Regulation of Acetylcholine Receptor Clustering by ADF/Cofilin- Directed Vesicular Trafficking Chi Wai Lee, Jianzhong Han, James R. Bamburg, Liang Han, Rachel Lynn, and James Q. Zheng Supplementary Figures Supplementary Figure 1: Endogenous expression of ADF/cofilin in Xenopus myotomal muscle tissues. (a) Western blot analysis showing the specificity of XAC and pxac antibodies in Xenopus myotomal muscle tissues. A single predominant band at the predicted molecular weight (~18 kd) of XAC or pxac was detected. (b) Expression of XAC in the muscle tissues as confirmed by RT-PCR analysis using a pair of specific primers for the XAC sequence. The presence of XAC mrna in the muscle tissues was evidenced by a band at a molecular weight of 236 base pairs (bp) in the PCR products. The band was absent in the negative control where no reverse transcriptase (-RT) was added during the cdna synthesis. M: markers. 1
Supplementary Figure 2: Preferential localization of non-phosphorylated XAC in AChR-poor perforations. Fluorescence images were taken with the same imaging settings in Rh-BTX-labeled muscle cells expressing wild-type (WT), constitutively active (3A) or inactive mutant (3E) of GFP- XAC. The ratio of GFP fluorescence intensities in the perforated (A) and other regions (B) of AChR clusters was quantified. The weak localization of GFP-XAC-3E is likely due to the effect of overexpression. Numbers indicate the number of cells measured in each group. Scale bars: 10 μm. 2
Supplementary Figure 3: Live confocal imaging of GFP-XAC in the spontaneous and agrin beadinduced AChR clusters. GFP-XAC-expressing muscle cells were labeled with Rh-BTX in live and imaged by Nikon C1 confocal system. Z-stack series were taken to show the differential subcellular localizations of AChR and GFP-XAC in the spontaneous (a) and agrin bead-induced clusters (b) in live cultured muscle cells. Scale bars: 20 μm. 3
Supplementary Figure 4: Specificity of latex beads coated with agrin C-terminal fragment in AChR clustering. Cultured Xenopus muscle cells were labeled with Rh-BTX for AChRs after control BSA-coated bead stimulation for 4 h. Neither AChR nor XAC was clustered at the bead-muscle contact sites (asterisks). The spontaneous AChR clusters retained in muscle cells (arrowhead). The AChR clustering activity by beads coated with BSA, full-length (Ag-FL) or C-terminal fragment of agrin (Ag- C 3,4,8 ) was quantified. Localization of AChR or XAC at the bead-muscle contacts was scored positive if its clusters were detected at or around the bead-contact regions after 4 h bead stimulation. Number indicates the number of bead-muscle contacts counted. 4
Supplementary Figure 5: Dynamic re-distribution of spontaneous AChR clusters in a cultured muscle cell. Muscle cultures were stained with Rh-BTX for AChR on day 1 before the start of the longterm time-lapse imaging and the labeled surface AChRs in a spontaneous cluster was monitored over a period of 7 d on the same muscle cell. The dynamic re-distribution of AChRs was reflected by the assembly and disassembly of spontaneous AChR clusters, marked as A, B, and C. Images were aligned in reference to the position of its cell nucleus (asterisks). The cell periphery was outlined with dotted lines in the AChR panels. 5
Supplementary Figure 6: Temporal difference between ADF/cofilin localization and AChR clustering induced by agrin beads. Cultured muscle cells were stimulated by agrin beads at 0 h to induce postsynaptic differentiation. (a) The cumulative percentage of bead contacts in association with localizations of GFP-XAC and AChR as revealed by quantification at a higher temporal resolution (20 min interval; 4 h duration) from 6 individual bead-muscle contacts. (b) Quantification of XAC localization in the spontaneous clusters and the bead-induced sites, showing an inverse relationship of ADF/cofilin localization between these two sites upon synaptic induction. Pool data were collected from over 20 muscle samples in each time point. 6
Supplementary Figure 7: Co-localization of ADF/cofilin and actin barbed ends in the perforated sites within the spontaneous AChR clusters. Triple staining of AChR, ADF/cofilin and actin barbed ends was performed by staining the muscle cells with Rh-BTX for AChRs in live, followed by labeling actin barbed ends with rhodamine-actin in a mild detergent saponin, and then fixed for XAC immunostaining. Quantification of multiple pairs of triple staining images showed the Pearson s colocalization coefficient between actin barbed ends and XAC was significantly higher than that between AChR and each of these markers. Asterisks indicate significant differences (t-test, * p < 0.005; ** p < 0.001). 7
Supplementary Figure 8: Suppression of agrin-induced AChR clustering by inhibition of membrane recycling. (a) Representative images showing the effects of an endocytosis inhibitor PAO (upper panels) and low temperature treatment (~4 o C; low panels) on agrin-induced AChR clustering. Arrows: bead-induced sites; arrowheads: spontaneous AChR clusters. (d) Quantification of the inhibitory effects on agrin-induced AChR clustering. The percentage of agrin beads in association with those markers were scored positive if the respective markers were enriched at or around the bead contact sites. Pool data were collected from over 90 bead contacts from 3 independent experiments. Asterisks indicate significant differences (t-test, * p < 0.005; ** p < 0.001). Scale bars: 10 μm. 8
Supplementary Figure 9: Experimental procedures to label different pools of AChRs. (a) An experimental protocol for the differential labeling of surface and internal AChRs in cultured muscle cells. Surface AChRs were labeled with Rh-BTX (red) and followed by a saturating dose of unlabeled BTX. The cells were fixed with 2% PFA and permeabilized with 0.5% Triton X-100. After blocking with 5% BSA for 1 h, the internal pool of AChRs was labeled with Alexa 488-BTX (green). (b) An experimental protocol for the sequential labeling of existing (old) and newly inserted (new) AChR clusters in live cultured muscle cells. Old AChRs were labeled with Rh-BTX (red) and then saturated with unlabeled BTX before agrin bead stimulation (if any). New AChRs were labeled with Alexa 488- BTX at 4 h after either bead stimulation or recovery. The changes in their localizations could be followed in multiple time points. 9
Supplementary Figure 10: Regulation of synaptic transmission by ADF/cofilin activity. (a) A set of example images showing the whole-cell patch-clamp recordings on a muscle cell over-expressed with one of the GFP-XAC constructs (M + ) that was innervated by a wild-type spinal neuron (N ). (b) Representative recording traces of SSCs (downward deflections of varying amplitudes) recorded from control as well as wild-type (WT), 3A, and 3E forms of GFP-XAC synapses. (c, d) Quantification of the effects of GFP-XAC manipulation in postsynaptic muscle cells on the amplitude (c) and frequency (d) of SSCs. Numbers indicate the number of nerve-muscle synapses measured from at least 3 independent experiments. Asterisks indicate significant differences (t-test, * p < 0.05). 10
Supplementary Figure 11: Working model on the spatial regulation of AChR clustering by ADF/cofilin-directed vesicular trafficking. Before nerve innervation, aneural AChR clusters are spontaneously formed and maintained on the surface of muscle membrane. Once the motoneuron approaches the target muscle cell, one of the nerve-derived factors, agrin, phosphorylates and activates the muscle-specific receptor tyrosine kinase (MuSK) via a putative protein called muscle-associated specific component (MASC). Recent studies have identified a low-density lipoprotein receptor-related protein Lrp4 as the receptor for agrin (Kim et al, Cell 135(2):334; Zhang et al, Neuron 60(2):285). The phosphorylation of MuSK signals through a cascade of downstream signaling pathways leading to the site-directed clustering of AChRs on the postsynaptic membrane, that involved the anchorage and stabilization of AChR clusters on a stable F-actin cytoskeletal scaffold through rapsyn. Concentration of synaptic AChRs at the postsynaptic sites is contributed, at least in part, by the re-distribution of AChRs from the spontaneous cluster and the delivery of newly synthesized AChRs in sub-synaptic 11
nuclei (as shown from the post-golgi vesicles). The re-distribution of AChRs from the aneural cluster to the synaptic site is likely regulated by a diffusion-trap mechanism on the cell surface and/or a regulated receptor trafficking mechanism by the vesicular machinery (transcytosis). In this study, we have identified a dynamic pool of cortical F-actin regulated by ADF/cofilin (AC) for the surface delivery of AChRs to the postsynaptic sites and to the spontaneous clusters. The activity of ADF/cofilin can be reversibly regulated on the serine-3 phosphorylation state by the counteracting actions of LIM kinase (LIMK) and Slingshot (SSH) or Chronophin (CIN) phosphatase. Non-phosphorylated active ADF/cofilin severs actin filaments and promotes actin assembly via generation of free barbed ends and monomeric G-actin. ADF/cofilin-associated dynamic actin turnover regulates the surface delivery of internal AChRs through the vesicular machinery. A scaffolding protein, 14-3-3ζ (ζ), is likely involved in the spatial localization of ADF/cofilin at the postsynaptic sites and in the aneural clusters. 12
Supplementary Figure 12: Knockdown expression of endogenous 14-3-3ζ/β by morpholino antisense oligos. Full-length western blots showed the reduction of 14-3-3ζ/β protein levels in cell lysates of 14-3-3ζ morpholino (MO) -injected embryos when compared to that of the control embryos. The membrane was stripped and re-probed for GADPH as a loading control. The predicted molecular weights of 14-3-3ζ/β and GADPH are ~29 kd and ~37 kd, respectively. Cropped blots were included in Fig. 7c. 13
Supplementary Videos Supplementary Video 1: Time-lapse movie of pagfp-actin in the spontaneous AChR clusters. A time-lapse series of fluorescence images was taken for 18 min at 1 min interval with using the same imaging settings. The movie was made at a playback rate of 2 frames per second (120 ). Supplementary Video 2: Time-lapse movie of pagfp-actin in the agrin bead-induced AChR clusters. A time-lapse series of fluorescence images was taken for 18 min at 1 min interval with using the same imaging settings. The movie was made at a playback rate of 2 frames per second (120 ). 14