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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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Analysis of cluster formation and detergent extractability of AChRs. (A) Representative images of Alexa 488–α-BTX–labeled AChR clusters forming spontaneously (no agrin; white arrowheads) or in the presence of agrin. Right, higher magnification of boxed regions in middle. Note that fragmented and less compact clusters (typical of plectin-deficient cells) are rarely seen in Plec+/+ cells. Bars, 30 μm (left and middle); 10 μm (right). (B) Statistically evaluated fluorescence intensities (recorded bellow fluorescence saturation) of large (≥5 μm2) clusters. Plec+/+, n = 114; Plec−/−, n = 218 clusters examined. (C, D) Quantification of large (C) and small (<5 μm2; micro, D) clusters per total myotube area visualized. Myotubes analyzed: Plec+/+, 271, and Plec−/−, 357 (C); and Plec+/+, 57, and Plec−/−, 38 (D). Note that upon agrin treatment, Plec−/− myotubes form approximately half as many (and ∼1.3-fold less dense) large clusters compared with Plec+/+ cells. (E) Quantification of agrin-induced clusters at different time points (presented as percentage increase over 0 h). Note that only Plec+/+ cells were responsive to agrin. Myotubes analyzed per time point: Plec+/+, ≥200; Plec−/−, ≥203. (F) Immunoblotting (using antibodies to AChR subunit α) of affinity-purified (biotin–α-BTX) AChRs after sequential extraction of Plec+/+ and Plec−/− myotubes with indicated concentrations of Triton X-100. (G) Bar diagram showing proportional extractability (%) of AChRs at various concentrations of detergent. Mean ± SEM, three experiments (B–E), five experiments (G), each. *p < 0.05 and ***p < 0.001 compared with Plec+/+ (D) or Plec+/+ agrin/no agrin/0 h (B, C ,E). Unpaired Student's t test.
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Figure 1: Analysis of cluster formation and detergent extractability of AChRs. (A) Representative images of Alexa 488–α-BTX–labeled AChR clusters forming spontaneously (no agrin; white arrowheads) or in the presence of agrin. Right, higher magnification of boxed regions in middle. Note that fragmented and less compact clusters (typical of plectin-deficient cells) are rarely seen in Plec+/+ cells. Bars, 30 μm (left and middle); 10 μm (right). (B) Statistically evaluated fluorescence intensities (recorded bellow fluorescence saturation) of large (≥5 μm2) clusters. Plec+/+, n = 114; Plec−/−, n = 218 clusters examined. (C, D) Quantification of large (C) and small (<5 μm2; micro, D) clusters per total myotube area visualized. Myotubes analyzed: Plec+/+, 271, and Plec−/−, 357 (C); and Plec+/+, 57, and Plec−/−, 38 (D). Note that upon agrin treatment, Plec−/− myotubes form approximately half as many (and ∼1.3-fold less dense) large clusters compared with Plec+/+ cells. (E) Quantification of agrin-induced clusters at different time points (presented as percentage increase over 0 h). Note that only Plec+/+ cells were responsive to agrin. Myotubes analyzed per time point: Plec+/+, ≥200; Plec−/−, ≥203. (F) Immunoblotting (using antibodies to AChR subunit α) of affinity-purified (biotin–α-BTX) AChRs after sequential extraction of Plec+/+ and Plec−/− myotubes with indicated concentrations of Triton X-100. (G) Bar diagram showing proportional extractability (%) of AChRs at various concentrations of detergent. Mean ± SEM, three experiments (B–E), five experiments (G), each. *p < 0.05 and ***p < 0.001 compared with Plec+/+ (D) or Plec+/+ agrin/no agrin/0 h (B, C ,E). Unpaired Student's t test.

Mentions: To study the molecular mechanism behind plectin's contribution to NMJ integrity, we first investigated cluster formation of AChRs in myotubes that had been differentiated ex vivo from immortalized plectin-positive (Plec+/+) and plectin-deficient (Plec−/−) myoblast cell lines (Winter et al., 2014). Plectin-deficient myotubes, like their normal counterparts, are multinucleated and show signs of full differentiation, such as spontaneous contraction (Winter et al., 2014). In addition, Plec−/− myotubes closely mimic the pathology of EBS-MD myofibers, including the development of desmin-positive protein aggregates (Winter et al., 2014). To induce AChR clustering, myotubes were exposed to recombinant neural agrin, and AChRs were visualized by Alexa 488–α-bungarotoxin (BTX) labeling (Figure 1A). A morphometric analysis revealed that in plectin-deficient myotubes, the formation of large clusters (≥5 μm2 in size) was strikingly reduced compared to wild-type (wt) cells (Figure 1C). Moreover, such clusters showed reduced fluorescence intensity compared with corresponding ones in plectin-positive myotubes (Figure 1B). On the other hand, in plectin-deficient myotubes, we observed a significantly increased number of (micro) clusters of sizes <5 μm2 (Figure 1D). To test the responsiveness of Plec−/− myotubes to agrin-induced AChR-clustering, we quantified large clusters at different time points after initiating the treatment. In contrast to Plec+/+ myotubes, where the number of AChR clusters increased consistently with time (Figure 1E), no increase was observed in Plec−/− myotubes. In control experiments in which AChR expression levels in myoblasts and myotubes were assessed by immunoblotting, no differences were observed between Plec+/+ and Plec−/− cells, regardless of whether or not they had been treated with agrin (Supplemental Figure S1).


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)

Analysis of cluster formation and detergent extractability of AChRs. (A) Representative images of Alexa 488–α-BTX–labeled AChR clusters forming spontaneously (no agrin; white arrowheads) or in the presence of agrin. Right, higher magnification of boxed regions in middle. Note that fragmented and less compact clusters (typical of plectin-deficient cells) are rarely seen in Plec+/+ cells. Bars, 30 μm (left and middle); 10 μm (right). (B) Statistically evaluated fluorescence intensities (recorded bellow fluorescence saturation) of large (≥5 μm2) clusters. Plec+/+, n = 114; Plec−/−, n = 218 clusters examined. (C, D) Quantification of large (C) and small (<5 μm2; micro, D) clusters per total myotube area visualized. Myotubes analyzed: Plec+/+, 271, and Plec−/−, 357 (C); and Plec+/+, 57, and Plec−/−, 38 (D). Note that upon agrin treatment, Plec−/− myotubes form approximately half as many (and ∼1.3-fold less dense) large clusters compared with Plec+/+ cells. (E) Quantification of agrin-induced clusters at different time points (presented as percentage increase over 0 h). Note that only Plec+/+ cells were responsive to agrin. Myotubes analyzed per time point: Plec+/+, ≥200; Plec−/−, ≥203. (F) Immunoblotting (using antibodies to AChR subunit α) of affinity-purified (biotin–α-BTX) AChRs after sequential extraction of Plec+/+ and Plec−/− myotubes with indicated concentrations of Triton X-100. (G) Bar diagram showing proportional extractability (%) of AChRs at various concentrations of detergent. Mean ± SEM, three experiments (B–E), five experiments (G), each. *p < 0.05 and ***p < 0.001 compared with Plec+/+ (D) or Plec+/+ agrin/no agrin/0 h (B, C ,E). Unpaired Student's t test.
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Figure 1: Analysis of cluster formation and detergent extractability of AChRs. (A) Representative images of Alexa 488–α-BTX–labeled AChR clusters forming spontaneously (no agrin; white arrowheads) or in the presence of agrin. Right, higher magnification of boxed regions in middle. Note that fragmented and less compact clusters (typical of plectin-deficient cells) are rarely seen in Plec+/+ cells. Bars, 30 μm (left and middle); 10 μm (right). (B) Statistically evaluated fluorescence intensities (recorded bellow fluorescence saturation) of large (≥5 μm2) clusters. Plec+/+, n = 114; Plec−/−, n = 218 clusters examined. (C, D) Quantification of large (C) and small (<5 μm2; micro, D) clusters per total myotube area visualized. Myotubes analyzed: Plec+/+, 271, and Plec−/−, 357 (C); and Plec+/+, 57, and Plec−/−, 38 (D). Note that upon agrin treatment, Plec−/− myotubes form approximately half as many (and ∼1.3-fold less dense) large clusters compared with Plec+/+ cells. (E) Quantification of agrin-induced clusters at different time points (presented as percentage increase over 0 h). Note that only Plec+/+ cells were responsive to agrin. Myotubes analyzed per time point: Plec+/+, ≥200; Plec−/−, ≥203. (F) Immunoblotting (using antibodies to AChR subunit α) of affinity-purified (biotin–α-BTX) AChRs after sequential extraction of Plec+/+ and Plec−/− myotubes with indicated concentrations of Triton X-100. (G) Bar diagram showing proportional extractability (%) of AChRs at various concentrations of detergent. Mean ± SEM, three experiments (B–E), five experiments (G), each. *p < 0.05 and ***p < 0.001 compared with Plec+/+ (D) or Plec+/+ agrin/no agrin/0 h (B, C ,E). Unpaired Student's t test.
Mentions: To study the molecular mechanism behind plectin's contribution to NMJ integrity, we first investigated cluster formation of AChRs in myotubes that had been differentiated ex vivo from immortalized plectin-positive (Plec+/+) and plectin-deficient (Plec−/−) myoblast cell lines (Winter et al., 2014). Plectin-deficient myotubes, like their normal counterparts, are multinucleated and show signs of full differentiation, such as spontaneous contraction (Winter et al., 2014). In addition, Plec−/− myotubes closely mimic the pathology of EBS-MD myofibers, including the development of desmin-positive protein aggregates (Winter et al., 2014). To induce AChR clustering, myotubes were exposed to recombinant neural agrin, and AChRs were visualized by Alexa 488–α-bungarotoxin (BTX) labeling (Figure 1A). A morphometric analysis revealed that in plectin-deficient myotubes, the formation of large clusters (≥5 μm2 in size) was strikingly reduced compared to wild-type (wt) cells (Figure 1C). Moreover, such clusters showed reduced fluorescence intensity compared with corresponding ones in plectin-positive myotubes (Figure 1B). On the other hand, in plectin-deficient myotubes, we observed a significantly increased number of (micro) clusters of sizes <5 μm2 (Figure 1D). To test the responsiveness of Plec−/− myotubes to agrin-induced AChR-clustering, we quantified large clusters at different time points after initiating the treatment. In contrast to Plec+/+ myotubes, where the number of AChR clusters increased consistently with time (Figure 1E), no increase was observed in Plec−/− myotubes. In control experiments in which AChR expression levels in myoblasts and myotubes were assessed by immunoblotting, no differences were observed between Plec+/+ and Plec−/− cells, regardless of whether or not they had been treated with agrin (Supplemental Figure S1).

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