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Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers.

Staszewska I, Fischer I, Wiche G - Hum. Mol. Genet. (2015)

Bottom Line: We show that P1-mediated targeting of desmin IFs to myonuclei is essential for maintenance of their typically spheroidal architecture as well as their proper positioning and movement along the myofiber.Mechanistically, P1 is shown to specifically interact with the myonuclear membrane-associated (BAR domain-containing) protein endophilin B.Our results open a new perspective on cytoskeleton-nuclear crosstalk via specific cytolinker proteins.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus

P1-specific rescue of nuclear morphology in differentiated myotubes. (A) Schematic representation of EGFP-tagged plectin isoform cDNA expression constructs. Exon organization (bar on top) and important molecular domains are indicated. ABD, actin binding domain; CH1/2, calponin homology domains; SH3, Src homology 3 domain; IFBD, intermediate filament binding domain. (B) P1−/− myoblasts transfected with EGFP-tagged plectin isoforms (P1-EGFP, P1b-EGFP or P1h-EGFP) were subjected (7 days) to differentiation into multinucleated myotubes and nuclei visualized with Hoechst dye; nuclei of non-transfected myotubes served as controls. Scale bars, 10 µm. (C) Statistical analysis of area, perimeter, shape factor and aspect ratio of nuclei in myotubes transfected with different plectin cDNA construct. Note rescue of nuclear morphology upon P1-EGFP expression and partial rescue upon P1b-EGFP expression. Error bars ± SEM, three experiments, n = 33 nuclei analyzed. *P < 0.05; **P < 0.005; ***P < 0.001.
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DDV438F4: P1-specific rescue of nuclear morphology in differentiated myotubes. (A) Schematic representation of EGFP-tagged plectin isoform cDNA expression constructs. Exon organization (bar on top) and important molecular domains are indicated. ABD, actin binding domain; CH1/2, calponin homology domains; SH3, Src homology 3 domain; IFBD, intermediate filament binding domain. (B) P1−/− myoblasts transfected with EGFP-tagged plectin isoforms (P1-EGFP, P1b-EGFP or P1h-EGFP) were subjected (7 days) to differentiation into multinucleated myotubes and nuclei visualized with Hoechst dye; nuclei of non-transfected myotubes served as controls. Scale bars, 10 µm. (C) Statistical analysis of area, perimeter, shape factor and aspect ratio of nuclei in myotubes transfected with different plectin cDNA construct. Note rescue of nuclear morphology upon P1-EGFP expression and partial rescue upon P1b-EGFP expression. Error bars ± SEM, three experiments, n = 33 nuclei analyzed. *P < 0.05; **P < 0.005; ***P < 0.001.

Mentions: To assess the potential of distinct plectin isoforms to restore normal nuclear morphology, plasmids encoding EGFP-tagged versions of full-length P1, P1b or P1h were transfected into P1−/− myoblasts that subsequently were differentiated into myotubes. All of the plectin expressed isoforms contained the C-terminal IF-binding domain, but differed at their N termini (Fig. 4A). P1 and P1b started with the short sequences encoded by their alternative first exons, whereas P1h (having its start codon in exon 6) represented an isoform without isoform-specific N terminal sequences and without a functional ABD (14) (Fig. 4A). As shown in Figure 4B and C, the forced expression of P1 and P1b, both resulted in a reduction of the size and the perimeters of nuclei, albeit P1 was significantly more effective than P1b. Statistically, the differences between wild-type and P1−/− myotubes in size and perimeters of nuclei were reduced upon expression of P1 by ∼64 and 50%, respectively. Remarkably, only full-length P1, but not P1b, was effecting a rescue of the elliptical shape typical of wild-type nuclei (Fig. 4C, aspect ratio). In contrast, overexpressed P1h showed no effect on any of the nuclear parameters measured, and thus was lacking any rescue potential (Fig. 4C).Figure 4.


Plectin isoform 1-dependent nuclear docking of desmin networks affects myonuclear architecture and expression of mechanotransducers.

Staszewska I, Fischer I, Wiche G - Hum. Mol. Genet. (2015)

P1-specific rescue of nuclear morphology in differentiated myotubes. (A) Schematic representation of EGFP-tagged plectin isoform cDNA expression constructs. Exon organization (bar on top) and important molecular domains are indicated. ABD, actin binding domain; CH1/2, calponin homology domains; SH3, Src homology 3 domain; IFBD, intermediate filament binding domain. (B) P1−/− myoblasts transfected with EGFP-tagged plectin isoforms (P1-EGFP, P1b-EGFP or P1h-EGFP) were subjected (7 days) to differentiation into multinucleated myotubes and nuclei visualized with Hoechst dye; nuclei of non-transfected myotubes served as controls. Scale bars, 10 µm. (C) Statistical analysis of area, perimeter, shape factor and aspect ratio of nuclei in myotubes transfected with different plectin cDNA construct. Note rescue of nuclear morphology upon P1-EGFP expression and partial rescue upon P1b-EGFP expression. Error bars ± SEM, three experiments, n = 33 nuclei analyzed. *P < 0.05; **P < 0.005; ***P < 0.001.
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Related In: Results  -  Collection

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DDV438F4: P1-specific rescue of nuclear morphology in differentiated myotubes. (A) Schematic representation of EGFP-tagged plectin isoform cDNA expression constructs. Exon organization (bar on top) and important molecular domains are indicated. ABD, actin binding domain; CH1/2, calponin homology domains; SH3, Src homology 3 domain; IFBD, intermediate filament binding domain. (B) P1−/− myoblasts transfected with EGFP-tagged plectin isoforms (P1-EGFP, P1b-EGFP or P1h-EGFP) were subjected (7 days) to differentiation into multinucleated myotubes and nuclei visualized with Hoechst dye; nuclei of non-transfected myotubes served as controls. Scale bars, 10 µm. (C) Statistical analysis of area, perimeter, shape factor and aspect ratio of nuclei in myotubes transfected with different plectin cDNA construct. Note rescue of nuclear morphology upon P1-EGFP expression and partial rescue upon P1b-EGFP expression. Error bars ± SEM, three experiments, n = 33 nuclei analyzed. *P < 0.05; **P < 0.005; ***P < 0.001.
Mentions: To assess the potential of distinct plectin isoforms to restore normal nuclear morphology, plasmids encoding EGFP-tagged versions of full-length P1, P1b or P1h were transfected into P1−/− myoblasts that subsequently were differentiated into myotubes. All of the plectin expressed isoforms contained the C-terminal IF-binding domain, but differed at their N termini (Fig. 4A). P1 and P1b started with the short sequences encoded by their alternative first exons, whereas P1h (having its start codon in exon 6) represented an isoform without isoform-specific N terminal sequences and without a functional ABD (14) (Fig. 4A). As shown in Figure 4B and C, the forced expression of P1 and P1b, both resulted in a reduction of the size and the perimeters of nuclei, albeit P1 was significantly more effective than P1b. Statistically, the differences between wild-type and P1−/− myotubes in size and perimeters of nuclei were reduced upon expression of P1 by ∼64 and 50%, respectively. Remarkably, only full-length P1, but not P1b, was effecting a rescue of the elliptical shape typical of wild-type nuclei (Fig. 4C, aspect ratio). In contrast, overexpressed P1h showed no effect on any of the nuclear parameters measured, and thus was lacking any rescue potential (Fig. 4C).Figure 4.

Bottom Line: We show that P1-mediated targeting of desmin IFs to myonuclei is essential for maintenance of their typically spheroidal architecture as well as their proper positioning and movement along the myofiber.Mechanistically, P1 is shown to specifically interact with the myonuclear membrane-associated (BAR domain-containing) protein endophilin B.Our results open a new perspective on cytoskeleton-nuclear crosstalk via specific cytolinker proteins.

View Article: PubMed Central - PubMed

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

No MeSH data available.


Related in: MedlinePlus