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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 deficiency leads to conformational changes of chromatin. (A) Triple labeling of nuclei and their cytoplasmic environment using Hoechst dye (nuclei) and antibodies to desmin and either euchromatin (H3K4ME3, left panels) or heterochromatin (H3K27ME3, right panels). Scale bars, 10 µm. (B) Bar graphs representing statistical analyses of euchromatin and heterochromatin content of nuclei shown in (A). Fluorescent signals of H3K4ME3 or H3K27ME3 were normalized to total nuclear areas. Error bars ± SEM, three experiments, n = 81 nuclei measured for each experiment. *P < 0.05; ***P < 0.001. (C) Quantitative IB analysis of euchromatin and heterochromatin in cell lysates derived from wild-type and P1−/− myotubes. Signals were normalized to GAPDH (see also Supplementary Material, Fig. S1). (D) Quantitation (as in C) of total amount of H3K9ac in wild-type and P1−/− myotube cell lysates. Note significant decrease in overall gene expression level in P1-deficient compared with wild-type cells.
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DDV438F6: P1 deficiency leads to conformational changes of chromatin. (A) Triple labeling of nuclei and their cytoplasmic environment using Hoechst dye (nuclei) and antibodies to desmin and either euchromatin (H3K4ME3, left panels) or heterochromatin (H3K27ME3, right panels). Scale bars, 10 µm. (B) Bar graphs representing statistical analyses of euchromatin and heterochromatin content of nuclei shown in (A). Fluorescent signals of H3K4ME3 or H3K27ME3 were normalized to total nuclear areas. Error bars ± SEM, three experiments, n = 81 nuclei measured for each experiment. *P < 0.05; ***P < 0.001. (C) Quantitative IB analysis of euchromatin and heterochromatin in cell lysates derived from wild-type and P1−/− myotubes. Signals were normalized to GAPDH (see also Supplementary Material, Fig. S1). (D) Quantitation (as in C) of total amount of H3K9ac in wild-type and P1−/− myotube cell lysates. Note significant decrease in overall gene expression level in P1-deficient compared with wild-type cells.

Mentions: It has been shown that myonuclei translocate along the long axis of the myofibers in order to minimize transport distances and ensure sufficient transcriptional capacity on-site (26–28). To assess whether P1 deficiency affected the movement of nuclei along the myotube, fully differentiated P1−/− and wild-type myotubes were subjected to live cell video microscopy. The parameters measured, total distance, velocity and pause frequency revealed that the nuclear dynamics in P1 cells were drastically decreased compared with wild-type. While control nuclei could easily translocate within the myotube, nuclei from P1-deficient cells migrate to a lesser extent or sometimes seem not to move at all (Fig. 5A). During a 2-h observation period, wild-type nuclei covered distances of up to 100 µm with an average velocity of 15 nm/s, while the corresponding parameters of P1−/− nuclei were reduced to ∼50% (Fig. 5B). Moreover, we noticed that P1−/− nuclei paused more frequently compared with their wild-type counterparts (Fig. 5B). Trajectory tracking of nuclei clearly demonstrated that within the same timeframe, wild-type nuclei migrated over longer distances than P1−/− nuclei (Fig. 5C). The latter also showed slower movements and, instead of migrating directionally, they often oscillated or were rotating in one place (Fig. 6A and C). The observed decrease in nuclear dynamics in P1−/− myotubes would be consistent with a potential role of P1 in force transmission from the cytoskeleton to the nuclear envelope.Figure 5.


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 deficiency leads to conformational changes of chromatin. (A) Triple labeling of nuclei and their cytoplasmic environment using Hoechst dye (nuclei) and antibodies to desmin and either euchromatin (H3K4ME3, left panels) or heterochromatin (H3K27ME3, right panels). Scale bars, 10 µm. (B) Bar graphs representing statistical analyses of euchromatin and heterochromatin content of nuclei shown in (A). Fluorescent signals of H3K4ME3 or H3K27ME3 were normalized to total nuclear areas. Error bars ± SEM, three experiments, n = 81 nuclei measured for each experiment. *P < 0.05; ***P < 0.001. (C) Quantitative IB analysis of euchromatin and heterochromatin in cell lysates derived from wild-type and P1−/− myotubes. Signals were normalized to GAPDH (see also Supplementary Material, Fig. S1). (D) Quantitation (as in C) of total amount of H3K9ac in wild-type and P1−/− myotube cell lysates. Note significant decrease in overall gene expression level in P1-deficient compared with wild-type cells.
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Related In: Results  -  Collection

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DDV438F6: P1 deficiency leads to conformational changes of chromatin. (A) Triple labeling of nuclei and their cytoplasmic environment using Hoechst dye (nuclei) and antibodies to desmin and either euchromatin (H3K4ME3, left panels) or heterochromatin (H3K27ME3, right panels). Scale bars, 10 µm. (B) Bar graphs representing statistical analyses of euchromatin and heterochromatin content of nuclei shown in (A). Fluorescent signals of H3K4ME3 or H3K27ME3 were normalized to total nuclear areas. Error bars ± SEM, three experiments, n = 81 nuclei measured for each experiment. *P < 0.05; ***P < 0.001. (C) Quantitative IB analysis of euchromatin and heterochromatin in cell lysates derived from wild-type and P1−/− myotubes. Signals were normalized to GAPDH (see also Supplementary Material, Fig. S1). (D) Quantitation (as in C) of total amount of H3K9ac in wild-type and P1−/− myotube cell lysates. Note significant decrease in overall gene expression level in P1-deficient compared with wild-type cells.
Mentions: It has been shown that myonuclei translocate along the long axis of the myofibers in order to minimize transport distances and ensure sufficient transcriptional capacity on-site (26–28). To assess whether P1 deficiency affected the movement of nuclei along the myotube, fully differentiated P1−/− and wild-type myotubes were subjected to live cell video microscopy. The parameters measured, total distance, velocity and pause frequency revealed that the nuclear dynamics in P1 cells were drastically decreased compared with wild-type. While control nuclei could easily translocate within the myotube, nuclei from P1-deficient cells migrate to a lesser extent or sometimes seem not to move at all (Fig. 5A). During a 2-h observation period, wild-type nuclei covered distances of up to 100 µm with an average velocity of 15 nm/s, while the corresponding parameters of P1−/− nuclei were reduced to ∼50% (Fig. 5B). Moreover, we noticed that P1−/− nuclei paused more frequently compared with their wild-type counterparts (Fig. 5B). Trajectory tracking of nuclei clearly demonstrated that within the same timeframe, wild-type nuclei migrated over longer distances than P1−/− nuclei (Fig. 5C). The latter also showed slower movements and, instead of migrating directionally, they often oscillated or were rotating in one place (Fig. 6A and C). The observed decrease in nuclear dynamics in P1−/− myotubes would be consistent with a potential role of P1 in force transmission from the cytoskeleton to the nuclear envelope.Figure 5.

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