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Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes.

Mihailovska E, Raith M, Valencia RG, Fischer I, Al Banchaabouchi M, Herbst R, Wiche G - Mol. Biol. Cell (2014)

Bottom Line: Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells.In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span.Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.

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Comparative morphometric analyses of NMJs in wt and various mutant mouse lines. (A) Confocal Z-stack image projections of Alexa 488–α-BTX–labeled AChR clusters on teased myofibers of soleus muscles. Note fragmented and dispersed NMJ structures in both cKO (Pax7- and MCK-Cre) and Des−/− but not in wt and P1c KO mice. (B) Densitometric analysis (voxel intensities) showing ∼50% decrement in intensity of Pax7-Cre/cKO in comparison to wt samples (wt, n = 283; Pax7-Cre/cKO, n = 803). (C, D) Number (C) and size (D) of discrete AChR-positive islands per soleus myofiber NMJs of 2-mo-old mice. NMJs analyzed: wt, 103; P1c KO, 36; MCK-Cre/cKO, 40; Pax7-Cre/cKO, 32; and Des−/−, 45. (E) Quantification of VGSC-AChR colocalization in wt and Pax7-Cre/cKO muscle. Mean ± SEM (B–D), three experiments. Mean ± SD (E), two experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with wt; unpaired Student's t test. (F, G) Teased myofibers (F) or longitudinal sections of soleus muscle (G) derived from wt and Pax7-Cre/cKO mice were double labeled as indicated. Arrowheads, pronounced nerve branching (F) and partial delocalization of VGSCs from receptors (G). Bars, 10 μm (A, F, G). (H) Time course of NMJ fragmentation frequency in plectin- and desmin-deficient soleus muscles. NMJs in soleus muscle fibers of 1-, 2-, and 4-mo-old mice were classified as fragmented (≥6 discontinuous fragments of AChR), partially fragmented (4–5 fragments), and normal (1–3 fragments). Note the early appearance of NMJ fragmentation in Pax7-Cre/cKO mice (1-mo-old) and more dramatic progression with age compared with Des−/− mice. A minimum of 30 NMJs were analyzed from each genotype per age.
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Figure 7: Comparative morphometric analyses of NMJs in wt and various mutant mouse lines. (A) Confocal Z-stack image projections of Alexa 488–α-BTX–labeled AChR clusters on teased myofibers of soleus muscles. Note fragmented and dispersed NMJ structures in both cKO (Pax7- and MCK-Cre) and Des−/− but not in wt and P1c KO mice. (B) Densitometric analysis (voxel intensities) showing ∼50% decrement in intensity of Pax7-Cre/cKO in comparison to wt samples (wt, n = 283; Pax7-Cre/cKO, n = 803). (C, D) Number (C) and size (D) of discrete AChR-positive islands per soleus myofiber NMJs of 2-mo-old mice. NMJs analyzed: wt, 103; P1c KO, 36; MCK-Cre/cKO, 40; Pax7-Cre/cKO, 32; and Des−/−, 45. (E) Quantification of VGSC-AChR colocalization in wt and Pax7-Cre/cKO muscle. Mean ± SEM (B–D), three experiments. Mean ± SD (E), two experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with wt; unpaired Student's t test. (F, G) Teased myofibers (F) or longitudinal sections of soleus muscle (G) derived from wt and Pax7-Cre/cKO mice were double labeled as indicated. Arrowheads, pronounced nerve branching (F) and partial delocalization of VGSCs from receptors (G). Bars, 10 μm (A, F, G). (H) Time course of NMJ fragmentation frequency in plectin- and desmin-deficient soleus muscles. NMJs in soleus muscle fibers of 1-, 2-, and 4-mo-old mice were classified as fragmented (≥6 discontinuous fragments of AChR), partially fragmented (4–5 fragments), and normal (1–3 fragments). Note the early appearance of NMJ fragmentation in Pax7-Cre/cKO mice (1-mo-old) and more dramatic progression with age compared with Des−/− mice. A minimum of 30 NMJs were analyzed from each genotype per age.

Mentions: To distinguish between presynaptic and postsynaptic contributions of plectin to the structural integrity of NMJs, we compared morphological features of NMJs in specimens isolated from wt and four different genetically modified mouse lines, namely Pax7-Cre/cKO, MCK-Cre cKO (Konieczny et al., 2008), desmin-knockout (Des−/−) mice (Li et al., 1996), and mice deficient in plectin isoform 1c (P1c), the major isoform expressed in motor neurons (Fuchs et al., 2009). Of interest, in soleus muscle of adult Pax7-Cre/cKO mice, we observed a dramatic deterioration of endplates into small islands of AChRs, in sharp contrast to the continuous (pretzel-like) appearance of AChR clusters in wt as well as in P1c-knockout mice (Figure 7A). The quantification of Alexa 488– α-BTX–specific signals in Pax7-Cre/cKO soleus muscle revealed a decrement of 51% compared to wt, suggesting a substantial reduction of AChR density in the absence of plectin (Figure 7B). Deterioration of the typical pretzel-like appearance was noticeable also in Des−/− and MCK-Cre/cKO mice, albeit in both of these cases it was less pronounced, as clearly revealed by statistical quantification of the number and sizes of fragmented clusters (Figure 7, C and D). Apart from soleus, severe fragmentation of NMJs was found in several other types of plectin-deficient muscle tested, such as extensor digitorum longus, gastrocnemius, tibialis, and diaphragm (Supplemental Figure S4). Concomitant with the fragmentation of NMJs, a disturbed topology of the nerve terminal was observed (Figure 7F). Moreover, we found a dispersion of voltage-gated sodium channels (VGSCs) in endplate regions of Pax7-Cre/cKO soleus muscle (Figure 7G), manifesting as approximately twofold reduced colocalization of VGSCs with AChRs (Figure 7E).


Neuromuscular synapse integrity requires linkage of acetylcholine receptors to postsynaptic intermediate filament networks via rapsyn-plectin 1f complexes.

Mihailovska E, Raith M, Valencia RG, Fischer I, Al Banchaabouchi M, Herbst R, Wiche G - Mol. Biol. Cell (2014)

Comparative morphometric analyses of NMJs in wt and various mutant mouse lines. (A) Confocal Z-stack image projections of Alexa 488–α-BTX–labeled AChR clusters on teased myofibers of soleus muscles. Note fragmented and dispersed NMJ structures in both cKO (Pax7- and MCK-Cre) and Des−/− but not in wt and P1c KO mice. (B) Densitometric analysis (voxel intensities) showing ∼50% decrement in intensity of Pax7-Cre/cKO in comparison to wt samples (wt, n = 283; Pax7-Cre/cKO, n = 803). (C, D) Number (C) and size (D) of discrete AChR-positive islands per soleus myofiber NMJs of 2-mo-old mice. NMJs analyzed: wt, 103; P1c KO, 36; MCK-Cre/cKO, 40; Pax7-Cre/cKO, 32; and Des−/−, 45. (E) Quantification of VGSC-AChR colocalization in wt and Pax7-Cre/cKO muscle. Mean ± SEM (B–D), three experiments. Mean ± SD (E), two experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with wt; unpaired Student's t test. (F, G) Teased myofibers (F) or longitudinal sections of soleus muscle (G) derived from wt and Pax7-Cre/cKO mice were double labeled as indicated. Arrowheads, pronounced nerve branching (F) and partial delocalization of VGSCs from receptors (G). Bars, 10 μm (A, F, G). (H) Time course of NMJ fragmentation frequency in plectin- and desmin-deficient soleus muscles. NMJs in soleus muscle fibers of 1-, 2-, and 4-mo-old mice were classified as fragmented (≥6 discontinuous fragments of AChR), partially fragmented (4–5 fragments), and normal (1–3 fragments). Note the early appearance of NMJ fragmentation in Pax7-Cre/cKO mice (1-mo-old) and more dramatic progression with age compared with Des−/− mice. A minimum of 30 NMJs were analyzed from each genotype per age.
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Figure 7: Comparative morphometric analyses of NMJs in wt and various mutant mouse lines. (A) Confocal Z-stack image projections of Alexa 488–α-BTX–labeled AChR clusters on teased myofibers of soleus muscles. Note fragmented and dispersed NMJ structures in both cKO (Pax7- and MCK-Cre) and Des−/− but not in wt and P1c KO mice. (B) Densitometric analysis (voxel intensities) showing ∼50% decrement in intensity of Pax7-Cre/cKO in comparison to wt samples (wt, n = 283; Pax7-Cre/cKO, n = 803). (C, D) Number (C) and size (D) of discrete AChR-positive islands per soleus myofiber NMJs of 2-mo-old mice. NMJs analyzed: wt, 103; P1c KO, 36; MCK-Cre/cKO, 40; Pax7-Cre/cKO, 32; and Des−/−, 45. (E) Quantification of VGSC-AChR colocalization in wt and Pax7-Cre/cKO muscle. Mean ± SEM (B–D), three experiments. Mean ± SD (E), two experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with wt; unpaired Student's t test. (F, G) Teased myofibers (F) or longitudinal sections of soleus muscle (G) derived from wt and Pax7-Cre/cKO mice were double labeled as indicated. Arrowheads, pronounced nerve branching (F) and partial delocalization of VGSCs from receptors (G). Bars, 10 μm (A, F, G). (H) Time course of NMJ fragmentation frequency in plectin- and desmin-deficient soleus muscles. NMJs in soleus muscle fibers of 1-, 2-, and 4-mo-old mice were classified as fragmented (≥6 discontinuous fragments of AChR), partially fragmented (4–5 fragments), and normal (1–3 fragments). Note the early appearance of NMJ fragmentation in Pax7-Cre/cKO mice (1-mo-old) and more dramatic progression with age compared with Des−/− mice. A minimum of 30 NMJs were analyzed from each genotype per age.
Mentions: To distinguish between presynaptic and postsynaptic contributions of plectin to the structural integrity of NMJs, we compared morphological features of NMJs in specimens isolated from wt and four different genetically modified mouse lines, namely Pax7-Cre/cKO, MCK-Cre cKO (Konieczny et al., 2008), desmin-knockout (Des−/−) mice (Li et al., 1996), and mice deficient in plectin isoform 1c (P1c), the major isoform expressed in motor neurons (Fuchs et al., 2009). Of interest, in soleus muscle of adult Pax7-Cre/cKO mice, we observed a dramatic deterioration of endplates into small islands of AChRs, in sharp contrast to the continuous (pretzel-like) appearance of AChR clusters in wt as well as in P1c-knockout mice (Figure 7A). The quantification of Alexa 488– α-BTX–specific signals in Pax7-Cre/cKO soleus muscle revealed a decrement of 51% compared to wt, suggesting a substantial reduction of AChR density in the absence of plectin (Figure 7B). Deterioration of the typical pretzel-like appearance was noticeable also in Des−/− and MCK-Cre/cKO mice, albeit in both of these cases it was less pronounced, as clearly revealed by statistical quantification of the number and sizes of fragmented clusters (Figure 7, C and D). Apart from soleus, severe fragmentation of NMJs was found in several other types of plectin-deficient muscle tested, such as extensor digitorum longus, gastrocnemius, tibialis, and diaphragm (Supplemental Figure S4). Concomitant with the fragmentation of NMJs, a disturbed topology of the nerve terminal was observed (Figure 7F). Moreover, we found a dispersion of voltage-gated sodium channels (VGSCs) in endplate regions of Pax7-Cre/cKO soleus muscle (Figure 7G), manifesting as approximately twofold reduced colocalization of VGSCs with AChRs (Figure 7E).

Bottom Line: Live imaging of acetylcholine receptors (AChRs) in cultured myotubes differentiated ex vivo from immortalized plectin-deficient myoblasts revealed them to be highly mobile and unable to coalesce into stable clusters, in contrast to wild-type cells.In their phenotypic behavior, mutant mice closely mimicked EBS-MD-MyS patients, including impaired body balance, severe muscle weakness, and reduced life span.Our study demonstrates that linkage to desmin IF networks via plectin is crucial for formation and maintenance of AChR clusters, postsynaptic NMJ organization, and body locomotion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria.

Show MeSH
Related in: MedlinePlus