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Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells.

Lammerding J, Hsiao J, Schulze PC, Kozlov S, Stewart CL, Lee RT - J. Cell Biol. (2005)

Bottom Line: Clin.Invest. 113:370-378).Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA. jlammerding@rics.bwh.harvard.edu

ABSTRACT
Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370-378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

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Emerin-deficient cells have apparently normal nuclear mechanics. (a) Emerin-deficient primary mouse embryo fibroblasts have normalized nuclear strain comparable to wild-type cells when subjected to biaxial strain. In contrast, A-type lamin-deficient cells have significantly increased normalized nuclear strain (normalized nuclear strain = 0.27 ± 0.044, 0.23 ± 0.066, and 0.57 ± 0.059 for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively; P < 0.001 for A-type lamin-deficient vs. wild-type cells). (b) Wild-type (left) and emerin-deficient (center) nuclei remain intact when microinjected with fluorescently labeled dextran, whereas A-type lamin-deficient nuclei (right) have more fragile nuclei that allow dextran to leak into the cytoplasm. Bars, 20 μm. (c) Emerin-deficient and wild-type cells have comparable frequency of nuclear rupture for nuclear microinjection at 500 hPa, whereas A-type lamin-deficient cells have a significantly increased fraction of ruptured nuclei (percentage of ruptured nuclei = 69 ± 4.9%, 64 ± 5.5%, and 96 ± 1.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively).
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fig4: Emerin-deficient cells have apparently normal nuclear mechanics. (a) Emerin-deficient primary mouse embryo fibroblasts have normalized nuclear strain comparable to wild-type cells when subjected to biaxial strain. In contrast, A-type lamin-deficient cells have significantly increased normalized nuclear strain (normalized nuclear strain = 0.27 ± 0.044, 0.23 ± 0.066, and 0.57 ± 0.059 for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively; P < 0.001 for A-type lamin-deficient vs. wild-type cells). (b) Wild-type (left) and emerin-deficient (center) nuclei remain intact when microinjected with fluorescently labeled dextran, whereas A-type lamin-deficient nuclei (right) have more fragile nuclei that allow dextran to leak into the cytoplasm. Bars, 20 μm. (c) Emerin-deficient and wild-type cells have comparable frequency of nuclear rupture for nuclear microinjection at 500 hPa, whereas A-type lamin-deficient cells have a significantly increased fraction of ruptured nuclei (percentage of ruptured nuclei = 69 ± 4.9%, 64 ± 5.5%, and 96 ± 1.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively).

Mentions: The nuclear shape changes observed in the time-lapse experiments can be caused by intracellular forces exerted from the cytoskeleton onto the nucleus, from intranuclear processes such as DNA synthesis and chromatin remodeling, or from nuclear envelope dynamics (remodeling) over the 8 h 20 min observation time. To explore the role of emerin on nuclear mechanics independently of intranuclear and cytoskeletal changes that occur over a relatively long time scale, cells were cultured on transparent silicone membranes and subjected to ∼5% biaxial strain. The applied membrane strain is transmitted to the cytoskeleton through membrane receptors such as integrins, resulting in intracellular forces applied to the nucleus (Maniotis et al., 1997; Caille et al., 1998). The induced nuclear deformations were calculated by tracking distinct features in the fluorescently labeled chromatin and normalized to membrane strain to compensate for small variations in the applied membrane strain. This method of strain induction allows quantitative measurements of nuclear stiffness compared with cytoskeletal stiffness in living cells without having to isolate the nuclei (Caille et al., 1998; Lammerding et al., 2004). In wild-type cells, the nucleus is much stiffer than the surrounding cytoskeleton and showed only minor deformations under strain (Fig. 4 a). Emerin-deficient nuclei exhibited deformations comparable to those of wild-type cells, indicating apparently normal nuclear mechanics (Fig. 4 a). In contrast, A-type lamin-deficient nuclei had significantly larger deformations compared with wild-type cells with increased normalized nuclear strain (Fig. 4 a). Experiments were performed in primary and immortalized mouse embryo fibroblasts, and we observed the same trend in all cell lines (data shown are for primary cells), with no significant difference between emerin-deficient and wild-type cells and A-type lamin-deficient cells showing significantly larger deformations.


Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells.

Lammerding J, Hsiao J, Schulze PC, Kozlov S, Stewart CL, Lee RT - J. Cell Biol. (2005)

Emerin-deficient cells have apparently normal nuclear mechanics. (a) Emerin-deficient primary mouse embryo fibroblasts have normalized nuclear strain comparable to wild-type cells when subjected to biaxial strain. In contrast, A-type lamin-deficient cells have significantly increased normalized nuclear strain (normalized nuclear strain = 0.27 ± 0.044, 0.23 ± 0.066, and 0.57 ± 0.059 for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively; P < 0.001 for A-type lamin-deficient vs. wild-type cells). (b) Wild-type (left) and emerin-deficient (center) nuclei remain intact when microinjected with fluorescently labeled dextran, whereas A-type lamin-deficient nuclei (right) have more fragile nuclei that allow dextran to leak into the cytoplasm. Bars, 20 μm. (c) Emerin-deficient and wild-type cells have comparable frequency of nuclear rupture for nuclear microinjection at 500 hPa, whereas A-type lamin-deficient cells have a significantly increased fraction of ruptured nuclei (percentage of ruptured nuclei = 69 ± 4.9%, 64 ± 5.5%, and 96 ± 1.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively).
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Related In: Results  -  Collection

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fig4: Emerin-deficient cells have apparently normal nuclear mechanics. (a) Emerin-deficient primary mouse embryo fibroblasts have normalized nuclear strain comparable to wild-type cells when subjected to biaxial strain. In contrast, A-type lamin-deficient cells have significantly increased normalized nuclear strain (normalized nuclear strain = 0.27 ± 0.044, 0.23 ± 0.066, and 0.57 ± 0.059 for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively; P < 0.001 for A-type lamin-deficient vs. wild-type cells). (b) Wild-type (left) and emerin-deficient (center) nuclei remain intact when microinjected with fluorescently labeled dextran, whereas A-type lamin-deficient nuclei (right) have more fragile nuclei that allow dextran to leak into the cytoplasm. Bars, 20 μm. (c) Emerin-deficient and wild-type cells have comparable frequency of nuclear rupture for nuclear microinjection at 500 hPa, whereas A-type lamin-deficient cells have a significantly increased fraction of ruptured nuclei (percentage of ruptured nuclei = 69 ± 4.9%, 64 ± 5.5%, and 96 ± 1.9% for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively).
Mentions: The nuclear shape changes observed in the time-lapse experiments can be caused by intracellular forces exerted from the cytoskeleton onto the nucleus, from intranuclear processes such as DNA synthesis and chromatin remodeling, or from nuclear envelope dynamics (remodeling) over the 8 h 20 min observation time. To explore the role of emerin on nuclear mechanics independently of intranuclear and cytoskeletal changes that occur over a relatively long time scale, cells were cultured on transparent silicone membranes and subjected to ∼5% biaxial strain. The applied membrane strain is transmitted to the cytoskeleton through membrane receptors such as integrins, resulting in intracellular forces applied to the nucleus (Maniotis et al., 1997; Caille et al., 1998). The induced nuclear deformations were calculated by tracking distinct features in the fluorescently labeled chromatin and normalized to membrane strain to compensate for small variations in the applied membrane strain. This method of strain induction allows quantitative measurements of nuclear stiffness compared with cytoskeletal stiffness in living cells without having to isolate the nuclei (Caille et al., 1998; Lammerding et al., 2004). In wild-type cells, the nucleus is much stiffer than the surrounding cytoskeleton and showed only minor deformations under strain (Fig. 4 a). Emerin-deficient nuclei exhibited deformations comparable to those of wild-type cells, indicating apparently normal nuclear mechanics (Fig. 4 a). In contrast, A-type lamin-deficient nuclei had significantly larger deformations compared with wild-type cells with increased normalized nuclear strain (Fig. 4 a). Experiments were performed in primary and immortalized mouse embryo fibroblasts, and we observed the same trend in all cell lines (data shown are for primary cells), with no significant difference between emerin-deficient and wild-type cells and A-type lamin-deficient cells showing significantly larger deformations.

Bottom Line: Clin.Invest. 113:370-378).Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Division, Brigham and Women's Hospital, Boston, MA 02115, USA. jlammerding@rics.bwh.harvard.edu

ABSTRACT
Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370-378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

Show MeSH
Related in: MedlinePlus