Correspondence to Jan Lammerding: jlammerding{at}rics.bwh.harvard.edu
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Introduction |
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Although emerin and A-type lamins (predominantly lamins A and/or C) are expressed in most human tissues, EDMD predominantly affects skeletal and cardiac muscles and tendons. The reason for this tissue-specific phenotype is not yet clear, but two alternative hypotheses for the disease mechanism have emerged. The "structural hypothesis" suggests that mutations in genes encoding emerin or A-type lamins lead to increased nuclear fragility and to eventual nuclear disruption in mechanically strained tissues, whereas the "gene regulation hypothesis" is based on the findings that lamin A/C and emerin can bind to a variety of transcriptional regulators that could exert tissue-specific effects. Lamins A/C are a major component of the nuclear lamina, and loss of A-type lamins leads to impaired nuclear mechanics and increased nuclear fragility (Broers et al., 2004; Lammerding et al., 2004). Emerin binds to several structural proteins such as lamin A/C, lamin B, nesprin-1/2, and nuclear actin, and has recently been demonstrated to promote actin polymerization in vitro (Mislow et al., 2002; Bengtsson and Wilson, 2004; Holaska et al., 2004; Zhang et al., 2005). Loss of emerin from the nuclear envelope could thus interfere with the normal function of these proteins and lead to nuclear structural abnormalities. At the same time, emerin can bind to the transcriptional repressors barrier-to-autointegration factor (BAF), germ cell-less (GCL), and Btf, and to the splicing factor YT521-B, suggesting an important role in gene regulation (Nili et al., 2001; Holaska et al., 2003; Wilkinson et al., 2003; Bengtsson and Wilson, 2004; Haraguchi et al., 2004). The structural hypothesis and the gene regulation hypothesis are not mutually exclusive, and in fact A-type lamin-deficient cells have increased nuclear fragility and abnormal nuclear mechanics as well as impaired signaling responses to mechanical strain or cytokine stimulation, indicating that tissue-specific effects observed in laminopathies could arise from varied degrees of impaired nuclear mechanics and transcriptional activation (Lammerding et al., 2004).
Here, we report independent measures of the structural and gene-regulatory functions of emerin-deficient, A-type lamin-deficient, and wild-type mouse embryo fibroblasts to explore the specific function of emerin on nuclear mechanics and gene regulation. We show that, in contrast to A-type lamin-deficient cells, emerin-deficient fibroblasts have apparently normal nuclear mechanics but display similar, although less profound, deficiencies in strain-induced gene regulation, leading to an increased rate of apoptosis in response to mechanical strain. These data suggest that emerin-associated laminopathies are predominantly caused by an impaired signaling response and not through direct strain-induced injury to the nuclear membrane.
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Results |
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Nuclear dynamics
Liu et al. (2000) previously demonstrated that nuclei of lamin-deficient Caenorhabditis elegans cells display significant shape changes over time. To assess dynamic changes in nuclear shape in A-type lamin-deficient and emerin-deficient mouse embryo fibroblasts, we analyzed nuclear shape stability using time-lapse imaging. Phase-contrast images were acquired every 5 min over an 8 h, 20 min period of time for a total of 100 frames. Nuclear motion and deformation were quantified by tracking individual nucleoli within a given nucleus over time (Fig. 3 a) and subsequently computing the translation, rotation, and deformation from these measurements. Cells that underwent mitosis during the observation period were excluded from the analysis. We defined the nuclear deformation as the average deviation from a linear affine transformation, i.e., a change in geometry that can be reduced to a combination of translation, rotation, and scaling in which relative positions to each other are maintained.
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Nuclear mechanics
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.
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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.
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Discussion |
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In contrast to the apparently normal nuclear mechanics, mechanosensitive gene regulation was deficient in emerin-deficient cells, resulting in a significantly increased rate of apoptosis in response to mechanical stimulation. The absence of changes in total cell death (propidium iodide uptake) in these cells could be due to the fact that the DNA contents assay measures early apoptotic chromatin breakdown while loss of plasma membrane integrity that is required for propidium iodide uptake occurs at a much later stage in apoptosis. Consistent with the apparently normal nuclear mechanics and the impaired activation of anti-apoptotic genes in response to mechanical stimulation, our findings indicate that emerin-deficient cells are more susceptible to strain-induced apoptosis, whereas both necrosis and apoptosis contribute to mechanically induced cell death in A-type lamin-deficient cells. Although the current study was narrowed to two representative mechanosensitive genes (egr-1 and iex-1) whose activation has previously been reported to be impaired in A-type lamin-deficient cells (Lammerding et al., 2004), we expect that the role of emerin (and A-type lamins) is not limited to these particular genes, and a more comprehensive gene array analysis of mechanically stimulated cells will provide more insight into the extent of lamin/emerin-dependent mechanotransduction.
The molecular mechanism that is responsible for the observed mechanotransduction deficiency in A-type lamin and emerin-deficient cells is not clear. We previously reported impaired NF-Bregulated transcriptional activation in A-type lamin-deficient cells, but emerin-deficient cells had apparently normal NF-
B signaling, indicating that alternative pathways are responsible for the observed mechanotransduction deficiencies. Emerin directly binds to the transcriptional regulators BAF, Btf, GCL, and YT521-B (Nili et al., 2001; Holaska et al., 2003; Wilkinson et al., 2003; Haraguchi et al., 2004). Btf is a transcription repressor that induces cell death when overexpressed (Kasof et al., 1999). Emerin is cleaved during apoptosis in proliferating mouse myoblasts and differentiating myotubes, and emerin may have an anti-apoptotic effect by sequestering Btf (Columbaro et al., 2001). This anti-apoptotic role of emerin is consistent with the increased rate of apoptosis observed in emerin-deficient cells after strain application. GCL is a repressor of E2F-DPregulated genes. Emerin cannot bind BAF and GCL simultaneously, and presence of BAF at the nuclear envelope might inhibit GCL binding to emerin in vivo (Bengtsson and Wilson, 2004). YT521-B is involved in determining sites for alternate mRNA splicing, and emerin influences splice site selection by YT521-B (Wilkinson et al., 2003). The role of emerin as a modulator for transcriptional regulation is further supported by DNA microarray analysis of fibroblasts from patients with the X-linked form of EDMD, which showed that at least 28 genes are specifically up-regulated in the emerin mutant cells (Tsukahara et al., 2002). The affected genes include both structural (lamin A/C,
II-spectrin, and filamin) and signal transductionassociated proteins such as protein phosphatase 2A (Tsukahara et al., 2002).
In addition to these direct interactions between emerin and transcriptional regulators, we cannot exclude the possibility that the impaired mechanotransduction response is caused by more indirect consequences of emerin deficiency. Abnormal nuclear shape and ultrastructure in emerin-deficient cells could affect force transmission from the cytoskeleton to the nucleus (Maniotis et al., 1997), and interaction of emerin with nuclear actin (Holaska et al., 2004) could affect both nuclear ultrastructure as well as transcription itself, as nuclear actin is emerging as a critical component of polymerase II transcription (Hofmann et al., 2004; Zhu et al., 2004).
In our experiments, emerin-deficient cells generally displayed a milder phenotype compared with the A-type lamin-deficient cells, including fewer and less extensive nuclear shape abnormalities, a less profound increase in apoptosis in response to strain, and less severe mechanotransduction deficiency. This observation is consistent with the milder phenotype found in emerin-deficient mice that don't display overt muscular dystrophy or cardiac problems but show signs of impaired muscle regeneration later on (unpublished data). Loss of function in lamin A/C mutants often leads to mislocalization of emerin from the nuclear envelope to the ER (Sullivan et al., 1999; Östlund et al., 2001; Raharjo et al., 2001; Muchir et al., 2003) and to a subsequent loss of normal emerin function at the nuclear envelope. Therefore, we expect that A-type lamin-deficient cells encompass phenotypes associated with emerin deficiency or loss of function. Loss of emerin, on the other hand, does not appear to grossly affect A-type lamin function, providing a possible explanation for the more severe phenotype in the A-type lamin-deficient cells compared with the emerin-deficient cells.
In conclusion, we found that A-type lamin and emerin deficiencies share common features such as abnormal nuclear shape and impaired mechanotransduction, but also selectively interfere with other structural and gene-regulatory functions. In the case of emerin deficiency, we found that emerin predominantly affects mechanosensitive gene regulation with only small effects on nuclear mechanics. By providing independent tests for measuring structural and gene-regulatory functions, our experiments can help clarify the effects of specific mutations in nuclear envelope proteins. Elucidating the molecular mechanisms will provide new insights into the specific disease mechanisms of X-linked recessive and autosomal dominant EDMD, and might eventually lead to new treatment courses.
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Materials and methods |
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Nuclear shape analysis
To quantify the overall fraction of irregularly shaped or blebbing nuclei, cells of various passages were incubated for 15 min with 1 µg/ml Hoechst 33342 (Molecular Probes) and washed with HBSS (Invitrogen). For each passage, fluorescence images of 100500 randomly selected nuclei were acquired at 20x on a microscope (model IX-70; Olympus) using a CoolSNAP camera (Roper Scientific), and were stored for subsequent image analysis. Nuclei of Lmna+/+, Lmna/, and Emd-/y cells were scored as normal, irregularly shaped (deviation from an oval or spherical shape), or as nuclei with nuclear blebs. These determinations were made using an observer blinded for the genotype. To quantify the variation in nuclear morphology, we measured nuclear area and perimeter in midsections of the fluorescent nuclei using custom-written MATLAB software. From these data, we computed the nuclear roundness or contour ratio (4 x area/perimeter2) (Goldman et al., 2004). The contour ratio reaches a maximum value of 1 for a circle and decreases with increasingly convoluted nuclear shapes. Elliptic Fourier analysis of nuclear shape was performed as described previously (Diaz et al., 1989). Custom-written MATLAB software was used to automatically trace the outline of fluorescently labeled nuclei and to compute the first 20 elliptic harmonics. Each single elliptic harmonic can be geometrically visualized as a pair of orthogonal semiaxes, and we used the ratio of the sum of the first major and minor semiaxes over the sum of the higher order semiaxes as indicator of irregular nuclear shape, as the higher order harmonics represent deviations from a purely elliptical shape.
Time-lapse experiments
Cells were plated at subconfluent density in 35-mm polystyrene cell culture dishes (Corning). Cells were grown in DME + 10% FCS at 37°C for at least 24 h before the start of the experiments. Subsequently, lidded culture dishes were sealed with parafilm M (American National Can) and placed at RT on the microscope stage (model IX-70; Olympus). After a brief equilibration period, images were automatically acquired every 5 min for a minimum of 8 h, 20 min (corresponding to 100 frames) using a digital CCD camera (CoolSNAP HQ; Roper Scientific) and were stored on a computer for subsequent analysis. Continued cell viability was confirmed by monitoring the cells for 24 h after the experiments. Nuclear rotation, translation, and shape changes were analyzed by tracking the centroid positions of 36 nucleoli for each nucleus using custom-written MATLAB software. For each frame, the linear conformal image transformation was computed that best mapped the current centroid positions to the original positions minimizing the least-square error. The linear conformal transformations can account for a combination of translation, rotation, and scaling, and preserves the relative position of objects to each other. The deviation from the best fit, i.e., the error between the least-square fit transformation and the actual nucleoli positions, was used as a measure of nuclear deformation as it describes the extent of nuclear deformation from its initial shape independent of absolute nuclear movement or uniform changes in size (see time-lapse videos, available at http://www.jcb.org/cgi/content/full/jcb.200502148/DC1). Nucleoli that fused over time or cells that underwent mitosis during or immediately after the observation period were excluded from the analysis. We analyzed 25 nuclei of each cell type and computed for each nucleus the time-averaged deformation, the time-averaged and maximal change in size, translation, and rotation.
Nuclear strain experiments
Experiments were performed as previously described (Lammerding et al., 2004). In brief, cells were plated at subconfluent cell density on fibronectin-coated silicone membranes in DME supplemented with 10% FCS, followed by serum starvation for 48 h in DME containing ITS supplement (Sigma-Aldrich). Preceding the strain experiments, cells were incubated with Hoechst 33342 nuclear stain (final concentration 1 µg/ml; Molecular Probes) in DME + ITS for 20 min. Membranes were placed on a custom-made strain device and uniform biaxial strain was applied to the silicone membrane. Membrane and nuclear strains were computed based on bright field and fluorescence images acquired before, during, and after strain application using a custom-written image analysis algorithm. Normalized nuclear strain was defined as the ratio of nuclear strain to membrane strain to compensate for small variations in applied membrane strain (range 4.55.5%).
Microinjection experiments
Cells were plated on fibronectin-coated glass dishes (WillCo Wells) and incubated overnight. Microinjections were performed using a microinjector (Eppendorf) with Femtotips (Eppendorf). In each dish, 3050 cells were injected with Texas redlabeled 70-kD dextran (dissolved at 10 mg/ml in PBS; Molecular Probes) into the cytoplasm or into the nucleus (injection pressure 500 hPa; injection time 0.6 s). After the microinjection, cells were washed with HBSS (Invitrogen) and intracellular localization of dextranTexas red was recorded under a fluorescent microscope using a CCD camera (CoolSNAP HQ; Roper Scientific) on a microscope (model IX-70; Olympus). Cells were considered as having defective nuclear envelope integrity in the case of uniform nuclear and cytoplasmic staining, and the fraction of defective nuclei was expressed as the ratio of total fluorescent cells.
Image acquisition and manipulation
Phase-contrast and fluorescence images were acquired as described above using a digital CCD-camera (CoolSNAP HQ; Roper Scientific) mounted on an inverted microscope (model IX-70; Olympus) using ImagePro image acquisition software (Media Cybernetics). Nuclear shape experiments, time-lapse studies, and microinjected cells were imaged using an Olympus LCPlanF 20x phase-contrast objective (NA 0.40), whereas nuclear strain experiments were conducted using an Olympus UApo/340 40x water immersion objective (NA 1.15). Cells were kept in HBSS+ buffer during imaging, except for time-lapse experiments that were performed on cells in full media. All experiments were performed at RT. Radiographs from Northern analysis were digitized on a scanner (Perfection 2450; Epson) using linear intensity settings. Digital images were processed using Adobe Photoshop (ver. 6.0) by adjusting the linear image intensity display range, and fluorescence grayscale images were colorized in Adobe Photoshop by selecting a colorplane (RGB) appropriate for the chromophore (i.e., blue for Hoechst 33342, red for Texas red).
Mechanotransduction experiments
Strain stimulation was performed as described previously (Cheng et al., 1997; Lammerding et al., 2004). In brief, cells were plated on fibronectin-coated silicone membranes (3,000 cells/cm2). After 72 h serum starvation, cells were subjected to bi-axial cyclic strain (4%, 1 Hz) for 2 or 4 h. For chemical stimulation, cells were incubated with IL-1ß (10 ng/ml; R&D Systems) or PMA (200 ng/ml; Sigma-Aldrich) in DME + ITS. Cellular mRNA of strained and unstrained control samples was isolated using the RNeasy Minikit (QIAGEN), and samples were subsequently analyzed by Northern analysis and real-time PCR.
Cell viability and apoptosis assay
Cells were plated on fibronectin-coated silicone membranes and maintained in full media for 48 h to provide sufficient cell attachment. After 24 h of cyclic biaxial strain application (1 Hz; 3, 5, or 10% strain), cells were incubated with propidium iodide (PI, final concentration 1 µg/ml; Sigma-Aldrich). Cells and culture media were collected, washed once in PBS and resuspend in HBSS. Each sample was divided into two equal parts to independently measure cell death (PI uptake) and apoptosis (DNA content analysis). One part was immediately analyzed for PI uptake using a Cytomics FC 500 flow cytometer (Beckman Coulter), counting 10,00030,000 events in each group. Thresholds for PI incorporation were determined based on negative (no PI staining) and positive (cells permeabilized by 50% ethanol) controls. The other cell fraction from the 24-h strain experiments was fixed in 80% ethanol and stored at 20°C. Samples were then spun down, resuspended in PBS and treated with Ribonuclease A (Sigma-Aldrich) for 1 h and subsequently stained with PI (final concentration 100 µg/ml). DNA content was measured on a flow cytometer and the apoptotic cell fraction was identified as cells with sub-G1 DNA content, counting 30,00050,000 events per sample as described previously (Walker et al., 1993).
Northern analysis
For Northern analysis of iex-1, egr-1, and GAPDH, mRNA from unstrained controls and from cells subjected to 2 and 4 h of biaxial cyclic strain was harvested using RNAeasy Mini Kit kit (QIAGEN). 712 µg of each collected RNA sample were separated by gel electrophoresis at 110130 V and mRNA was transferred overnight to a transfer membrane (MAGNA, Nylon, 0.45 micron; Osmonics, Inc.). Expression of iex-,1egr-1, and GAPDH mRNA was assessed by Northern analysis as described previously (De Keulenaer et al., 2002).
Real-time polymerase chain reaction
For further analysis of iex-1 expression, cells were prepared for strain experiments with 2 and 4 h biaxial strain. Cells were harvested using the QIAGEN RNAeasy kit. For each sample, 1 µl of collected RNA was added to RT Mixes of Stratagene Light Cycler kit with iex-1 and ß-tubulin primers from Integrated DNA Technologies, Inc. (De Keulenaer et al., 2002). The polymerase reaction was conducted in Roche Molecular Biochemicals Light Cycler Version 5.32 with 45 cycles. Results were normalized with ß-tubulin expression and expressed as percent increase from baseline (unstrained controls).
Luciferase experiments
Cells were transfected with plasmids for NF-Bcontrolled luciferase expression and SV40-regulated ß-galactosidase (Promega) using FuGENE6 (Roche). After transfection, cells were serum starved in DME + ITS medium for 48 h, followed by overnight stimulation with 200 ng/ml PMA or 10 ng/ml IL-1ß. Luciferase assays were quantified in a Victor2 Multilabel Counter (PerkinElmer). Results were normalized for ß-galactosidase activity and expressed as percentage of wild-type control.
Statistical analysis
All experiments were performed at least three independent times. Data are expressed as mean ± SEM. Statistical analysis was performed using the PRISM 3.0 and INSTAT software (GraphPad). The data were analyzed by unpaired t test (allowing different SD), one-way ANOVA (followed by Tukey's multiple comparison test) or, in case of non-Gaussian distribution, the Mann-Whitney or Kruskal Wallis tests (the latter when comparing more than two groups, followed by Dunn's multiple comparison test). For nuclear shape analysis, two-way ANOVA was performed on datasets from three different passages to evaluate the source of variation. In contrast to cell type, passage number was not a significant source of variation, and thus datasets from different passages were pooled and analyzed using the Kruskal-Wallis test for non-Gaussian distributions with Dunn's post test. Real-time PCR data were analyzed using a paired test and comparing induction of iex-1 (as percentage of baseline levels) at 2 and 4 h to wild-type induction. A two-tailed P-value of <0.05 was considered significant.
Online supplemental material
Full-length time-lapse sequences of A-type lamin-deficient, emerin-deficient, and wild-type mouse embryo fibroblasts corresponding to the still images presented in Fig. 3 can be found in the supplemental material. Each video consists of a sequence of images acquired every 5 min over an 8 h, 20 min period for a total of 100 frames. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200502148/DC1.
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Acknowledgments |
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This work was supported by grants from National Heart, Lung, and Blood Institute (HL073809, HL64858) and a post-doctoral fellowship from the American Heart Association for J. Lammerding.
Submitted: 24 February 2005
Accepted: 25 July 2005
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References |
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