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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 and A-type lamin-deficient cells are more sensitive to mechanical strain. (a) A-type lamin-deficient cells have a significantly increased number of propidium iodide–positive cells after 24 h of cyclic, 10% biaxial strain application. Emerin-deficient cells are not significantly different from wild-type cells. (Percentage of propidium iodide–positive cells at rest vs. after 24 h strain application = 0.13 ± 0.03% vs. 0.38 ± 0.12%, 0.2 ± 0.00% vs. 0.37 ± 0.04%, and 0.68 ± 0.18% vs. 2.94 ± 0.47% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.01 for wild-type vs. A-type lamin-deficient strained cells; P < 0.05 for wild-type vs. A-type lamin-deficient controls). (b) Emerin and A-type lamin-deficient cells have increased fractions of apoptotic cells after 24 h of cyclic, 10% biaxial strain application (percentage of apoptotic cells at rest vs. after 24 h of strain application = 0.37 ± 0.05% vs. 0.30 ± 0.04% for wild-type, 0.43 ± 0.02% vs. 0.70 ± 0.06% for emerin-deficient, and 0.72 ± 0.05% vs. 1.04 ± 0.11% for A-type lamin-deficient cells; P < 0.001 for strained emerin and A-type lamin-deficient vs. wild-type cells).
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fig5: Emerin and A-type lamin-deficient cells are more sensitive to mechanical strain. (a) A-type lamin-deficient cells have a significantly increased number of propidium iodide–positive cells after 24 h of cyclic, 10% biaxial strain application. Emerin-deficient cells are not significantly different from wild-type cells. (Percentage of propidium iodide–positive cells at rest vs. after 24 h strain application = 0.13 ± 0.03% vs. 0.38 ± 0.12%, 0.2 ± 0.00% vs. 0.37 ± 0.04%, and 0.68 ± 0.18% vs. 2.94 ± 0.47% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.01 for wild-type vs. A-type lamin-deficient strained cells; P < 0.05 for wild-type vs. A-type lamin-deficient controls). (b) Emerin and A-type lamin-deficient cells have increased fractions of apoptotic cells after 24 h of cyclic, 10% biaxial strain application (percentage of apoptotic cells at rest vs. after 24 h of strain application = 0.37 ± 0.05% vs. 0.30 ± 0.04% for wild-type, 0.43 ± 0.02% vs. 0.70 ± 0.06% for emerin-deficient, and 0.72 ± 0.05% vs. 1.04 ± 0.11% for A-type lamin-deficient cells; P < 0.001 for strained emerin and A-type lamin-deficient vs. wild-type cells).

Mentions: To evaluate the effect of emerin deficiency on cell survival in response to prolonged mechanical stimulation, mouse embryo fibroblasts were subjected to 24 h cyclic biaxial strain (1 Hz; 3, 5, or 10% strain). Total cell death and apoptosis were subsequently measured by propidium iodide uptake and DNA content analysis, respectively. Early apoptotic events can be detected by a characteristic pattern of DNA strand breaks through endonucleolysis, resulting in increased amounts of DNA fragments that are visible by flow cytometry as the sub-G1 phase in the DNA content distribution (Walker et al., 1993). Strain application at the two lowest settings (3 and 5% biaxial strain) had no significant effect on cell death or apoptosis in either cell line, but at the highest strain rate (10% biaxial strain), A-type lamin-deficient fibroblasts had a significantly increased fraction of dead cells compared with nonstretched controls and to strained wild-type cells (Fig. 5 a). Total cell death in emerin-deficient cells was not significantly increased compared with nonstretched controls and to wild-type cells. DNA content analysis of samples from the same experiments revealed a large increase in apoptotic cell fraction in the A-type lamin-deficient cells compared with nonstretched controls and strained wild-type cells. Interestingly, we also found a significantly increased apoptotic cell fraction in the emerin-deficient fibroblasts (Fig. 5 b). Baseline levels of apoptotic cells were comparable for emerin-deficient and wild-type cells, but significantly elevated in the A-type lamin-deficient cells.


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 and A-type lamin-deficient cells are more sensitive to mechanical strain. (a) A-type lamin-deficient cells have a significantly increased number of propidium iodide–positive cells after 24 h of cyclic, 10% biaxial strain application. Emerin-deficient cells are not significantly different from wild-type cells. (Percentage of propidium iodide–positive cells at rest vs. after 24 h strain application = 0.13 ± 0.03% vs. 0.38 ± 0.12%, 0.2 ± 0.00% vs. 0.37 ± 0.04%, and 0.68 ± 0.18% vs. 2.94 ± 0.47% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.01 for wild-type vs. A-type lamin-deficient strained cells; P < 0.05 for wild-type vs. A-type lamin-deficient controls). (b) Emerin and A-type lamin-deficient cells have increased fractions of apoptotic cells after 24 h of cyclic, 10% biaxial strain application (percentage of apoptotic cells at rest vs. after 24 h of strain application = 0.37 ± 0.05% vs. 0.30 ± 0.04% for wild-type, 0.43 ± 0.02% vs. 0.70 ± 0.06% for emerin-deficient, and 0.72 ± 0.05% vs. 1.04 ± 0.11% for A-type lamin-deficient cells; P < 0.001 for strained emerin and A-type lamin-deficient vs. wild-type cells).
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fig5: Emerin and A-type lamin-deficient cells are more sensitive to mechanical strain. (a) A-type lamin-deficient cells have a significantly increased number of propidium iodide–positive cells after 24 h of cyclic, 10% biaxial strain application. Emerin-deficient cells are not significantly different from wild-type cells. (Percentage of propidium iodide–positive cells at rest vs. after 24 h strain application = 0.13 ± 0.03% vs. 0.38 ± 0.12%, 0.2 ± 0.00% vs. 0.37 ± 0.04%, and 0.68 ± 0.18% vs. 2.94 ± 0.47% for wild-type, emerin-deficient, and A-type lamin-deficient cells respectively, P < 0.01 for wild-type vs. A-type lamin-deficient strained cells; P < 0.05 for wild-type vs. A-type lamin-deficient controls). (b) Emerin and A-type lamin-deficient cells have increased fractions of apoptotic cells after 24 h of cyclic, 10% biaxial strain application (percentage of apoptotic cells at rest vs. after 24 h of strain application = 0.37 ± 0.05% vs. 0.30 ± 0.04% for wild-type, 0.43 ± 0.02% vs. 0.70 ± 0.06% for emerin-deficient, and 0.72 ± 0.05% vs. 1.04 ± 0.11% for A-type lamin-deficient cells; P < 0.001 for strained emerin and A-type lamin-deficient vs. wild-type cells).
Mentions: To evaluate the effect of emerin deficiency on cell survival in response to prolonged mechanical stimulation, mouse embryo fibroblasts were subjected to 24 h cyclic biaxial strain (1 Hz; 3, 5, or 10% strain). Total cell death and apoptosis were subsequently measured by propidium iodide uptake and DNA content analysis, respectively. Early apoptotic events can be detected by a characteristic pattern of DNA strand breaks through endonucleolysis, resulting in increased amounts of DNA fragments that are visible by flow cytometry as the sub-G1 phase in the DNA content distribution (Walker et al., 1993). Strain application at the two lowest settings (3 and 5% biaxial strain) had no significant effect on cell death or apoptosis in either cell line, but at the highest strain rate (10% biaxial strain), A-type lamin-deficient fibroblasts had a significantly increased fraction of dead cells compared with nonstretched controls and to strained wild-type cells (Fig. 5 a). Total cell death in emerin-deficient cells was not significantly increased compared with nonstretched controls and to wild-type cells. DNA content analysis of samples from the same experiments revealed a large increase in apoptotic cell fraction in the A-type lamin-deficient cells compared with nonstretched controls and strained wild-type cells. Interestingly, we also found a significantly increased apoptotic cell fraction in the emerin-deficient fibroblasts (Fig. 5 b). Baseline levels of apoptotic cells were comparable for emerin-deficient and wild-type cells, but significantly elevated in the A-type lamin-deficient cells.

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