Grp94 Regulates the Recruitment of Aneural AChR Clusters for the Assembly of Postsynaptic Specializations by Modulating ADF/Cofilin Activity and Turnover

HSP90 regulates AChR recruitment from aneural clusters to agrin-induced clusters. A, Representative images showing the differential contribution of diffuse and aneurally clustered AChRs to agrin bead-induced synaptic AChR clusters in control or 17-AAG-treated muscle cells using laser-based photobleaching approach. Green boxes indicate the magnified view of muscle cells with agrin bead contacts at different time-points for clarity. Yellow dotted-line boxes indicate the photobleaching region of aneural AChR clusters before agrin bead stimulation. Dotted lines highlight the periphery of muscle cells. 8-bit pseudo-color images highlight the relative fluorescence intensity of preexisting (old) and newly inserted (new) AChR signals in muscle cells contacted by agrin beads for 1 and 3 d. B, C, Quantification showing the fluorescence intensity of preexisting (B) and newly inserted (C) AChR signals at agrin bead-muscle contacts in control or 17-AAG-treated muscle cells, either with or without photobleaching of aneural AChR clusters before agrin bead stimulation; n = 11 (control, without photobleaching), n = 16 (control, photobleaching of aneural AChR clusters), n = 17 (17-AAG-treated, without photobleaching), and n = 21 (17-AAG-treated, photobleaching of aneural AChR clusters) muscle cells from three independent experiments. Scale bars: 10 μm. Data are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test. * and ** represent p ≤ 0.05 and 0.01, respectively. n.s.: non-significant.

Grp94 knock-down inhibits agrin bead-induced AChR clustering by modulating ADF/cofilin localization. A, B, Western blot analysis (A) and quantification (B) showing the expression level of Grp94 in WT, Control MO, and Grp94 MO embryos. β-Tubulin was used as the loading control for normalization. C, Representative images showing the intensity and complexity of aneural AChR clusters in response to Grp94 knock-down. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR clusters in control versus Grp94 knock-down muscle cells. Insets show the fluorescent dextran (Dex) signals, indicating the presence of MO. D, Quantification showing the effects of MO-mediated Grp94 knock-down on the formation of aneural AChR clusters in cultured muscles; n = 150 (WT), n = 143 (Control MO), and n = 150 (Grp94 MO) muscle cells from three independent experiments. E, Quantification showing the effects of MO-mediated Grp94 knock-down on the intensity and complexity of aneural AChR clusters; n = 64 (WT), n = 47 (Control MO), and n = 52 (Grp94 MO) muscle cells from four independent experiments for fluorescence intensity measurement (left y-axis); n = 44 (WT), n = 42 (Control MO), and n = 44 (Grp94 MO) muscle cells from four independent experiments for cluster complexity measurement (right y-axis). F, Representative sets of time-lapse images showing the effects of Grp94 knock-down on GFP-XAC localization in association with the dispersal of aneural AChR clusters (left panels) and with the formation of agrin bead-induced AChR clusters (right panels). 8-bit pseudo-color images highlight the change in fluorescence intensity of aneural AChR clusters after agrin bead stimulation for 4 h. G, H, Individual value plots showing the percentage change in the fluorescence intensities of AChRs (G) and GFP-XAC (H) in the same aneural AChR clusters among different conditions after agrin bead stimulation for 4 h; n = 12 (GFP-XAC alone), n = 9 (Control MO + GFP-XAC), and n = 11 (Grp94 MO + GFP-XAC) muscle cells from three independent experiments. I, Quantification showing the effects of Grp94 knock-down on agrin bead-induced AChR clustering (left y-axis) and GFP-XAC localization (right y-axis); n = 24 (GFP-XAC alone), n = 24 (Control MO + GFP-XAC), and n = 21 (Grp94 MO + GFP-XAC) muscle cells from four independent experiments. Scale bars: 5 μm. Data are shown as mean ± SEM (B, D, E, I) or mean ± SD (G, H). One-way ANOVA with Dunnett’s multiple comparisons test (B, E), two-way ANOVA with Tukey’s multiple comparisons test (D), two-way ANOVA with Sidak’s multiple comparison test (G, H) and one-way ANOVA with Turkey’s multiple comparison test (I). *, **, ***, and **** represent p ≤ 0.05, 0.01, 0.001, and 0.0001, respectively. n.s.: non-significant.

HSP90 inhibition accelerates ADF/cofilin turnover at different regions of aneural AChR clusters. A, Representative time-lapse TIRF images showing the fluorescence recovery of GFP-XAC signals after photobleaching the region of aneural AChR clusters (yellow rectangles, which are magnified in bottom rows with multiple timepoints) in control or 17-AAG-treated muscle cells. 8-bit pseudo-color images highlight the relative fluorescence intensity of GFP-XAC signals. B, C, Quantification showing the FRAP curves (B) and the calculated recovery half-time (C) of GFP-XAC signals at perforated and AChR-rich regions within aneural AChR clusters in control versus 17-AAG-treated muscle cells; n = 12 (Control) and n = 7 (17-AAG) muscle cells from three independent experiments. Scale bars: 5 μm. Data are shown as mean ± SEM. Student’s t test. * and ** represent p ≤ 0.05 and 0.01, respectively.

Postsynaptic Grp94 knock-down impairs synaptic structures and functions at developing NMJs. A, Representative images showing the effects of muscle Grp94 knock-down on nerve-induced AChR clustering. Fluorescent dextran signals indicate the presence of MO. Arrows indicate sites of nerve-muscle contacts. B, C, Quantification showing the effects of muscle Grp94 knock-down on the percentage of nerve-muscle contacts with AChR clusters (B) and the fluorescence intensity of nerve-induced AChR clusters (C) in 1-d-old Xenopus nerve-muscle co-cultures; n = 150 (WT), n = 146 (Control MO), and n = 191 (Grp94 MO) nerve-muscle contacts from three independent experiments for quantifying the percentage of nerve-muscle contacts with synaptic AChR clusters (B); n = 33 (WT), n = 31 (Control MO), and n = 33 (Grp94 MO) nerve-muscle contacts from three independent experiments for measuring AChR fluorescence intensity (C). D, Representative images showing the whole-cell patch-clamp recording on a Grp94 MO muscle cell innervated by a WT spinal neuron. Fluorescent dextran signals indicate the presence of MO. E, Representative electrophysiological recording traces of SSCs recorded from WT, Control MO, or Grp94 MO muscles that were innervated by WT spinal neurons. F, G, Quantification showing the effects of muscle Grp94 knock-down on the amplitude (F) and frequency (G) of SSCs. H–J, Cumulative distribution plots of the inter-event interval (H), 10–90% rise time (I), and decay time (J) of SSCs recorded from WT, Control MO, or Grp94 MO muscles innervated by WT spinal neurons. n = 10 (WT), n = 9 (Control MO), and n = 9 (Grp94 MO) nerve-muscle pairs from three independent experiments (F–J). Scale bars: 10 μm. “M”: muscle; “N”: neuron. Data are represented as mean ± SEM (B, C) and mean ± SD (F, G). One-way ANOVA with Tukey’s multiple comparisons test (B, C). Kruskal–Wallis ANOVA test with Dunn's multiple comparison test (F, G). * and *** represent p ≤ 0.05 and 0.001, respectively.