Limits...
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.

Show MeSH

Related in: MedlinePlus

Time-lapse video microscopy of desmin-GFP–expressing Plec+/+ and Plec−/− myotubes. (A) Top, confocal image showing part of a desmin-GFP–expressing Plec+/+ myotube; Rows below, time-lapse images of the central area (delineated by dashed lines). Bracket indicates region of a representative large AChR cluster embedded in a dense network of desmin IFs. Note the nearly unchanged position and compactness of AChR cluster over the entire observation period (0–52.5 min; see also Supplemental Video S1). (B) As in A, except that a Plec−/− myotube is shown. Cell margins are outlined in white. Note the prominent desmin IF network aggregate, distant from clusters. White and yellow arrowheads, microclusters separating from a large cluster; note their repeated transient mutual interaction during traveling and final disassembly of the one labeled white in three even smaller microclusters. Purple arrowhead, separation of another microcluster (magenta; time point 75 min) that drifts away and eventually vanishes completely (see also Supplemental Video S2). Note also positional shift of the large cluster as a whole relative to its initial position (bracket). Bars, 10 μm (A, B). (C–E) Statistical quantification of mobile cluster parameters (number, velocity, and total distance traveled) in Plec+/+ cells (before or after induction of IF network collapse using OA, WFA, or P1f-Ins16) and in unreconstituted or P1f-reconstituted Plec−/− cells. Myotubes analyzed in C: Plec+/+, untreated, 6, and treated, 7; Plec−/−, 5; and P1f-GFP, 5. Clusters analyzed in D and E: Plec+/+, untreated, 202, and treated, 174; Plec−/−, 217; and P1f-GFP, 21. Mean ± SEM, three independent experiments. *p < 0.05 and ***p < 0.001 compared with Plec+/+, untreated; unpaired Student's t test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4263455&req=5

Figure 3: Time-lapse video microscopy of desmin-GFP–expressing Plec+/+ and Plec−/− myotubes. (A) Top, confocal image showing part of a desmin-GFP–expressing Plec+/+ myotube; Rows below, time-lapse images of the central area (delineated by dashed lines). Bracket indicates region of a representative large AChR cluster embedded in a dense network of desmin IFs. Note the nearly unchanged position and compactness of AChR cluster over the entire observation period (0–52.5 min; see also Supplemental Video S1). (B) As in A, except that a Plec−/− myotube is shown. Cell margins are outlined in white. Note the prominent desmin IF network aggregate, distant from clusters. White and yellow arrowheads, microclusters separating from a large cluster; note their repeated transient mutual interaction during traveling and final disassembly of the one labeled white in three even smaller microclusters. Purple arrowhead, separation of another microcluster (magenta; time point 75 min) that drifts away and eventually vanishes completely (see also Supplemental Video S2). Note also positional shift of the large cluster as a whole relative to its initial position (bracket). Bars, 10 μm (A, B). (C–E) Statistical quantification of mobile cluster parameters (number, velocity, and total distance traveled) in Plec+/+ cells (before or after induction of IF network collapse using OA, WFA, or P1f-Ins16) and in unreconstituted or P1f-reconstituted Plec−/− cells. Myotubes analyzed in C: Plec+/+, untreated, 6, and treated, 7; Plec−/−, 5; and P1f-GFP, 5. Clusters analyzed in D and E: Plec+/+, untreated, 202, and treated, 174; Plec−/−, 217; and P1f-GFP, 21. Mean ± SEM, three independent experiments. *p < 0.05 and ***p < 0.001 compared with Plec+/+, untreated; unpaired Student's t test.

Mentions: To visualize the dynamic interplay of AChR clusters and IFs, we performed live imaging of desmin–green fluorescent protein (GFP)–transfected Plec+/+ and Plec−/− myotubes that after withdrawal of agrin were additionally labeled with Rhodamine Red–streptavidin-biotin-α-BTX. Monitoring Plec+/+ cells for up to 2 h in the absence of agrin, we observed compact and relatively immobile AChR clusters enclosed in a lattice of desmin IFs (Figure 3A and Supplemental Video S1). In contrast, in Plec−/− myotubes, collapsed desmin IF networks were visualized as aggregates that were distant from receptor clusters without showing any overlap. Of interest, already as early as 6 min after initiating the imaging period, fragmentation and a drifting away from IF-disconnected larger clusters could be observed in this case (Figure 3B and Supplemental Video S2). Moreover, large clusters as a whole were changing their position within the plane of the sarcolemma (Figure 3B and Supplemental Video S2). Finally, transient and apparently unstable interactions between microclusters were detected, suggesting that these structures were unable to fuse into more stable larger clusters (Figure 3B and Supplemental Video S2). When agrin-induced AChR cluster formation was monitored by live imaging of transfected GFP-AChR-ε-subunit instead of α-BTX labeling, similar results were obtained (Supplemental Videos S5 and S6).


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)

Time-lapse video microscopy of desmin-GFP–expressing Plec+/+ and Plec−/− myotubes. (A) Top, confocal image showing part of a desmin-GFP–expressing Plec+/+ myotube; Rows below, time-lapse images of the central area (delineated by dashed lines). Bracket indicates region of a representative large AChR cluster embedded in a dense network of desmin IFs. Note the nearly unchanged position and compactness of AChR cluster over the entire observation period (0–52.5 min; see also Supplemental Video S1). (B) As in A, except that a Plec−/− myotube is shown. Cell margins are outlined in white. Note the prominent desmin IF network aggregate, distant from clusters. White and yellow arrowheads, microclusters separating from a large cluster; note their repeated transient mutual interaction during traveling and final disassembly of the one labeled white in three even smaller microclusters. Purple arrowhead, separation of another microcluster (magenta; time point 75 min) that drifts away and eventually vanishes completely (see also Supplemental Video S2). Note also positional shift of the large cluster as a whole relative to its initial position (bracket). Bars, 10 μm (A, B). (C–E) Statistical quantification of mobile cluster parameters (number, velocity, and total distance traveled) in Plec+/+ cells (before or after induction of IF network collapse using OA, WFA, or P1f-Ins16) and in unreconstituted or P1f-reconstituted Plec−/− cells. Myotubes analyzed in C: Plec+/+, untreated, 6, and treated, 7; Plec−/−, 5; and P1f-GFP, 5. Clusters analyzed in D and E: Plec+/+, untreated, 202, and treated, 174; Plec−/−, 217; and P1f-GFP, 21. Mean ± SEM, three independent experiments. *p < 0.05 and ***p < 0.001 compared with Plec+/+, untreated; unpaired Student's t test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4263455&req=5

Figure 3: Time-lapse video microscopy of desmin-GFP–expressing Plec+/+ and Plec−/− myotubes. (A) Top, confocal image showing part of a desmin-GFP–expressing Plec+/+ myotube; Rows below, time-lapse images of the central area (delineated by dashed lines). Bracket indicates region of a representative large AChR cluster embedded in a dense network of desmin IFs. Note the nearly unchanged position and compactness of AChR cluster over the entire observation period (0–52.5 min; see also Supplemental Video S1). (B) As in A, except that a Plec−/− myotube is shown. Cell margins are outlined in white. Note the prominent desmin IF network aggregate, distant from clusters. White and yellow arrowheads, microclusters separating from a large cluster; note their repeated transient mutual interaction during traveling and final disassembly of the one labeled white in three even smaller microclusters. Purple arrowhead, separation of another microcluster (magenta; time point 75 min) that drifts away and eventually vanishes completely (see also Supplemental Video S2). Note also positional shift of the large cluster as a whole relative to its initial position (bracket). Bars, 10 μm (A, B). (C–E) Statistical quantification of mobile cluster parameters (number, velocity, and total distance traveled) in Plec+/+ cells (before or after induction of IF network collapse using OA, WFA, or P1f-Ins16) and in unreconstituted or P1f-reconstituted Plec−/− cells. Myotubes analyzed in C: Plec+/+, untreated, 6, and treated, 7; Plec−/−, 5; and P1f-GFP, 5. Clusters analyzed in D and E: Plec+/+, untreated, 202, and treated, 174; Plec−/−, 217; and P1f-GFP, 21. Mean ± SEM, three independent experiments. *p < 0.05 and ***p < 0.001 compared with Plec+/+, untreated; unpaired Student's t test.
Mentions: To visualize the dynamic interplay of AChR clusters and IFs, we performed live imaging of desmin–green fluorescent protein (GFP)–transfected Plec+/+ and Plec−/− myotubes that after withdrawal of agrin were additionally labeled with Rhodamine Red–streptavidin-biotin-α-BTX. Monitoring Plec+/+ cells for up to 2 h in the absence of agrin, we observed compact and relatively immobile AChR clusters enclosed in a lattice of desmin IFs (Figure 3A and Supplemental Video S1). In contrast, in Plec−/− myotubes, collapsed desmin IF networks were visualized as aggregates that were distant from receptor clusters without showing any overlap. Of interest, already as early as 6 min after initiating the imaging period, fragmentation and a drifting away from IF-disconnected larger clusters could be observed in this case (Figure 3B and Supplemental Video S2). Moreover, large clusters as a whole were changing their position within the plane of the sarcolemma (Figure 3B and Supplemental Video S2). Finally, transient and apparently unstable interactions between microclusters were detected, suggesting that these structures were unable to fuse into more stable larger clusters (Figure 3B and Supplemental Video S2). When agrin-induced AChR cluster formation was monitored by live imaging of transfected GFP-AChR-ε-subunit instead of α-BTX labeling, similar results were obtained (Supplemental Videos S5 and S6).

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