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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 fibroblasts have abnormal nuclear shape. (a) Fluorescently labeled nuclei of emerin-deficient mouse embryo fibroblasts. Asterisk denotes nucleus with chromatin protruding from the nucleus (nuclear bleb). Arrows indicate nuclei that have mild deviations from the typical round shape. Bar, 20 μm. (b) The fraction of abnormally shaped nuclei and nuclear blebs was significantly increased in emerin and A-type lamin-deficient fibroblasts, with emerin-deficient cells displaying a milder phenotype (cell fractions with abnormal nuclear shape were 0.077 ± 0.006 for wild-type, 0.163 ± 0.009 for emerin-deficient, and 0.378 ± 0.011 for A-type lamin-deficient cells, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; cell fractions with nuclear blebs were 0.012 ± 0.002, 0.042 ± 0.005, and 0.093 ± 0.006 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; n ∼1,800 for each cell type). (c) Emerin-deficient cells have a significantly decreased contour ratio compared with wild-type cells, but display a milder phenotype compared with A-type lamin-deficient cells (contour ratio = 0.89 ± 0.004 for wild-type, 0.86 ± 0.006 for emerin-deficient, and 0.80 ± 0.007 for A-type lamin-deficient cells, P < 0.001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type; n ∼1500 for each cell type in three independent experiments). (d) Relative frequency distribution of the contour ratio for wild-type, emerin-deficient, and A-type lamin-deficient cells (median values were 0.896, 0.869, and 0.817 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively). The difference in the medians was statistically significant (P < 0.001 for emerin-deficient and A-type lamin-deficient vs. wild-type). (e) Emerin and A-type lamin-deficient cells have significantly increased nuclear cross-sectional areas compared with wild-type cells (Nuclear cross-sectional area = 178 ± 4.5 μm2, 252 ± 24.7 μm2, and 259 ± 25.6 μm2 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.01 for emerin and A-type lamin-deficient compared with wild-type cells, n ∼1500 for each cell type in three independent experiments). (f) Frequency distribution of the cross-sectional area for wild-type, emerin-deficient, and A-type lamin-deficient cells.
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fig2: Emerin and A-type lamin-deficient fibroblasts have abnormal nuclear shape. (a) Fluorescently labeled nuclei of emerin-deficient mouse embryo fibroblasts. Asterisk denotes nucleus with chromatin protruding from the nucleus (nuclear bleb). Arrows indicate nuclei that have mild deviations from the typical round shape. Bar, 20 μm. (b) The fraction of abnormally shaped nuclei and nuclear blebs was significantly increased in emerin and A-type lamin-deficient fibroblasts, with emerin-deficient cells displaying a milder phenotype (cell fractions with abnormal nuclear shape were 0.077 ± 0.006 for wild-type, 0.163 ± 0.009 for emerin-deficient, and 0.378 ± 0.011 for A-type lamin-deficient cells, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; cell fractions with nuclear blebs were 0.012 ± 0.002, 0.042 ± 0.005, and 0.093 ± 0.006 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; n ∼1,800 for each cell type). (c) Emerin-deficient cells have a significantly decreased contour ratio compared with wild-type cells, but display a milder phenotype compared with A-type lamin-deficient cells (contour ratio = 0.89 ± 0.004 for wild-type, 0.86 ± 0.006 for emerin-deficient, and 0.80 ± 0.007 for A-type lamin-deficient cells, P < 0.001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type; n ∼1500 for each cell type in three independent experiments). (d) Relative frequency distribution of the contour ratio for wild-type, emerin-deficient, and A-type lamin-deficient cells (median values were 0.896, 0.869, and 0.817 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively). The difference in the medians was statistically significant (P < 0.001 for emerin-deficient and A-type lamin-deficient vs. wild-type). (e) Emerin and A-type lamin-deficient cells have significantly increased nuclear cross-sectional areas compared with wild-type cells (Nuclear cross-sectional area = 178 ± 4.5 μm2, 252 ± 24.7 μm2, and 259 ± 25.6 μm2 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.01 for emerin and A-type lamin-deficient compared with wild-type cells, n ∼1500 for each cell type in three independent experiments). (f) Frequency distribution of the cross-sectional area for wild-type, emerin-deficient, and A-type lamin-deficient cells.

Mentions: Fibroblasts derived from EDMD patients often have irregularly shaped nuclei and show blebbing of the nuclear membrane (Fidzianska et al., 1998; Fidzianska and Hausmanowa-Petrusewicz, 2003). Emerin-deficient mouse embryo fibroblasts had a significantly increased fraction of abnormally shaped nuclei (Fig. 2, a and b) and nuclei with membrane and chromatin protrusions (nuclear blebs) when compared with wild-type fibroblasts. These nuclear shape abnormalities were less pronounced compared with those of A-type lamin-deficient cells (Fig. 2 b). To assess the degree of irregular nuclear shape more quantitatively, we measured nuclear cross-sectional area and perimeter of Hoechst 33342 stained nuclei and computed the nuclear contour ratio (4π × area/perimeter2), which yields a quantitative measure of nuclear roundness. For a circular shape that maximizes the area-to-perimeter ratio the contour ratio has a value of 1, whereas more convoluted outlines lead to smaller values. We found that emerin-deficient cells had a significantly lower mean contour ratio compared with wild-type cells (Fig. 2 c), and the frequency distribution showed that the overall distribution was shifted toward lower values of the contour ratio, indicating a higher prevalence of abnormally shaped nuclei (Fig. 2 d). A-type lamin-deficient cells had an even lower mean contour ratio, with very few cells reaching normal values (∼0.90) and a much wider distribution overall, indicating that both the frequency as well as the extent of irregular nuclear shape is more severe in A-type lamin-deficient cells compared with wild-type and emerin-deficient cells (Fig. 2 d). These findings were confirmed when analyzing the nuclear length/width ratio (unpublished data) that was close to unity for wild-type cells and significantly lower for emerin and especially for A-type lamin-deficient fibroblasts (unpublished data). Fourier shape analysis of nuclear cross-sectional outlines (Diaz et al., 1989) revealed that both emerin and A-type lamin-deficient cells had a significantly higher contribution of higher order harmonics to the nuclear shape, again confirming that these cells have a more irregular nuclear shape compared with wild-type cells (ratio of 1st order harmonics to higher order harmonics was 11.9 ± 0.08 for wild-type, 11.3 ± 0.11 for emerin-deficient, and 7.4 ± 0.07 for A-type lamin-deficient cells; P < 0.001 for wild-type vs. emerin and 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 fibroblasts have abnormal nuclear shape. (a) Fluorescently labeled nuclei of emerin-deficient mouse embryo fibroblasts. Asterisk denotes nucleus with chromatin protruding from the nucleus (nuclear bleb). Arrows indicate nuclei that have mild deviations from the typical round shape. Bar, 20 μm. (b) The fraction of abnormally shaped nuclei and nuclear blebs was significantly increased in emerin and A-type lamin-deficient fibroblasts, with emerin-deficient cells displaying a milder phenotype (cell fractions with abnormal nuclear shape were 0.077 ± 0.006 for wild-type, 0.163 ± 0.009 for emerin-deficient, and 0.378 ± 0.011 for A-type lamin-deficient cells, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; cell fractions with nuclear blebs were 0.012 ± 0.002, 0.042 ± 0.005, and 0.093 ± 0.006 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; n ∼1,800 for each cell type). (c) Emerin-deficient cells have a significantly decreased contour ratio compared with wild-type cells, but display a milder phenotype compared with A-type lamin-deficient cells (contour ratio = 0.89 ± 0.004 for wild-type, 0.86 ± 0.006 for emerin-deficient, and 0.80 ± 0.007 for A-type lamin-deficient cells, P < 0.001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type; n ∼1500 for each cell type in three independent experiments). (d) Relative frequency distribution of the contour ratio for wild-type, emerin-deficient, and A-type lamin-deficient cells (median values were 0.896, 0.869, and 0.817 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively). The difference in the medians was statistically significant (P < 0.001 for emerin-deficient and A-type lamin-deficient vs. wild-type). (e) Emerin and A-type lamin-deficient cells have significantly increased nuclear cross-sectional areas compared with wild-type cells (Nuclear cross-sectional area = 178 ± 4.5 μm2, 252 ± 24.7 μm2, and 259 ± 25.6 μm2 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.01 for emerin and A-type lamin-deficient compared with wild-type cells, n ∼1500 for each cell type in three independent experiments). (f) Frequency distribution of the cross-sectional area for wild-type, emerin-deficient, and A-type lamin-deficient cells.
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fig2: Emerin and A-type lamin-deficient fibroblasts have abnormal nuclear shape. (a) Fluorescently labeled nuclei of emerin-deficient mouse embryo fibroblasts. Asterisk denotes nucleus with chromatin protruding from the nucleus (nuclear bleb). Arrows indicate nuclei that have mild deviations from the typical round shape. Bar, 20 μm. (b) The fraction of abnormally shaped nuclei and nuclear blebs was significantly increased in emerin and A-type lamin-deficient fibroblasts, with emerin-deficient cells displaying a milder phenotype (cell fractions with abnormal nuclear shape were 0.077 ± 0.006 for wild-type, 0.163 ± 0.009 for emerin-deficient, and 0.378 ± 0.011 for A-type lamin-deficient cells, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; cell fractions with nuclear blebs were 0.012 ± 0.002, 0.042 ± 0.005, and 0.093 ± 0.006 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.0001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type cells; n ∼1,800 for each cell type). (c) Emerin-deficient cells have a significantly decreased contour ratio compared with wild-type cells, but display a milder phenotype compared with A-type lamin-deficient cells (contour ratio = 0.89 ± 0.004 for wild-type, 0.86 ± 0.006 for emerin-deficient, and 0.80 ± 0.007 for A-type lamin-deficient cells, P < 0.001 for emerin-deficient and A-type lamin-deficient cells compared with wild-type; n ∼1500 for each cell type in three independent experiments). (d) Relative frequency distribution of the contour ratio for wild-type, emerin-deficient, and A-type lamin-deficient cells (median values were 0.896, 0.869, and 0.817 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively). The difference in the medians was statistically significant (P < 0.001 for emerin-deficient and A-type lamin-deficient vs. wild-type). (e) Emerin and A-type lamin-deficient cells have significantly increased nuclear cross-sectional areas compared with wild-type cells (Nuclear cross-sectional area = 178 ± 4.5 μm2, 252 ± 24.7 μm2, and 259 ± 25.6 μm2 for wild-type, emerin-deficient, and A-type lamin-deficient cells, respectively, P < 0.01 for emerin and A-type lamin-deficient compared with wild-type cells, n ∼1500 for each cell type in three independent experiments). (f) Frequency distribution of the cross-sectional area for wild-type, emerin-deficient, and A-type lamin-deficient cells.
Mentions: Fibroblasts derived from EDMD patients often have irregularly shaped nuclei and show blebbing of the nuclear membrane (Fidzianska et al., 1998; Fidzianska and Hausmanowa-Petrusewicz, 2003). Emerin-deficient mouse embryo fibroblasts had a significantly increased fraction of abnormally shaped nuclei (Fig. 2, a and b) and nuclei with membrane and chromatin protrusions (nuclear blebs) when compared with wild-type fibroblasts. These nuclear shape abnormalities were less pronounced compared with those of A-type lamin-deficient cells (Fig. 2 b). To assess the degree of irregular nuclear shape more quantitatively, we measured nuclear cross-sectional area and perimeter of Hoechst 33342 stained nuclei and computed the nuclear contour ratio (4π × area/perimeter2), which yields a quantitative measure of nuclear roundness. For a circular shape that maximizes the area-to-perimeter ratio the contour ratio has a value of 1, whereas more convoluted outlines lead to smaller values. We found that emerin-deficient cells had a significantly lower mean contour ratio compared with wild-type cells (Fig. 2 c), and the frequency distribution showed that the overall distribution was shifted toward lower values of the contour ratio, indicating a higher prevalence of abnormally shaped nuclei (Fig. 2 d). A-type lamin-deficient cells had an even lower mean contour ratio, with very few cells reaching normal values (∼0.90) and a much wider distribution overall, indicating that both the frequency as well as the extent of irregular nuclear shape is more severe in A-type lamin-deficient cells compared with wild-type and emerin-deficient cells (Fig. 2 d). These findings were confirmed when analyzing the nuclear length/width ratio (unpublished data) that was close to unity for wild-type cells and significantly lower for emerin and especially for A-type lamin-deficient fibroblasts (unpublished data). Fourier shape analysis of nuclear cross-sectional outlines (Diaz et al., 1989) revealed that both emerin and A-type lamin-deficient cells had a significantly higher contribution of higher order harmonics to the nuclear shape, again confirming that these cells have a more irregular nuclear shape compared with wild-type cells (ratio of 1st order harmonics to higher order harmonics was 11.9 ± 0.08 for wild-type, 11.3 ± 0.11 for emerin-deficient, and 7.4 ± 0.07 for A-type lamin-deficient cells; P < 0.001 for wild-type vs. emerin and 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