Fig 2: Aneural versus synaptic AChR clusters in MT1-MMP-/- diaphragm muscles at E15.5.(A) Representative confocal images showing the AChR pre-patterns (red) and synaptic AChR clusters (yellow) in whole-mount diaphragms from control and MT1-MMP-/- mice at E15.5. Whole-mount tissues were stained for AChR and neurofilament (NF). (B-E) Quantification on the density of aneural (B) versus synaptic (C) AChR clusters, the width of end-plate bands (D), and the length of axonal branches (E) in diaphragm muscles between wild-type and MT1-MMP-/- mouse embryos. n = 3 (WT) and 1 (MT1-MMP-/-) embryos from 1 experiment only. Data are represented as mean ± SEM (WT). Scale bar represents 50 µm.

Fig 3: Postsynaptic MT1-MMP is required for the recruitment of aneural AChR clusters to the assembly of synaptic AChR clusters.(A) A schematic diagram illustrating the use of laser-based photobleaching approach to differentially identify the contribution of aneural AChR clusters and diffuse AChRs for the assembly of nerve-induced synaptic AChR clusters. (B) Representative images showing the differential contribution of aneural AChR clusters and diffuse AChRs to nerve-induced synaptic AChR clusters in control co-cultures (WT) and in the chimeric co-cultures of wild-type neurons and muscle cells with control MO or MT1-MMP MO. Yellow boxes (right panels) indicate the region of photobleaching. White boxes show a magnified view of nerve-muscle contacts in 1-d old co-cultures. Dotted lines highlight the periphery of muscle cells. Fluorescent dextran signals in the insets indicate the muscle cells with microinjected MO. 8-bit pseudo-color images highlight the relative fluorescence intensity of AChR signals. Arrows indicate synaptic AChR clusters at nerve-muscle contact sites. Arrowheads indicate the original location of aneural AChR clusters. (C) Quantification on the intensity of AChR signals at the nerve-muscle contacts in control co-cultures and in chimeric co-cultures either with or without photobleaching. The control groups without photobleaching indicate the contribution from aneural AChR clusters + diffuse AChRs, n = 15 (WT), 9 (Control MO), and 9 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. The experimental groups with photobleaching of aneural AChR clusters indicate the contribution from diffuse AChRs only, n = 23 (WT), 13 (Control MO), and 8 (MT1-MMP MO) nerve-muscle pairs from 3 independent experiments. Scale bars represent 10 µm. Data are represented as mean ± SEM. Two-way ANOVA with Sidak’s multiple comparison test, *, ** represent p=0.05 and 0.01 respectively.

Fig 4: Localization of PLS core and cortex markers at AChR clusters in postnatal mice.Representative confocal images showing the enrichment of PLS core marker, ADF/cofilin (top panels), and cortex marker, talin (bottom panels), at synaptic AChR clusters in longitudinal cryosections of hindlimb muscles isolated from P11 wild-type or MT1-MMP-/- mice. Maximum projection intensity images of 12 z-stack confocal frames with 0.48 µm intervals were constructed using ZEN 2.3 (Carl Zeiss). Scale bar represents 10 µm.

Fig 5: Intracellular trafficking of MT1-MMP is directed to PLS-associated AChR clusters.(A) Representative images showing the intracellular trafficking of MT1-MMP vesicles in a cultured muscle cell expressing both MT1-MMP-mCherry (MT1-mCherry) and EB1-GFP. The time-lapse series of two regions (i and ii) outlined in the merge image showed (i) the search-and-capture of MT1-mCherry vesicles (arrows) by an EB1-GFP comet (arrowheads); and (ii) the bidirectional movement of MT1-mCherry vesicles (arrows) along the microtubules (arrowheads). (B) Kymographs showing the spatiotemporal correlation between MT1-mCherry and EB1-GFP signals along K1 and K2 lines indicated in the merge image. The maximal projection of MT1-mCherry and EB1-GFP signals was constructed from 40 frames in a 40 s time-lapse series. Arrows indicate the examples of lateral displacement of initially stationary MT1-mCherry vesicles after EB1-GFP comets had passed through. (C) Representative TIRF-FRAP images showing the local capturing of MT1-mCherry vesicles at AChR clusters. After photobleaching, the recovery of MT1-mCherry signals and their trajectories in two regions of interest indicate (i) MT1-mCherry vesicles (arrows) were transported to and captured at the perforation of AChR clusters; and (ii) two groups of MT1-mCherry vesicles (red and green arrows) were transported to and captured at the same site of AChR cluster periphery over a period of 120 s. (D) Kymographs showing the spatiotemporal capture of MT1-mCherry vesicles at AChR clusters. Two kymographs were constructed from 120 time-lapse images along K3 and K4 lines, as indicated in the merge image (top panels in C). Arrowheads and arrows indicate the sites of MT1-mCherry capture at the perforated and peripheral regions of AChR clusters, respectively. Scale bars represent 10 µm, unless stated otherwise.