Regulation of acetylcholine receptor clustering by ADF/cofilin-directed vesicular trafficking

Abstract

Postsynaptic receptor localization is crucial for synapse development and function, but the underlying cytoskeletal mechanisms remain elusive. Using Xenopus neuromuscular junctions as a model, we found that actin depolymerizing factor (ADF)/cofilin regulated actin-dependent vesicular trafficking of acetylcholine receptors (AChRs) to the postsynaptic membrane. Active ADF/cofilin was concentrated in small puncta adjacent to AChR clusters and was spatiotemporally correlated with the formation and maintenance of surface AChR clusters. Notably, increased actin dynamics, vesicular markers and intracellular AChRs were all enriched at the sites of ADF/cofilin localization. Furthermore, a substantial amount of new AChRs was detected at these ADF/cofilin-enriched sites. Manipulation of either ADF/cofilin activity through its serine-3 phosphorylation or ADF/cofilin localization via 14-3-3 proteins markedly attenuated AChR insertion and clustering. These results suggest that spatiotemporally restricted ADF/cofilin-mediated actin dynamics regulate AChR trafficking during the development of neuromuscular synapses.

Figure 1. Localization of ADF/cofilin in spontaneous and synaptic AChR clusters

(a) Representative DIC and fluorescent images of a 1-d old cultured Xenopus muscle cell showing spontaneous AChR clusters after Rh-BTX labeling. Insets: magnified regions. Arrow: striation; arrowheads: yolk granules. (b) The spatial pattern of spontaneous AChR clusters and XAC in Xenopus muscle cells after 5 d in culture. First and second rows: the distribution of AChRs (Rh-BTX labeling) and endogenous XAC and pXAC (immunostaining). Third row: the distribution of AChRs and GFP-XAC in a live muscle cell. Last row: AChR distribution and the cell volume labeled by DTAF. Asterisk: a site of volume reduction caused by a yolk granule. (c) Agrin bead-induced AChR clustering and XAC localization as revealed by immunostaining in fixed cells or live imaging. Arrow: GFP-XAC accumulation around the AChR clusters induced by an agrin bead. Arrowhead: GFP-XAC accumulation at an agrin bead contact even without AChRs. (d) The spatial distributions of AChR clusters and GFP-XAC at developing neuromuscular junctions in culture. GFP-XAC-expressing muscle cells (M⁺) were co-cultured with wild-type spinal neurons (N⁻) for 3 d. The nerve-muscle contacts were outlined by the dotted lines in the DIC image, which was overlaid with Rh-BTX-labeled AChR signals (red). Another example of AChR clusters and GFP-XAC signals from a different cell was shown in the bottom row. Insets: the boxed region was magnified and pseudo-colored after an intensity threshold. Scale bars: 40 µm (a, d); 10 µm (b, c).

Figure 2. Dynamics of ADF/cofilin in spontaneous and agrin-induced re-distribution of AChRs

(a) A time-lapse series showing the dynamic re-distribution of GFP-XAC and AChRs in two spontaneous clusters. AChRs were labeled with Rh-BTX before the start of recordings. For a better clarity, pseudo-colored images after an intensity threshold were shown in the insets. Arrowheads: the position of the original spontaneous cluster; arrows: the position of a newly formed spontaneous cluster. Numbers indicate elapsed time (in hour) in the recordings. (b) A time-lapse series showing the dynamics of GFP-XAC in the formation and dispersal of agrin-induced and spontaneous AChR clusters, respectively. Muscle cells were labeled with Rh-BTX and then stimulated by agrin beads at 0 h. Pairs of pseudo-colored images after intensity threshold were shown in the insets. Gray boxed regions were focused on the top of muscle cells where agrin beads made contacts with the muscle membrane. DIC images from the start and end of the recordings were included in the last column to show a slight lateral movement of beads (asterisks) on the muscle surface during the 40 h time-lapse recordings. Arrowheads: the position of the spontaneous cluster; arrows: the position of the bead-induced specialization. Numbers indicate time (in hour) after bead stimulation. Scale bars: 20 µm.

Figure 3. Regulation of actin dynamics by ADF/cofilin in spontaneous and synaptic AChR clusters

(a) Actin dynamics in the spontaneous AChR clusters as studied by dissecting different forms of F-actin: total F-actin, newly polymerized F-actin, actin barbed ends, and G-actin (top to bottom rows). Arrows: enrichment of F-actin in the AChR clusters; arrowhead: cell periphery. (b – d) Actin dynamics as revealed by paGFP-actin photoactivation. A cultured muscle cell expressing paGFP-actin was globally stimulated by UV light, resulting in a dramatic increase in paGFP-actin fluorescence intensity in the whole cell (b). When paGFP-actin was locally activated at a region (blue circle) containing the spontaneous AChR clusters in a muscle cell (c), fluorescent time-lapse imaging on photoactivated paGFP-actin revealed different rates of changes in fluorescence over time at three different regions as presented in the intensity plot (d; n=4). Analysis boxes: 1, AChR-rich region; 2, AChR-poor perforations; 3, background. (e) Spatial distribution of different forms of actin cytoskeleton in cultured muscle cells stimulated with agrin beads for 4 h. (f, g) Fluorescent images of paGFP-actin photoactivated in a region (blue circle) enclosing the bead-muscle contact (grey circle) in a muscle cell (f). The changes in fluorescence intensity of photoactivated paGFP-actin at three different regions are shown in the intensity plot (g, n=4). paGFP-actin signals in a region adjacent to the bead-induced AChR clusters were found on a different focal plane with that associated with AChR clusters, thus we used paGFP-actin in the striation structure for comparison. Analysis boxes: 1, striation region, 2: region adjacent to the bead-induced AChR clusters; 3: background. Scale bars: 10 µm. Error bars in (d) and (g) represent s.e.m.

Figure 4. Local enrichment of vesicular trafficking machinery and intracellular AChRs in spontaneous and agrin-induced AChR clusters

(a) Vesicular components in spontaneous AChR clusters as labeled with FM4-64 in live cells or EEA1 antibodies in fixed cultures. Pseudo-colored FM4-64 signals were highlighted and magnified in the inset. In the case of double staining with FM4-64, which emits red fluorescence, we used Alexa 488-BTX for AChRs. For the purpose of consistency, we reverted the colors such that FM4-64 is shown as green and AChRs as red. (b) Vesicular components surrounding the agrin bead-induced AChR clusters. After 4 h agrin bead stimulation, the muscle cells were stained with either FM4-64 or EEA1 antibodies. Insets: DIC images showing locations of bead-muscle contacts. (c, d) Surface and internal AChRs as revealed by differential double labeling. Cultured muscle cells were stimulated without (c) or with (d) agrin beads for 4 h. Surface AChRs were labeled with Rh-BTX and then saturated with unlabeled BTX. The cells were fixed, permeabilized and then the intracellular pool of AChRs (internal) was labeled with Alexa 488-BTX. Dotted lines represent the periphery of the cells where an agrin bead landed between two muscle cells. The spatial segregation of surface and internal pools of AChRs was clearly presented in the 3D intensity plots of their fluorescence intensities in the last column of each panel. Scale bars: 20 µm (a); 10 µm (b-d).

Figure 5. Time-dependent incorporation of newly inserted AChRs into the existing surface AChR clusters

(a) Existing and new AChRs as revealed by sequential double labeling. The existing AChRs (Old) were first labeled with Rh-BTX followed by a saturating dose of unlabeled BTX. After 0 or 4 h, newly inserted AChRs (New) were labeled with Alexa 488-BTX. Inset: a merged image of old and new AChRs highlighted by pseudocolors after an intensity threshold. (b) Paired images showing old and new AChRs at multiple time points after the sequential double labeling. (c) Quantification of the time-dependent incorporation of new AChRs into the old AChR clusters. A: area of the old AChR clusters. B: AChR-poor perforated regions in the clusters. The percentage of area with new AChRs at the perforated region (Green in Merge ÷ B) and the percentage of area with new AChR insertion and/or incorporation into existing AChR region (Yellow in Merge ÷ A) were plotted. Pearson’s co-localization coefficients between old and new AChR clusters at different time points were plotted. (d) Representative images showing old and new AChR clusters at the agrin bead contact. Paired images of old and new AChR clusters were taken at 4 and 20 h. (e) Pearson’s co-localization coefficients between old and new AChR clusters at multiple time points were plotted. Asterisks indicate significant differences (ttest, * p < 0.005; ** p < 0.001). Scale bars: 20 µm (a, b); 5 µm (d). Error bars in (c) and (e) represent s.e.m.

Figure 6. Regulation of agrin- and nerve-induced AChR clustering by ADF/cofilin activity

The cultured muscle cells over-expressed with wild-type or serine-3 phosphorylation mutant forms of GFP-XAC were stimulated with agrin beads (a, b) or spinal neurons (c, d). (a) A representative set of images showing AChR clusters induced by 4 h agrin bead stimulation in GFP-expressing muscle cells. Locations of agrin beads were outlined with dotted circles. (b) Quantification of the effects of XAC activity on agrin-induced AChR clustering. The percentage of agrin beads in association with those markers were scored if the respective markers were enriched at or around the bead contact sites. (c) A representative set of images showing AChR clustering on GFP-XAC-expressed muscles (M⁺) induced by co-culturing with wild-type spinal neurons (N⁻) for 1 d. The nerve-muscle contacts were outlined with dotted lines for clarity. (d) Quantification of the effects of XAC activity on nerve-induced AChR clustering by plotting the area of nerve-induced AChR clusters per a unit length of nerve-muscle contact. Numbers indicate the number of bead-muscle contacts (b) or nerve-muscle contacts (d) counted from at least 3 independent experiments. Asterisks indicate significant differences (t-test, * p < 0.005; ** p < 0.001). Scale bars: 20 µm. Error bars in (b) and (d) represent s.e.m.

Figure 7. Involvement of 14-3-3ζ in AChR clustering and ADF/cofilin localization. (a, b)

Representative sets of images showing the effects of different over-expression levels of GFP-14-3-3ζ on spontaneous (a) and agrin bead-induced (b) AChR clusters. Please note that the images of high GFP-14-3-3ζ expressing cells were taken using a reduced exposure to allow the examination of subcellular localization. The insets represent the images of the same cells, but acquired using the same exposure as that for cells expressing a low level of GFP-14-3-3ζ. One thus can appreciate the huge difference in the expression level of GFP-14-3-3ζ between these two groups. The magnified regions were pseudo-colored after an intensity threshold to show the differential localization of GFP-14-3-3ζ and AChRs (color insets). XAC immunostaining was performed in GFP-14-3-3ζ-expressed muscle cells stimulated with agrin beads. (c) Western blotting analysis on the 14-3-3ζ/β protein levels in the 14-3-3ζ morpholino (MO) knockdown experiment with GADPH as a loading control. Full-length blots are presented in Supplementary Fig. 12. (d) A similar disorganization of the spontaneous AChR clusters cultured from 14-3-3ζ morpholino embryos, as identified by fluorescent dextran signals. Ellipses (a, d) were drawn to outline the periphery of AChR clusters for the quantitative analysis in panel f. (e) A representative set of images showing the effects of 14-3-3ζ morpholino on AChR clustering and XAC localization induced by agrin beads. (f, g) Quantifications of spontaneous (f) and agrin bead-induced (g) AChR clusters in response to 14-3-3ζ over-expression or morpholino knockdown. Numbers indicate the number of samples counted from 2 independent experiments. Asterisks indicate significant differences (t-test, * p < 0.005; ** p < 0.001). Scale bars: 20 µm (a, d); 10 µm (b, e). Error bars in (f) and (g) represent s.e.m.

Figure 8. Regulation of surface targeting of new AChRs by ADF/cofilin activity and 14-3-3ζ

(a, c) Representative images showing newly inserted AChRs in agrin bead-induced (a) or spontaneous (c) AChR clusters in muscle cells expressing different XAC or 14-3-3ζ morpholino knockdown. The pre-existing surface AChRs were first masked with a saturating dose of unlabeled BTX. After 4 h, the cells were labeled with Rh-BTX and fixed to allow precise and reliable quantification of the newly inserted AChRs (New AChR) in a large number of cells at this particular time point. The muscle cells over-expressing wild-type or mutants of GFP-XAC were identified by GFP expression, whereas 14-3-3ζ morpholino knockdown was identified by fluorescent dextran signals. It should be noted that the exact subcellular localization of GFP-tagged proteins may be altered after fixation. The area of new AChRs was highlighted with red pseudocolors through the application of an intensity threshold in merge images (bottom rows). Locations of agrin beads were outlined with dotted circles in top panels. (b, d) Quantifications of XAC activity and 14-3-3ζ manipulations on surface targeting of new AChRs in agrin-induced (b) or spontaneous clusters (d). Areas of new AChR clusters at the bead-muscle contacts and at the spontaneous clusters were measured in the threshold images. Numbers indicate the number of bead-muscle contacts (b) or spontaneous clusters (d) measured from 2 independent experiments. Asterisks indicate significant differences (t-test, * p < 0.005; ** p < 0.001). Error bars in (b) and (d) represent s.e.m.