Liprin-α-1 is a novel component of the murine neuromuscular junction and is involved in the organization of the postsynaptic machinery

Abstract

Neuromuscular junctions (NMJs) are specialized synapses that connect motor neurons to skeletal muscle fibers and orchestrate proper signal transmission from the nervous system to muscles. The efficient formation and maintenance of the postsynaptic machinery that contains acetylcholine receptors (AChR) are indispensable for proper NMJ function. Abnormalities in the organization of synaptic components often cause severe neuromuscular disorders, such as muscular dystrophy. The dystrophin-associated glycoprotein complex (DGC) was shown to play an important role in NMJ development. We recently identified liprin-α-1 as a novel binding partner for one of the cytoplasmic DGC components, α-dystrobrevin-1. In the present study, we performed a detailed analysis of localization and function of liprin-α-1 at the murine NMJ. We showed that liprin-α-1 localizes to both pre- and postsynaptic compartments at the NMJ, and its synaptic enrichment depends on the presence of the nerve. Using cultured muscle cells, we found that liprin-α-1 plays an important role in AChR clustering and the organization of cortical microtubules. Our studies provide novel insights into the function of liprin-α-1 at vertebrate neuromuscular synapses.

Figure 1

Liprin-α-1 localizes to Z-discs and NMJs in skeletal muscles. (a–d) Liprin-α-1 localized to the Z-discs of contractile machinery. Tibialis anterior muscle fibers from P30 mice were stained with antibody against liprin-α-1 protein (green) and indicated markers for contractile machinery (red). (b) Shows a higher magnification of an area in (a). (e) Liprin-α-1 (green) was enriched mainly at the postsynaptic machinery of the NMJ. Fluorescently labeled BTX was used to visualize AChR (red), and anti-synaptophysin and anti-neurofilament were used to visualize nerve terminals (blue). (f) Enrichment of liprin-α-1 at the postsynaptic machinery. Fluorescence intensity for liprin-α-1 immunochemistry (green) and AChR (BTX, red) along the line shown in (e) was plotted using RGB profiler plugin to the ImageJ/Fiji software. (g) Liprin-α-1-GFP fusion protein localized to the NMJ on transfected fiber. AChR (red); liprin-α-1-GFP (green). Scale bar 10 μm.

Figure 2

Localization of liprin-α-1 is similar in various muscles and maintained throughout the development. (a) Liprin-α-1 immunoreactivity in different muscles. TS, triangularis sterni; TA, tibialis anterior; SOL, soleus; GAS, gastrocnemius. Muscles were collected on P30. (b) Immunohistochemical analysis of TA muscle fibers on the indicated embryonic (E) and postnatal (P) days. Anti-liprin-α-1 antibody (green); AChR (red). Scale bar 10 μm.

Figure 3

Liprin-α-1 NMJ localization is nerve-dependent. Fibers were isolated from the tibialis anterior muscle from P30 mice on the indicated days after the nerve cut procedure. (a) Liprin-α-1 (green) localisation at different days after nerve cut. The bottom panel represents higher magnification of the box in the upper panel (D14). (b) α-dystrobrevin-1 immunostaining (green) 14 days after nerve cut. AChR are visualized with BTX in red and anti-synaptophysin/anti-neurofilament immunostaining is shown in blue. All of the images were collected using the same exposure and imaging parameters. Scale bar 10 μm.

Figure 4

Liprin-α-1 is required for the formation of AChR clusters. (a) Validation of liprin-α-1 knockdown efficiency by Western blot. Hek-293 cells were co-transfected with liprin-α-1-GFP and the indicated siRNAs that targeted liprin-α-1 or indicated control siRNAs. Western blot for tubulin was used as a loading control. (b) Validation of liprin-α-1 knockdown efficiency by qRT-PCR. C2C12 differentiated myotubes were transfected with indicated siRNAs and 2 days later the total RNA was isolated from cell extract followed by qRT-PCR analysis of liprin-α-1 expression. (c) C2C12 myotubes cultured on laminin-coated surfaces were transfected with siRNAs that targeted liprin-α-1 and AChR clusters were visualised with BTX. (d) Quantification of the total number of clusters. (e) Quantification of topologically complex (perforated) clusters. (f,g) Liprin-α-1 was essential for the formation of agrin-induced AChR clusters. C2C12 myotubes were cultured on gelatin-coated surfaces, transfected with siRNAs that targeted liprin-α-1, and stimulated with soluble agrin (Z-fragment) to form AChR clusters. (g) Quantification of AChR clusters in (f). (h) Western Blot analysis of AChR and rapsyn expression levels after liprin-α-1 knockdown. Lysates from C2C12 myotubes transfected with liprin-α-1 targeting siRNAs or non-targeting RNAs were probed with antibodies against specified proteins. Levels of tubulin were used as a loading control. Negative control -non-targeting siRNA; positive control - muscle-specific kinase (MuSK) siRNA. Statistical significance was analysed using 2-tailed t-test. *p < 0.05; **p < 0.01; ****p < 0.0001. Error bars represent standard error of the mean (SEM). Scale bar 20 μm.

Figure 5

Liprin-α-1 regulates clustering of AChRs by organizing microtubules. (a–c) C2C12 myotubes depleted of liprin-α-1 have substantial amounts of surface AChR. (a) Surface AChR (green) were stained with Alexa-488-BTX and internal AChR were labelled with Alexa-555-BTX. Lower panel shows control experiment for labelling specificity of the Alexa-488-BTX staining shown in the middle panel. Unlabelled BTX was applied to live cells prior to Alexa-488-BTX labelling to mask BTX binding sites on AChR. (b) Fluorescence intensity quantification of surface AChR labelling, values normalized to control. (c) Analysis of surface AChRs in liprin-α-1-depleted myotubes by AChR precipitation and Western blotting. S-surface and T-total AChR precipitates. (d,e) Liprin-α-1 knockdown reduces the number of plasma membrane-attached microtubules in myoblasts. (d) Cells were transfected with indicated siRNAs and microtubules were visualized with anti-α-tubulin antibody. Lower panel represents higher magnifications of areas in the upper panels. Arrows indicate examples of microtubules in contact with cell surface. (e) Quantification of cell surface-attached microtubules per micrometre of the cell edge from 60 cells in three independent experiments. (f,g) Liprin-α-1 knockdown reduces the number of EB1 foci at the AChR clusters in myotubes. Microtubule tips were visualised with anti-EB1 antibody. Columns at the right represent higher magnifications of boxed areas. Asterisks mark the nucleous of an undifferentiated myoblast. (f) Quantification of EB1 foci at from 20 AChR clusters from three independent experiments. (h,i) Microtubules are dispensable for maintenance of AChR assemblies but are required for the cluster expansion. (h) Pre-existing AChR were labelled in live cells with Alexa-555-BTX (Old AChR) and nocodazole or DMSO (control) was added to the cells for 6 h and live cells were labelled again with Alexa-488-BTX (New AChR). (i) Fluoresence intensity quantification of surface AChRs. (j) Nocodazole treatment does not significantly affect AChR exocytosis. Western blot analysis of surface and total AChR in cell lysates (Lys), flow-through (FL) and elution (EL) fractions from control (DMSO) and nocodazole experiments. Scale bar 10 μm. Statistical significance was analysed using 2-tailed t-test. ****p < 0.0001. Error bars represent SEM.