Nerve-independent formation of a topologically complex postsynaptic apparatus

Abstract

As the mammalian neuromuscular junction matures, its acetylcholine receptor (AChR)-rich postsynaptic apparatus is transformed from an oval plaque into a pretzel-shaped array of branches that precisely mirrors the branching pattern of the motor nerve terminal. Although the nerve has been believed to direct postsynaptic maturation, we report here that myotubes cultured aneurally on matrix-coated substrates form elaborately branched AChR-rich domains remarkably similar to those seen in vivo. These domains share several characteristics with the mature postsynaptic apparatus, including colocalization of multiple postsynaptic markers, clustering of subjacent myonuclei, and dependence on the muscle-specific kinase and rapsyn for their formation. Time-lapse imaging showed that branched structures arise from plaques by formation and fusion of AChR-poor perforations through a series of steps mirroring that seen in vivo. Multiple fluorophore imaging showed that growth occurs by circumferential, asymmetric addition of AChRs. Analysis in vivo revealed similar patterns of AChR addition during normal development. These results reveal the sequence of steps by which a topologically complex domain forms on a cell and suggest an unexpected nerve-independent role for the postsynaptic cell in generating this topological complexity.

Figure 1.

Topologically complex AChR clusters in myotubes cultured aneurally on laminin. (A–B) C2 myotubes grown on a gelatin-coated substrate form simple plaque-like AChR clusters, revealed by labeling with Btx (A) whereas myotubes grown on a laminin-coated substrate form elaborate, branched aggregates (B). A and B are shown at the same magnification. (C–D) Similarity of AChR aggregates made by C2 myotubes grown on laminin (C) to those at adult NMJs from a mouse with YFP-positive motor axons (D). D′ shows precise opposition of AChRs (red) to nerve terminal branches (green). Both aneural aggregates and NMJs have an “open” side (asterisks). At the NMJ, this is the side from which the axon enters. (E) Primary myotubes, cultured from neonatal mice, also form branched aggregates, showing that this complexity is not a peculiarity of cell lines. Bars: (B) 50 μm for A and B; (C–E) 10 μm.

Figure 2.

Requirements for formation of branched AChR aggregates on laminin-coated substrates. (A–B) MuSK is required. Myotubes from a MuSK^−/− cell line form no AChR aggregates when cultured on laminin-coated substrates. AChRs are instead distributed uniformly over their surface (cells stained uniformly red with Btx). When transfected with GFP-MuSK (green), such cells form receptor aggregates (red) with complex morphology (A). High power view shows colocalization of these aggregates (B) with GFP-MuSK (B′). Arrowheads mark corresponding points in B and B′. (C and D) Rapsyn is required. Myotubes from a control cell line form complex AChR aggregates when cultured on laminin (C), but myotubes from a rapsyn^−/− cell line form no clusters (D). (E and F) Agrin is not required. Primary myotubes from controls (E, agrin^+/?, meaning heterozygous or wild type) and agrin^−/− (F) mice both form complex clusters on laminin-coated substrates. (G) Polyornithine-coated substrates do not support complex AChR aggregate formation in C2 cells. AChR aggregates in these cultures are plaque shaped. (H) Laminin is not required. C2 myotubes form branched aggregates on fibronectin-coated substrates, although they are generally smaller and less complex than those that develop on laminin-coated substrates. (I) When C2 myotubes are mechanically detached from laminin-coated substrata, postsynaptic “ghosts” labeled with Btx (red) remain attached to the substrate. Nuclear staining with DAPI (blue) shows that there is no myotube over the receptor aggregate. Dotted lines indicate approximate borders of the former myotube. Bars: (A) 100 μm; (B′–I) 20 μm.

Figure 3.

Colocalization of AChRs and other postsynaptic markers in aneural myotubes cultured on laminin. Myotubes were double stained with Btx (A–E) and antibodies to rapsyn (A′), phosphotyrosine (B′), laminin β2 (C′), laminin α5 (D′), or laminin γ1 (E′). Bar, 20 μm for all panels.

Figure 4.

Molecularly specialized nuclei cluster at AChR aggregates in aneural myotubes cultured on laminin. (A) DAPI-stained myonuclei (blue) cluster in apposition to branched aggregates (red); fewer nuclei are present elsewhere in the myotube (arrowheads). Dashed lines indicate myotube borders. (B) Nuclear density was quantified beneath an elliptical region defined by the extent of the AChR aggregate and in regions lacking AChR aggregates. The means of these distributions (± SEM) shows that average nuclear density is nearly threefold higher in association with aggregates (n = 230) than in AChR poor regions (n = 40). (C) Syne-1 (green) is more abundant in aggregate-associated nuclei (arrows) than in nuclei displaced from AChR aggregates (arrowheads). AChRs are red and nuclei blue, as in A. Dashed lines indicate the myotube borders. Bars: (A and C′′) 20 μm.

Figure 5.

Transitional AChR aggregate morphologies in vivo and in vitro. (A–D) AChR-rich postsynaptic specializations at the NMJ are initially plaque-shaped (A), perforated (B), C-shaped (C), and branched (D) structures become successively more prevalent as development proceeds. Postnatal ages (P) are indicated in each panel. (E–H) AChR aggregates formed on laminin-coated substrates in the absence of innervation also display plaque-shaped, perforated, C-shaped, and branched shapes. PF, days after initiating fusion. (I) The diameter of myotubes bearing aggregates of the four morphological types shown in A–H. n = 32–147 clusters per point, from a 5-d postfusion culture. Diameter was measured at the cluster. The schematic applies to I and J. (J) The prevalence of morphologies in vitro as a function of days PF. n = 205–232 clusters per time point. Bar, 20 μm for all panels.

Figure 6.

Time-lapse imaging of AChR aggregates on aneural myotubes. (A–C) Cultures were stained with Btx and imaged immediately, then after two successive 12-h intervals. Before the last time point, cultures were restained with Btx. This AChR plaque acquired a central AChR-poor hole (B) that broke through the annulus to form an “open” configuration (C). (D) Overlay of the first (red) and final images (green) shows growth of the AChR aggregate and disproportionate enlargement of the central hole. (E–G) This initially perforated aggregate developed branches by partial fusion of AChR-poor holes along part of their perimeter. Numbered perforations are sketched in the insets to show how branches form through partial fusion (red lines). (H) Overlay of the first and last time points as in D. Bars: (A–D) 10 μm; (E–H) 20 μm.

Figure 7.

Patterns of AChR addition to aneural aggregates. (A–D) Circumferential addition of AChRs. Myotubes cultured on a laminin-coated substrate were stained with Alexa 594–Btx at 5 d after fusion (A, red), incubated 12 h, restained with Alexa 488–Btx (B, green), incubated an additional 12 h, and then stained with Alexa 647–Btx (C, blue) and imaged. Thus, each color marks AChRs that have spent a different amount of time in the membrane. Overlay (D), emphasizes the distinct distribution of each population: the oldest receptors (red) are most central, whereas the youngest (blue) are most peripheral. Inset in D shows relative intensities of the three labels in the boxed region. (E–I) Limited AChR mobility within aggregates. Cultures were stained with Alexa 594–Btx and imaged immediately (E, pop. 1). After 24 h, the cultures were stained with Alexa 488–Btx and both the original population (F, pop. 1′) and the new population (G, pop. 2) of AChRs were imaged. Overlay of population 1 (t = 0) and population 2 (t = 24 h) illustrates the growth of the AChR aggregate (H). Despite this growth, the lateral extent of the original population changed little (I; pop. 1′ is pseudocolored blue). Numerous AChR-rich vesicles near the aggregate (F and I) may indicate endocytotic removal of AChRs. Bars: (D and I) 20 μm.

Figure 8.

Circumferential growth of AChR clusters at the NMJ. (A) Btx was injected above the sternomastoid muscle of a postnatal day five pup that expressed YFP in its motor axons. After 1 d, the pup was killed, and unlabeled AChRs were stained with a second, spectrally distinct Btx conjugate. AChRs carrying the first tag (red) are found preferentially near the center of aggregates, whereas younger AChRs (blue) are concentrated at the periphery. Note that in “open” aggregates (arrowheads) new AChRs are preferentially concentrated along the convex margin. The nerve invariably enters the synapse from the opposite side (inset, overlay with YFP to show pattern of innervation). (B) High resolution image of a single junction labeled as above. (C) Relative intensities of the two labels in the region boxed in B. Bars: (A) 20 μm; (B) 10 μm.