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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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Phenotypic characterization of Pax7-Cre/cKO mice. (A) Longitudinal cryosections of soleus muscles derived from adult wt, MCK-Cre/cKO, and Pax7-Cre/cKO mice were immunolabeled for plectin (yellow) and desmin (red) and labeled for AChR with Alexa 488–α-BTX (green). Note the complete absence of plectin from myofibers (including endplate regions) of Pax7-Cre/cKO mice, whereas myofibers from adult MCK-Cre/cKO mice showed some reexpression and accumulation of plectin at endplate regions (arrowhead). (B) X-radiography of wt and Pax7-Cre/cKO mice. Note severe kyphosis in Pax7-Cre/cKO mice. (C) Survival rates of wt and Pax7-Cre/cKO mice. Note greatly reduced lifespan of mutant mice, showing a 50% survival rate of only 19 wk (n > 35 per genotype).
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Figure 6: Phenotypic characterization of Pax7-Cre/cKO mice. (A) Longitudinal cryosections of soleus muscles derived from adult wt, MCK-Cre/cKO, and Pax7-Cre/cKO mice were immunolabeled for plectin (yellow) and desmin (red) and labeled for AChR with Alexa 488–α-BTX (green). Note the complete absence of plectin from myofibers (including endplate regions) of Pax7-Cre/cKO mice, whereas myofibers from adult MCK-Cre/cKO mice showed some reexpression and accumulation of plectin at endplate regions (arrowhead). (B) X-radiography of wt and Pax7-Cre/cKO mice. Note severe kyphosis in Pax7-Cre/cKO mice. (C) Survival rates of wt and Pax7-Cre/cKO mice. Note greatly reduced lifespan of mutant mice, showing a 50% survival rate of only 19 wk (n > 35 per genotype).

Mentions: The inefficient AChR clustering observed in ex vivo–differentiated plectin-deficient myotubes, together with the grossly disorganized structure of NMJs typical for patients suffering from plectinopathy-associated MyS (Engel, 2012), strongly suggested that postsynaptic plectin plays a role in maintaining dense AChR clusters at the crests of sarcolemma infoldings. To examine on the organismal level whether the lack of postsynaptic plectin causes defects in NMJ morphology with consequences for muscle strength and body balance, we generated a mouse line (Pax7-Cre/cKO) in which plectin gene disruption was controlled by Pax7, a transcription factor that is expressed in muscle stem cells (Relaix et al., 2005). In contrast to a previously generated MCK-Cre/cKO mouse line (Konieczny et al., 2008), in which precursor satellite cells were exempt from ablation (allowing reexpression of plectin in adult myofibers during regeneration), Pax7-Cre/cKO mice were plectin negative not only in mature myofibers of limb muscles, but also in satellite cells (Supplemental Figure S3) and consequently at NMJs (Figure 6A). Whereas MCK-Cre/cKO mice were hardly distinguishable from their wt littermates until reaching adulthood, Pax7-Cre/cKO mice suffered from body weakness, manifesting as small size, profound kyphosis, and a survival rate of only 50% at the age of 19 wk (Figure 6, B and C).


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)

Phenotypic characterization of Pax7-Cre/cKO mice. (A) Longitudinal cryosections of soleus muscles derived from adult wt, MCK-Cre/cKO, and Pax7-Cre/cKO mice were immunolabeled for plectin (yellow) and desmin (red) and labeled for AChR with Alexa 488–α-BTX (green). Note the complete absence of plectin from myofibers (including endplate regions) of Pax7-Cre/cKO mice, whereas myofibers from adult MCK-Cre/cKO mice showed some reexpression and accumulation of plectin at endplate regions (arrowhead). (B) X-radiography of wt and Pax7-Cre/cKO mice. Note severe kyphosis in Pax7-Cre/cKO mice. (C) Survival rates of wt and Pax7-Cre/cKO mice. Note greatly reduced lifespan of mutant mice, showing a 50% survival rate of only 19 wk (n > 35 per genotype).
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Related In: Results  -  Collection

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Figure 6: Phenotypic characterization of Pax7-Cre/cKO mice. (A) Longitudinal cryosections of soleus muscles derived from adult wt, MCK-Cre/cKO, and Pax7-Cre/cKO mice were immunolabeled for plectin (yellow) and desmin (red) and labeled for AChR with Alexa 488–α-BTX (green). Note the complete absence of plectin from myofibers (including endplate regions) of Pax7-Cre/cKO mice, whereas myofibers from adult MCK-Cre/cKO mice showed some reexpression and accumulation of plectin at endplate regions (arrowhead). (B) X-radiography of wt and Pax7-Cre/cKO mice. Note severe kyphosis in Pax7-Cre/cKO mice. (C) Survival rates of wt and Pax7-Cre/cKO mice. Note greatly reduced lifespan of mutant mice, showing a 50% survival rate of only 19 wk (n > 35 per genotype).
Mentions: The inefficient AChR clustering observed in ex vivo–differentiated plectin-deficient myotubes, together with the grossly disorganized structure of NMJs typical for patients suffering from plectinopathy-associated MyS (Engel, 2012), strongly suggested that postsynaptic plectin plays a role in maintaining dense AChR clusters at the crests of sarcolemma infoldings. To examine on the organismal level whether the lack of postsynaptic plectin causes defects in NMJ morphology with consequences for muscle strength and body balance, we generated a mouse line (Pax7-Cre/cKO) in which plectin gene disruption was controlled by Pax7, a transcription factor that is expressed in muscle stem cells (Relaix et al., 2005). In contrast to a previously generated MCK-Cre/cKO mouse line (Konieczny et al., 2008), in which precursor satellite cells were exempt from ablation (allowing reexpression of plectin in adult myofibers during regeneration), Pax7-Cre/cKO mice were plectin negative not only in mature myofibers of limb muscles, but also in satellite cells (Supplemental Figure S3) and consequently at NMJs (Figure 6A). Whereas MCK-Cre/cKO mice were hardly distinguishable from their wt littermates until reaching adulthood, Pax7-Cre/cKO mice suffered from body weakness, manifesting as small size, profound kyphosis, and a survival rate of only 50% at the age of 19 wk (Figure 6, B and C).

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