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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

Nuclear alterations of plectin-deficient myofibers are mimicked by myotubes differentiated ex vivo from P1-deficient myoblasts. (A) Upper panels, double IFM of wild-type and P1−/− myoblasts using anti-P1 and anti-desmin antibodies, and visualization of nuclei with Hoechst dye; lower panels, higher magnification of stained nuclei from three different cells. Scale bars, 5 µm (upper panels), 10 µm (lower panels). (B) IB of total cell lysates prepared from wild-type or P1–/– myotubes using anti-pan plectin and anti-P1 antibodies. (C) Statistical analysis of the length of wild-type and P1−/− myotubes differentiated for the time indicated. Error bars ± SEM. *P < 0.05. Myotubes analyzed: WT, n = 76, P1–/–, n = 79 (day 2); WT, n = 285, P1–/–, n = 127 (day 4); WT, n = 335, P1–/–, n = 270 (day 6); WT, n = 539, P1–/–, n = 399 (day 8); WT, n = 363, P1–/–, n = 260 (day 10). (D) Double IFM of wild-type and P1−/− myotubes using antibodies to desmin and α-actinin; nuclei visualized with Hoechst dye are highlighted by a dashed line. Arrowheads indicate aggregates of desmin IFs at the nuclear periphery in P1−/− cells. Scale bars, 10 µm. (E) Upper panel: IFM of wild-type and P1−/− myotubes (day 7) using antibodies to desmin; nuclei were visualized with Hoechst dye. Lower panel: bar graphs representing statistical analyses of areas, perimeters, shape factors and aspect ratios of nuclei derived from undifferentiated myoblasts and multinucleated myotubes of wild-type and P1−/− cell lines. Error bars ± SEM, three experiments, n = 60 nuclei analyzed from undifferentiated and differentiated cells. **P < 0.005; ***P < 0.001. (F) Wild-type and P1−/− myotubes were immunolabeled using antibodies to desmin, and nuclei (highlighted by dashed lines) were visualized with Hoechst dye. 3D reconstructions of single WT and P1−/−cells were prepared from confocal Z-stack images using Huygens software. Images are shown from two perspectives: above the cells (upper panels), and sideways (lower panels). Image orientations (yellow asterisk) and rotation axes are indicated. Scale bars, 10 µm. (G) Statistical evaluation of the volume of nuclei present in wild-type and P1−/− myotubes. Error bars ± SEM, three experiments. Nuclei analyzed in each experiment: WT, n = 97; P1−/−, n = 101. ***P < 0.001.
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DDV438F3: Nuclear alterations of plectin-deficient myofibers are mimicked by myotubes differentiated ex vivo from P1-deficient myoblasts. (A) Upper panels, double IFM of wild-type and P1−/− myoblasts using anti-P1 and anti-desmin antibodies, and visualization of nuclei with Hoechst dye; lower panels, higher magnification of stained nuclei from three different cells. Scale bars, 5 µm (upper panels), 10 µm (lower panels). (B) IB of total cell lysates prepared from wild-type or P1–/– myotubes using anti-pan plectin and anti-P1 antibodies. (C) Statistical analysis of the length of wild-type and P1−/− myotubes differentiated for the time indicated. Error bars ± SEM. *P < 0.05. Myotubes analyzed: WT, n = 76, P1–/–, n = 79 (day 2); WT, n = 285, P1–/–, n = 127 (day 4); WT, n = 335, P1–/–, n = 270 (day 6); WT, n = 539, P1–/–, n = 399 (day 8); WT, n = 363, P1–/–, n = 260 (day 10). (D) Double IFM of wild-type and P1−/− myotubes using antibodies to desmin and α-actinin; nuclei visualized with Hoechst dye are highlighted by a dashed line. Arrowheads indicate aggregates of desmin IFs at the nuclear periphery in P1−/− cells. Scale bars, 10 µm. (E) Upper panel: IFM of wild-type and P1−/− myotubes (day 7) using antibodies to desmin; nuclei were visualized with Hoechst dye. Lower panel: bar graphs representing statistical analyses of areas, perimeters, shape factors and aspect ratios of nuclei derived from undifferentiated myoblasts and multinucleated myotubes of wild-type and P1−/− cell lines. Error bars ± SEM, three experiments, n = 60 nuclei analyzed from undifferentiated and differentiated cells. **P < 0.005; ***P < 0.001. (F) Wild-type and P1−/− myotubes were immunolabeled using antibodies to desmin, and nuclei (highlighted by dashed lines) were visualized with Hoechst dye. 3D reconstructions of single WT and P1−/−cells were prepared from confocal Z-stack images using Huygens software. Images are shown from two perspectives: above the cells (upper panels), and sideways (lower panels). Image orientations (yellow asterisk) and rotation axes are indicated. Scale bars, 10 µm. (G) Statistical evaluation of the volume of nuclei present in wild-type and P1−/− myotubes. Error bars ± SEM, three experiments. Nuclei analyzed in each experiment: WT, n = 97; P1−/−, n = 101. ***P < 0.001.

Mentions: To study the mechanisms leading to the nuclear phenotype of isoform P1-deficient myofibers on molecular and cellular levels, we generated a P1 isoform-deficient immortalized myoblast cell line, using a previously established protocol that is based on the isolation of myoblast cultures from mutant mouse lines crossed into a p53−/− background (20). As shown by immunostaining and immunoblotting (IB) using isoform-specific and anti-pan plectin antibodies, cultures of immortalized myoblasts isolated from P1−/−/p53−/− double knockout mice (from now on referred to as P1−/− myoblasts) were devoid of P1, while other isoforms of plectin seemed to be expressed normally (Fig. 3A and B). Immunofluorescence microscopy (IFM) of P1+/+/p53−/− myoblasts (referred to as wild-type) using isoform P1 and desmin-specific antibodies revealed partial colocalization of both proteins predominantly in perinuclear areas (Fig. 3A). Moreover, a trend toward a more spherical shape of P−/− compared with wild-type nuclei became apparent at higher magnifications (Fig. 3A, lower panels; see also Fig. 3E).Figure 3.


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)

Nuclear alterations of plectin-deficient myofibers are mimicked by myotubes differentiated ex vivo from P1-deficient myoblasts. (A) Upper panels, double IFM of wild-type and P1−/− myoblasts using anti-P1 and anti-desmin antibodies, and visualization of nuclei with Hoechst dye; lower panels, higher magnification of stained nuclei from three different cells. Scale bars, 5 µm (upper panels), 10 µm (lower panels). (B) IB of total cell lysates prepared from wild-type or P1–/– myotubes using anti-pan plectin and anti-P1 antibodies. (C) Statistical analysis of the length of wild-type and P1−/− myotubes differentiated for the time indicated. Error bars ± SEM. *P < 0.05. Myotubes analyzed: WT, n = 76, P1–/–, n = 79 (day 2); WT, n = 285, P1–/–, n = 127 (day 4); WT, n = 335, P1–/–, n = 270 (day 6); WT, n = 539, P1–/–, n = 399 (day 8); WT, n = 363, P1–/–, n = 260 (day 10). (D) Double IFM of wild-type and P1−/− myotubes using antibodies to desmin and α-actinin; nuclei visualized with Hoechst dye are highlighted by a dashed line. Arrowheads indicate aggregates of desmin IFs at the nuclear periphery in P1−/− cells. Scale bars, 10 µm. (E) Upper panel: IFM of wild-type and P1−/− myotubes (day 7) using antibodies to desmin; nuclei were visualized with Hoechst dye. Lower panel: bar graphs representing statistical analyses of areas, perimeters, shape factors and aspect ratios of nuclei derived from undifferentiated myoblasts and multinucleated myotubes of wild-type and P1−/− cell lines. Error bars ± SEM, three experiments, n = 60 nuclei analyzed from undifferentiated and differentiated cells. **P < 0.005; ***P < 0.001. (F) Wild-type and P1−/− myotubes were immunolabeled using antibodies to desmin, and nuclei (highlighted by dashed lines) were visualized with Hoechst dye. 3D reconstructions of single WT and P1−/−cells were prepared from confocal Z-stack images using Huygens software. Images are shown from two perspectives: above the cells (upper panels), and sideways (lower panels). Image orientations (yellow asterisk) and rotation axes are indicated. Scale bars, 10 µm. (G) Statistical evaluation of the volume of nuclei present in wild-type and P1−/− myotubes. Error bars ± SEM, three experiments. Nuclei analyzed in each experiment: WT, n = 97; P1−/−, n = 101. ***P < 0.001.
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DDV438F3: Nuclear alterations of plectin-deficient myofibers are mimicked by myotubes differentiated ex vivo from P1-deficient myoblasts. (A) Upper panels, double IFM of wild-type and P1−/− myoblasts using anti-P1 and anti-desmin antibodies, and visualization of nuclei with Hoechst dye; lower panels, higher magnification of stained nuclei from three different cells. Scale bars, 5 µm (upper panels), 10 µm (lower panels). (B) IB of total cell lysates prepared from wild-type or P1–/– myotubes using anti-pan plectin and anti-P1 antibodies. (C) Statistical analysis of the length of wild-type and P1−/− myotubes differentiated for the time indicated. Error bars ± SEM. *P < 0.05. Myotubes analyzed: WT, n = 76, P1–/–, n = 79 (day 2); WT, n = 285, P1–/–, n = 127 (day 4); WT, n = 335, P1–/–, n = 270 (day 6); WT, n = 539, P1–/–, n = 399 (day 8); WT, n = 363, P1–/–, n = 260 (day 10). (D) Double IFM of wild-type and P1−/− myotubes using antibodies to desmin and α-actinin; nuclei visualized with Hoechst dye are highlighted by a dashed line. Arrowheads indicate aggregates of desmin IFs at the nuclear periphery in P1−/− cells. Scale bars, 10 µm. (E) Upper panel: IFM of wild-type and P1−/− myotubes (day 7) using antibodies to desmin; nuclei were visualized with Hoechst dye. Lower panel: bar graphs representing statistical analyses of areas, perimeters, shape factors and aspect ratios of nuclei derived from undifferentiated myoblasts and multinucleated myotubes of wild-type and P1−/− cell lines. Error bars ± SEM, three experiments, n = 60 nuclei analyzed from undifferentiated and differentiated cells. **P < 0.005; ***P < 0.001. (F) Wild-type and P1−/− myotubes were immunolabeled using antibodies to desmin, and nuclei (highlighted by dashed lines) were visualized with Hoechst dye. 3D reconstructions of single WT and P1−/−cells were prepared from confocal Z-stack images using Huygens software. Images are shown from two perspectives: above the cells (upper panels), and sideways (lower panels). Image orientations (yellow asterisk) and rotation axes are indicated. Scale bars, 10 µm. (G) Statistical evaluation of the volume of nuclei present in wild-type and P1−/− myotubes. Error bars ± SEM, three experiments. Nuclei analyzed in each experiment: WT, n = 97; P1−/−, n = 101. ***P < 0.001.
Mentions: To study the mechanisms leading to the nuclear phenotype of isoform P1-deficient myofibers on molecular and cellular levels, we generated a P1 isoform-deficient immortalized myoblast cell line, using a previously established protocol that is based on the isolation of myoblast cultures from mutant mouse lines crossed into a p53−/− background (20). As shown by immunostaining and immunoblotting (IB) using isoform-specific and anti-pan plectin antibodies, cultures of immortalized myoblasts isolated from P1−/−/p53−/− double knockout mice (from now on referred to as P1−/− myoblasts) were devoid of P1, while other isoforms of plectin seemed to be expressed normally (Fig. 3A and B). Immunofluorescence microscopy (IFM) of P1+/+/p53−/− myoblasts (referred to as wild-type) using isoform P1 and desmin-specific antibodies revealed partial colocalization of both proteins predominantly in perinuclear areas (Fig. 3A). Moreover, a trend toward a more spherical shape of P−/− compared with wild-type nuclei became apparent at higher magnifications (Fig. 3A, lower panels; see also Fig. 3E).Figure 3.

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