Supplemental Materials
This article contains the following supporting material:
- Supplemental Materials
- Movie03 - Video 3. Time-lapse images of Rhodamine Red-streptavidin-biotin-α-BTX-labeled AChR clusters in Plec+/+ myotubes upon induction of IF network collapse. Composite time-lapse image series (each in 15 s intervals) of agrin-induced AChR clusters (Rhodamine Red-streptavidin-biotin-α-BTX-labeled) in Plec+/+ myotubes upon addition of OA (top panel), addition of WFA (middle), or forced expression of P1f-Ins16-GFP (bottom). Myotubes shown in top and middle (but not bottom) panels were expressing desmin-GFP. Note immobile (collapsed) desmin networks visualized by desmin-GFP, and of similar structures visualized by P1f-Ins16-GFP. In contrast, the majority of microclusters were mobile in all 3 cases. White arrowheads (appearing once or sequentially) monitor examples of highly mobile microclusters that constantly undergo shape changes (apparently due to repeated collisions or short-lived fusions with other clusters) during time intervals 00:00-28:30 (top), 00:00-10:00 and 23:00-28:30 (middle), and 00:00-24:45 (bottom). Magenta arrowheads mark vanishing microclusters in time intervals 04:00-08:45 (top), 10:45-23:00 (middle), and 25:00-28:30 bottom).
- Movie10 - Video 10. Recording of bottom, and left and right side views of a Pax7-Cre/cKO mouse swimming along an elongated rectangular tank. Note, in this case the mouse is swimming along the wall of the tank and, instead of primarily using its HLs (like its wt counterpart, see Video 9), it extensively uses its FLs for swimming. Note also the stiff tale (kept mostly outside the water) had lost its navigating function, probably due to complete absence of oscillating movements.
- Movie09 - Video 9. Recording of bottom, and left and right side views of a wt mouse swimming along an elongated rectangular tank. The movement of the mouse is characterized by well-coordinated HL strokes, continuous sinusoidal tail oscillations, and only sporadic FL strokes. Note, the animal swims predominantly in the central region of the tank.
- Movie02 - Video 2. Time-lapse images of Rhodamine Red-streptavidin-biotin-α-BTX-labeled AChR clusters in a desmin-GFP-expressing Plec-/- myotube. Images were taken as in Video 1 during a period of 2 h following the withdrawal of agrin. The first sequence shows two microclusters (white and yellow arrowheads) dissociating from a larger cluster (time points 00:03:30 to 00:06:45), transiently coalescing with each other (time points 00:10:45 to 00:12:00) and finally independently drifting away from the original cluster (time points 00:24:15 to 00:28:00). During diffusion, one of the microclusters (white arrowhead, time point 00:27:00) becomes fragmented into three independent smaller clusters (time point 00:27:30) that re-fuse (00:28:00), and migrate further away, transiently assembling with other microclusters (white arrowhead, interval between 00:33:30 and 00:57:15). Between time points 00:59:45 to 01:13:15, another microcluster (magenta arrowhead) separates from the large cluster and rapidly migrates away from it (01:13:30 to 01:20:45) and eventually disappears (time point 01:21:15). Shortly afterwards, an additional microcluster (cyan arrowhead, time interval 01:28:45 to 01:54:00) splits off from the large cluster and transiently aggregates with other microclusters, while continuously alternating its shape and location.
- Movie07 - Video 7. Spatial (3D) network organization of NMJ-proximal desmin IFs in a wt muscle fiber. 3D reconstruction of confocal Z-stack images of wt soleus myofibers labeled for desmin (red), the nerve terminal marker synaptophysin (blue), and AChR (Alexa 488-α- BTX, green), using Huygens software. Bar, 10 μm.
- Movie01 - Video 1. Time-lapse images of Rhodamine Red-streptavidin-biotin-α-BTX-labeled AChR clusters in a desmin-GFP-expressing Plec+/+ myotube. Images were taken (at 15 s intervals) during a period of >50 min after withdrawal of agrin. Arrowhead, large AChR cluster (red) embedded in desmin-GFP network. Note compact AChR cluster (stably incorporated into the desmin IF lattice), maintaining its condensed morphology over the entire time period.
- Movie04 - Video 4. Time-lapse images (15 s intervals) of agrin-induced Rhodamine Redstreptavidin- biotin-α-BTX-labeled AChR clusters in a P1f-GFP expressing Plec-/- myotube. Note recruitment and immobilization of AChR clusters in the P1f-GFPenriched region of the sarcolemma. White arrowhead, large cluster remaining immobile over entire observation period (01:38:00). Cyan arrowhead, a formerly P1f-unlinked highly mobile microcluster (time points 00:18:45-00:20:45) becomes “trapped” and immobilized at the P1f-enriched region (time points 00:26:30-01:38:00).
- Movie05 - Video 5. Time-lapse imaging of AChRϵ-GFP-labeled AChR clusters in a Plec+/+ myotube. Images were collected in 2 s intervals upon agrin addition. Stepwise fusion of a larger cluster with a smaller cluster (outlined in yellow) is shown (time interval 00:00- 08:00). After fusion, the cluster remains in a compact state during the rest of the observation period (>19 min).
- Movie06 - Video 6. Time-lapse imaging of AChRϵ-GFP-labeled AChR clusters in a Plec-/- myotube. Conditions were as in Video 5. Two concatenated versions of one video sequence are shown. In the first version yellow circles outline two loose receptor aggregates undergoing continuous shape changes due to fragmentation into smaller, mobile particles. Note upon coalescence of both clusters (time interval 03:10 06:00) the structure breaks apart into two smaller clusters (time point 06:42) migrating away from each other. In the second version, a different cluster gradually reducing its size and density (outlined in yellow).
- Movie08 - Video 8. Spatial (3D) network organization of NMJ-proximal desmin IFs in a plectindeficient muscle fiber. As Video 7, but showing images of plectin-deficient soleus.