.
Figure 6. Diffusion-mediated trapping of QD-AChRs on the surface of a muscle cell exposed to DC electric field.FITC-BTX was used to mark the position of the developing cluster (B). QD-labeled AChRs within the developing cluster were followed (region marked by rectangle in C). (D) Time-lapse recording of QD-AChRs within the region. During this approximately 5 min recording, some QD-AChRs were stationary and never moved (encircled). A moving receptor (marked by white arrow) showing Brownian motion during the first 3 min became immobilized and remained stationary after 3 min (indicated by white arrowhead). A stationary AChR (indicated by a star) was used as a reference point from which the distance of the mobile receptor (arrow or arrowhead in each panel) was measured (shown as d values, in pixels). (E) The trajectory of the mobile AChR shown in panel D is shown in higher magnification with the starting and stopping positions shown by arrow and arrowhead, respectively.
Figure 7. The role of rapsyn in DC-electric field induced AChR clustering.(A-C) Colocalization of rapsyn with electric field-induced AChR cluster along the cathodal edge of the muscle cell. (E-G) GFP expression did not perturb AChR clustering and rapsyn colocalization. (H-J) Exogenous expression of a full-length GFP-tagged rapsyn did not affect AChR clustering. (K-M) Expression of a GFP-tagged rapsyn whose coiled-coil AChR-interacting domain was deleted. This abolished the electric field-induced AChR clustering. (N) Quantification of these results. Number of cells scored: 89 for control cells, 76 for GFP-tagged full length rapsyn and 70 for GFP-tagged truncated rapsyn. Error bars are SEM. * p<0.001.
Figure 8. The effect of perturbing actin polymerization on electric field-induced AChR clustering.(A, B) A control cell showing cathodal AChR clustering. (C, D) Latrunculin A (LtnA) at 50 µM abolished electric field-induced cluster formation. (E, F) Jasplalinolide (Jas) at 50 µM also inhibited this process. Cells were pretreated for 1 hr and then maintained in medium containing LtnA or Jas throughout electric field application. (G) Quantification shows dose-dependent inhibition of cluster formation by these actin-disrupting toxins. More than 50 cells were examined in each treatment. * p<0.001.
Figure 9. Association of phosphotyrosine proteins at electric field-induced ACh clusters.(A-C) Localization of phosphotyrosine labeling at cathodal AChR clusters. Mab4G10 was used to label phosphotyrosine-containing proteins in C. (D-F) AChR β-subunit phosphorylated on Y390 (F) was prominently localized at cathodal cluster after two hours of exposure to electric field. (G-I) Localization of phospho-Src (pSrc) at cathodal clusters. An antibody that recognizes Src with its Y416 phosphorylated was used to localize active Src. pSrc was localized clearly at cathodal clusters (H and I).
Figure 10. The function of MuSK in electric field-induced AChR clustering.(A-C) Xenopus muscle cells expressing GFP only showed normal cathodal clusters (pointed by arrow on the right edge for the cell). (D-F) The expression of a kinase-dead MuSK abolished AChR clustering. (G-I) Full-length MuSK did not affect the cluster formation on the cathodal side. The expression of Trk B, either with its kinase domain truncated (J-L) or in full length (M-O), did not affect the cell's response to electric field. (P) Quantification based on the following number of cells examined, nâ=â105 (control), 77 (truncated MuSK), 86 (full-length MuSK), 94 (truncated TrkB) and 72 (full-length TrkB). Error bars are SEM. * p<0.01.
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