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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

Altered mRNA expression and protein homeostasis in P1-deficient muscle cells. (A) Quantitative analysis of mechanotransducer transcripts in wild-type and P1-deficient myotubes. Graphs show the relative expression levels of YAP, TAZ, STAT1/3, ERK1/2 and MAPK p38 in wild-type versus P1-deficient myotubes. RNA was isolated from at least three independent samples of (each) wild-type and P1-deficient cell cultures. RT-PCR analyses were performed in duplicates and measurements made using 96-well optical plates as described in the text. The housekeeping gene HPRT was used as a reference. Note significant downregulation of mRNA expression of STAT1, ERK1 and ERK2 in P1-deficient cells compared with wild-type controls. Error bars, ±SEM. *P < 0.05; ***P < 0.001. (B–E) Equivalent amounts of cell lysates derived from wild-type and P1–/– muscles were subjected to IB analysis using antibodies to the signal transducers and activators of transcriptions YAP/TAZ and STAT1 (B), the protein kinases ERK1/2 and MAPK p38 (C), AMP-activated protein kinase (AMPK) (D) and heat shock protein (hsp) 27 (E). GAPDH was used as loading control. Error bars ± SEM. *P < 0.05; **P < 0.005; ***P < 0.001. Note striking (5-fold) upregulation of hsp27 in P1-deficient cells (D). (F) Double IFM of the nuclear area of wild-type and P1-KO teased EDL muscle fibers using antibodies to desmin and hsp27; nuclei were visualized with Hoechst dye. Note accumulation and colocalization of desmin and hsp27 in the perinuclear area of the P1-KO fiber (arrowheads). Scale bars, 10 µm.
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DDV438F7: Altered mRNA expression and protein homeostasis in P1-deficient muscle cells. (A) Quantitative analysis of mechanotransducer transcripts in wild-type and P1-deficient myotubes. Graphs show the relative expression levels of YAP, TAZ, STAT1/3, ERK1/2 and MAPK p38 in wild-type versus P1-deficient myotubes. RNA was isolated from at least three independent samples of (each) wild-type and P1-deficient cell cultures. RT-PCR analyses were performed in duplicates and measurements made using 96-well optical plates as described in the text. The housekeeping gene HPRT was used as a reference. Note significant downregulation of mRNA expression of STAT1, ERK1 and ERK2 in P1-deficient cells compared with wild-type controls. Error bars, ±SEM. *P < 0.05; ***P < 0.001. (B–E) Equivalent amounts of cell lysates derived from wild-type and P1–/– muscles were subjected to IB analysis using antibodies to the signal transducers and activators of transcriptions YAP/TAZ and STAT1 (B), the protein kinases ERK1/2 and MAPK p38 (C), AMP-activated protein kinase (AMPK) (D) and heat shock protein (hsp) 27 (E). GAPDH was used as loading control. Error bars ± SEM. *P < 0.05; **P < 0.005; ***P < 0.001. Note striking (5-fold) upregulation of hsp27 in P1-deficient cells (D). (F) Double IFM of the nuclear area of wild-type and P1-KO teased EDL muscle fibers using antibodies to desmin and hsp27; nuclei were visualized with Hoechst dye. Note accumulation and colocalization of desmin and hsp27 in the perinuclear area of the P1-KO fiber (arrowheads). Scale bars, 10 µm.

Mentions: To assess whether proteins involved in mechanotransduction were affected, we measured mRNA and protein expression levels of the mechanosignaling-linked transcription factors YAP, TAZ/WWTR1, STAT1 and STAT3 (30–32), and of the MAP kinase pathway constituents ERK1/2, and MAPK p38. Using quantitative RT-PCR (qRT-PCR), we found mRNA levels of STAT1 as well as ERK1 and ERK2 to be significantly reduced in P1−/− compared with wild-type cells (by 48, 38, and 45%, respectively), whereas no significant differences were found for the remainder of the transcripts tested (Fig. 7A; and Supplementary Material, Fig. S2). Interestingly, however, on the protein level, not only STAT1 and ERK1/2, but all of these proteins tested, including YAP, TAZ and MAPK p38, were found to be significantly reduced, with STAT1 (70% reduction) and MAP kinase ERK1 (75%) showing the most dramatic effects (Fig. 7A and B). On the other hand, AMP-activated protein kinase (AMPK), a protein responsible for energy homeostasis that is recruited to Z-disks by plectin during myogenesis (33), was found to be upregulated in P1-deficient versus wild-type cells by 40% (Fig. 7C). Similarly, a striking (5-fold compared with wild-type) upregulation of heat shock protein hsp27 was observed for P1−/− myotube lysates (Fig. 7D), indicative of a massive chaperone activation in response to P1 deficiency-inflicted IF network collapse. A comparably strong upregulation of hsp27 had previously been observed in plectin- myotubes (20). As expected, double IFM of teased P1−/− myofibers using antibodies to hsp27 and desmin revealed colocalization of hsp27 with desmin-positive perinuclear aggregates (Fig. 7E). In all, these results supported the notion that P1 not only is important for specific morphological features, translocation and compartmentalization of myonuclei, but also affects functional properties of myonuclei, including the expression of some genes involved in mechanotransduction, metabolism and stress response.Figure 7.


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)

Altered mRNA expression and protein homeostasis in P1-deficient muscle cells. (A) Quantitative analysis of mechanotransducer transcripts in wild-type and P1-deficient myotubes. Graphs show the relative expression levels of YAP, TAZ, STAT1/3, ERK1/2 and MAPK p38 in wild-type versus P1-deficient myotubes. RNA was isolated from at least three independent samples of (each) wild-type and P1-deficient cell cultures. RT-PCR analyses were performed in duplicates and measurements made using 96-well optical plates as described in the text. The housekeeping gene HPRT was used as a reference. Note significant downregulation of mRNA expression of STAT1, ERK1 and ERK2 in P1-deficient cells compared with wild-type controls. Error bars, ±SEM. *P < 0.05; ***P < 0.001. (B–E) Equivalent amounts of cell lysates derived from wild-type and P1–/– muscles were subjected to IB analysis using antibodies to the signal transducers and activators of transcriptions YAP/TAZ and STAT1 (B), the protein kinases ERK1/2 and MAPK p38 (C), AMP-activated protein kinase (AMPK) (D) and heat shock protein (hsp) 27 (E). GAPDH was used as loading control. Error bars ± SEM. *P < 0.05; **P < 0.005; ***P < 0.001. Note striking (5-fold) upregulation of hsp27 in P1-deficient cells (D). (F) Double IFM of the nuclear area of wild-type and P1-KO teased EDL muscle fibers using antibodies to desmin and hsp27; nuclei were visualized with Hoechst dye. Note accumulation and colocalization of desmin and hsp27 in the perinuclear area of the P1-KO fiber (arrowheads). Scale bars, 10 µm.
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DDV438F7: Altered mRNA expression and protein homeostasis in P1-deficient muscle cells. (A) Quantitative analysis of mechanotransducer transcripts in wild-type and P1-deficient myotubes. Graphs show the relative expression levels of YAP, TAZ, STAT1/3, ERK1/2 and MAPK p38 in wild-type versus P1-deficient myotubes. RNA was isolated from at least three independent samples of (each) wild-type and P1-deficient cell cultures. RT-PCR analyses were performed in duplicates and measurements made using 96-well optical plates as described in the text. The housekeeping gene HPRT was used as a reference. Note significant downregulation of mRNA expression of STAT1, ERK1 and ERK2 in P1-deficient cells compared with wild-type controls. Error bars, ±SEM. *P < 0.05; ***P < 0.001. (B–E) Equivalent amounts of cell lysates derived from wild-type and P1–/– muscles were subjected to IB analysis using antibodies to the signal transducers and activators of transcriptions YAP/TAZ and STAT1 (B), the protein kinases ERK1/2 and MAPK p38 (C), AMP-activated protein kinase (AMPK) (D) and heat shock protein (hsp) 27 (E). GAPDH was used as loading control. Error bars ± SEM. *P < 0.05; **P < 0.005; ***P < 0.001. Note striking (5-fold) upregulation of hsp27 in P1-deficient cells (D). (F) Double IFM of the nuclear area of wild-type and P1-KO teased EDL muscle fibers using antibodies to desmin and hsp27; nuclei were visualized with Hoechst dye. Note accumulation and colocalization of desmin and hsp27 in the perinuclear area of the P1-KO fiber (arrowheads). Scale bars, 10 µm.
Mentions: To assess whether proteins involved in mechanotransduction were affected, we measured mRNA and protein expression levels of the mechanosignaling-linked transcription factors YAP, TAZ/WWTR1, STAT1 and STAT3 (30–32), and of the MAP kinase pathway constituents ERK1/2, and MAPK p38. Using quantitative RT-PCR (qRT-PCR), we found mRNA levels of STAT1 as well as ERK1 and ERK2 to be significantly reduced in P1−/− compared with wild-type cells (by 48, 38, and 45%, respectively), whereas no significant differences were found for the remainder of the transcripts tested (Fig. 7A; and Supplementary Material, Fig. S2). Interestingly, however, on the protein level, not only STAT1 and ERK1/2, but all of these proteins tested, including YAP, TAZ and MAPK p38, were found to be significantly reduced, with STAT1 (70% reduction) and MAP kinase ERK1 (75%) showing the most dramatic effects (Fig. 7A and B). On the other hand, AMP-activated protein kinase (AMPK), a protein responsible for energy homeostasis that is recruited to Z-disks by plectin during myogenesis (33), was found to be upregulated in P1-deficient versus wild-type cells by 40% (Fig. 7C). Similarly, a striking (5-fold compared with wild-type) upregulation of heat shock protein hsp27 was observed for P1−/− myotube lysates (Fig. 7D), indicative of a massive chaperone activation in response to P1 deficiency-inflicted IF network collapse. A comparably strong upregulation of hsp27 had previously been observed in plectin- myotubes (20). As expected, double IFM of teased P1−/− myofibers using antibodies to hsp27 and desmin revealed colocalization of hsp27 with desmin-positive perinuclear aggregates (Fig. 7E). In all, these results supported the notion that P1 not only is important for specific morphological features, translocation and compartmentalization of myonuclei, but also affects functional properties of myonuclei, including the expression of some genes involved in mechanotransduction, metabolism and stress response.Figure 7.

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