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The actin-driven movement and formation of acetylcholine receptor clusters.
Dai Z
,
Luo X
,
Xie H
,
Peng HB
.
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A new method was devised to visualize actin polymerization induced by postsynaptic differentiation signals in cultured muscle cells. This entails masking myofibrillar filamentous (F)-actin with jasplakinolide, a cell-permeant F-actin-binding toxin, before synaptogenic stimulation, and then probing new actin assembly with fluorescent phalloidin. With this procedure, actin polymerization associated with newly induced acetylcholine receptor (AChR) clustering by heparin-binding growth-associated molecule-coated beads and by agrin was observed. The beads induced local F-actin assembly that colocalized with AChR clusters at bead-muscle contacts, whereas both the actin cytoskeleton and AChR clusters induced by bath agrin application were diffuse. By expressing a green fluorescent protein-coupled version of cortactin, a protein that binds to active F-actin, the dynamic nature of the actin cytoskeleton associated with new AChR clusters was revealed. In fact, the motive force generated by actin polymerization propelled the entire bead-induced AChR cluster with its attached bead to move in the plane of the membrane. In addition, actin polymerization is also necessary for the formation of both bead and agrin-induced AChR clusters as well as phosphotyrosine accumulation, as shown by their blockage by latrunculin A, a toxin that sequesters globular (G)-actin and prevents F-actin assembly. These results show that actin polymerization induced by synaptogenic signals is necessary for the movement and formation of AChR clusters and implicate a role of F-actin as a postsynaptic scaffold for the assembly of structural and signaling molecules in neuromuscular junction formation.
Figure 1. Masking of preexistent F-actin structures by jasplakinolide pretreatment. (A–C) Competition between jasplakinolide and phalloidin for F-actin binding. (A) Rh-phalloidin labeling of myofibrils. (B and C) Pretreating the culture with 1 μM jasplakinolide followed by Rh-phalloidin blocked myofibril labeling, thus showing that these two toxins compete for F-actin binding. (D–G) Jasplakinolide pretreatment does not abolish new F-actin assembly in motile muscle cells. This cell was treated with 10 μM jasplakinolide for 3 h and rinsed with drug-free medium. The lamellipodia developed after jasplakinolide removal (compare D and E) exhibited F-actin assembly as shown by Rh-phalloidin labeling (G) at the end of the 24-h period. D and E are live images and F and G are fixed cell images. Phase–contrast of the cell at 24 h is shown in F. (H–K) In contrast to motile cells, this nonmotile cell showed no new F-actin assembly after jasplakinolide pretreatment. H and I are live images and J and K are fixed cell images.
Figure 2. F-actin assembly at AChR clusters induced by HB-GAM beads. A, C, F, and I, Rh-phalloidin labeling; B, D, G, and J, OG-BTX labeling. (A and B) F-actin at an AChR hot spot in a thin lamella region. Phalloidin-labeled myofibrils and F-actin at AChR cluster. (C, D, and F–J) Jasplakinolide pretreatment to mask preexisting F-actin before bead stimulation. The beads induced spatially discrete actin polymerization and AChR clustering as shown in two examples (C and D and F–H). In addition to sites of bead–muscle contacts, new F-actin assembly was also seen at filopodia and lamellipodia at the cell periphery (F, arrows). H is a phase image of the cell shown in F and G. At higher magnification (I and J), the punctate and circumferential pattern of new F-actin can be seen. The F-actin and AChR patterns are not congruent. (E) Quantification of F-actin and AChR induction by beads. The majority of beads induced both specializations (correlation coefficient 0.987). Data from 40 cells are pooled.
Figure 3. F-actin assembly induced by agrin after jasplakinolide pretreatment. Unlike beads, agrin induced a more diffuse F-actin assembly which paralleled the scattered AChR clustering pattern. (A and B) Examples of colocalization of agrin-induced F-actin and AChR patches (circles). However, the patterns of these two specializations are different despite their colocalization. (C and D) An example of the general lack of precise registration in these two specializations.
Figure 4. Cortactin-EGFP expression. (A and B) In unstimulated cells, cortactin was concentrated at membrane lamellae and often showed no colocalized with AChR hot spots. (C and D) Bead-induced AChR clusters were marked by cortactin localization. (E and F) Agrin induced association of cortactin with membrane areas encompassing AChR clusters.
Figure 5. Movement of bead-induced AChR clusters. One day after cluster formation induced by 5-μm beads, the clusters were followed for an additional 40 h. (A) Superimposed time-lapse image of the positions of four AChR clusters. (B–D) Corresponding phase–contrast images to show bead positions. Arrows in B point to two reference markers which showed little displacement during the 40-h period.
Figure 6. Trajectories of bead/AChR movement. (A and B) Four clusters and their associated beads were followed during a 24-h period. This image was taken at the end of the recording. The trajectories of these four beads are shown in C with the beginning and end points indicated. (D) Except for slow diffusional drift, the bead movement was abolished after Ltn A (1 μM) treatment. (E) MSD plotted against time. The linear slope of Ltn A data provides an estimate of the diffusion coefficient of the bead/AChR complex in the plane of the membrane. The solid curve is a regression fit of the control data using the equation described. From this, an actin-driven bead/cluster movement velocity of 0.1 nm/s is calculated. The R coefficient of the regression is 0.9985.
Figure 7. The effect of Ltn A on AChR clustering. (A and B) Rh-phalloidin labeling after jasplakinolide masking. Ltn A at 1 μM diminished bead-induced actin polymerization. (C) Change in fluorescence intensity of bead-induced AChR clusters as shown by OG-BTX labeling resulting from Ltn A treatment.
Figure 8. Inhibition of AChR clustering by Ltn A. (A–D) Effect on bead-induced cluster formation. (A and B) In the untreated culture, clusters were induced by the majority of beads. Asterisks in B indicate cluster-positive beads. At the same time, preexistent AChR hot spots were dispersed as shown by the lack of non-bead-associated clusters. (C and D) Ltn A at 40 μM completely abolished the effect of beads in inducing new clusters, whereas preexisting hot spots were retained (C, arrow). (E–H) Effect on agrin-induced cluster formation. Control culture exhibited AChR hot spots (E) that generally were much larger than agrin-induced punctate clusters (F). Ltn A blocked the effect of agrin in a dose-dependent manner (G and H). Hot spots (arrows) were retained in the presence of Ltn A. (I) Quantification of the Ltn A effect. Cytochalasin D at comparable concentrations was ineffective in inhibiting cluster formation (only bead data shown). (J) Reversal of the Ltn A effect. While Ltn A inhibited both bead and agrin-induced AChR clustering (middle bars), cells recovered from this blockage and responded to these stimuli to the same level as control cells following its removal (right bars).
Figure 9. Inhibition of AChR clustering by jasplakinolide. (A–D) Agrin-induced cluster formation was inhibited by jasplakinolide, but the hot spot was retained. (E and F) Bead-induced clustering was also inhibited. Clusters that formed in the presence of jasplakinolide (50 μM) were much smaller than control clusters. (G) Quantification of the inhibitory effect.
Figure 10. Disruption of PY accumulation by Ltn A. (A–D) Two examples of PY accumulation at HB-GAM bead-induced AChR clusters. (E–G) Ltn A (50 μM) treatment abolished AChR clustering (F) and PY accumulation (E) at bead–muscle contacts (G, asterisks), but left both AChRs and PY intact at preexisting hot spots (arrows).
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