JCB: ARTICLE Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells Jan Lammerding,1 Janet Hsiao,2 P. Christian Schulze,1 Serguei Kozlov,3 Colin L. Stewart,3 and Richard T. Lee1 1Cardiovascular Division, Brigham and Women’s Hospital, Boston, MA 02115 2HST Division, Massachusetts Institute of Technology, Cambridge, MA 02139 3Cancer and Developmental Biology Lab, National Cancer Institute, Frederick, MD 21702 mery-Dreifuss muscular dystrophy can be caused type cells in cellular strain experiments, and the integrity by mutations in the nuclear envelope proteins lamin of emerin-deficient nuclear envelopes appeared normal E A/C and emerin. We recently demonstrated that in a nuclear microinjection assay. Interestingly, expres- A-type lamin-deficient cells have impaired nuclear me- sion of mechanosensitive genes in response to mechani- chanics and altered mechanotransduction, suggesting cal strain was impaired in emerin-deficient cells, and two potential disease mechanisms (Lammerding, J., P.C. prolonged mechanical stimulation increased apoptosis in Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, emerin-deficient cells. Thus, emerin-deficient mouse em- C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113: bryo fibroblasts have apparently normal nuclear me- 370–378). Here, we examined the function of emerin on chanics but impaired expression of mechanosensitive nuclear mechanics and strain-induced signaling. Emerin- genes in response to strain, suggesting that emerin muta- deficient mouse embryo fibroblasts have abnormal nu- tions may act through altered transcriptional regulation clear shape, but in contrast to A-type lamin-deficient and not by increasing nuclear fragility. cells, exhibit nuclear deformations comparable to wild- Introduction Emerin is a small (34 kD) nuclear envelope protein with a sin- emerin from the nuclear periphery and mislocalization of gle transmembrane domain spanning the inner nuclear mem- emerin to the cytoplasm (Ellis et al., 1998; Vaughan et al., brane and a large nucleoplasmic domain that can interact with 2001). Although X-linked EDMD is associated with a mutation other nuclear envelope proteins such as lamins (Bengtsson and in the EMD gene, the autosomal dominant form of EDMD is Wilson, 2004). Emerin is not essential for cell viability, but it caused by missense mutations in the LMNA gene that encodes contributes to shared vital functions, including nuclear assem- the nuclear A-type lamins (Bonne et al., 1999). These muta- bly and cell cycle progression. Emerin is encoded by the EMD tions often result in misfolding or failure of the A-type lamins THE JOURNAL OF CELL BIOLOGY gene (also known as STA) located on the X-chromosome to correctly assemble, leading to partial or complete loss of (Bione et al., 1994). Mutations in this gene can cause the function (Burke and Stewart, 2002). Historically, X-linked X-linked recessive form of Emery-Dreifuss muscular dystrophy EDMD was the first disorder to be recognized as a nuclear (EDMD), a disease characterized by early onset in childhood membrane disease and opened a new area of research on the with slowly progressive skeletal muscle wasting, contractures role of the nuclear envelope in disease. Currently, more than of the elbow, neck, and Achilles tendons, a rigid spine, abnor- 180 mutations have been identified in the LMNA and EMD mal heart rhythms, heart block, and cardiomyopathy leading to genes that are causally linked to at least 10 distinct diseases, in- increased risk of cardiac arrest (Bione et al., 1994). The major- cluding EDMD, dilated cardiomyopathy, familial partial lipo- ity of the disease-causing mutations are nonsense mutations dystrophy, and Hutchinson Gilford progeria syndrome (Bonne that produce soluble forms of the protein, leading to loss of et al., 1999; Fatkin et al., 1999; Cao and Hegele, 2000; Shackle- ton et al., 2000; Burke and Stewart, 2002; De Sandre-Giovan- noli et al., 2002, 2003; Eriksson et al., 2003). Correspondence to Jan Lammerding: [email protected] Although emerin and A-type lamins (predominantly Abbreviations used in this paper: BAF, barrier-to-autointegration factor; EDMD, lamins A and/or C) are expressed in most human tissues, Emery-Dreifuss muscular dystrophy; GAPDH, glyceraldehyde 3-phosphate dehy- drogenase; GCL, germ cell-less. EDMD predominantly affects skeletal and cardiac muscles and The online version of this article contains supplemental material. tendons. The reason for this tissue-specific phenotype is not yet The Journal of Cell Biology, Vol. 170, No. 5, August 29, 2005 781–791 http://www.jcb.org/cgi/doi/10.1083/jcb.200502148 JCB 781 clear, but two alternative hypotheses for the disease mechanism have emerged. The “structural hypothesis” suggests that muta- tions in genes encoding emerin or A-type lamins lead to in- creased nuclear fragility and to eventual nuclear disruption in mechanically strained tissues, whereas the “gene regulation hy- pothesis” is based on the findings that lamin A/C and emerin can bind to a variety of transcriptional regulators that could ex- ert tissue-specific effects. Lamins A/C are a major component of the nuclear lamina, and loss of A-type lamins leads to im- paired 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 in- terfere 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 regula- tion (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 me- Figure 1. Characterization of emerin null fibroblasts. (a) Western analysis of homozygous (Ϫ/Ϫ and Ϫ/Y) and heterozygous (ϩ/Ϫ) emerin null and chanics as well as impaired signaling responses to mechanical wild-type (ϩ/ϩ and ϩ/Y) adult muscle fibroblasts, showing a complete strain or cytokine stimulation, indicating that tissue-specific ef- lack of emerin in the homozygous cells. Protein expression of lamin A/C fects observed in laminopathies could arise from varied degrees and lamin B1 appeared normal in the emerin null homozygous and het- erozygous cells. (b) Female heterozygous (i.e., Emdϩ/Ϫ) muscle fibroblasts of impaired nuclear mechanics and transcriptional activation stained with anti-emerin antibody (left) and DAPI (right). Two cells (*) (Lammerding et al., 2004). clearly lack emerin staining (as visualized by comparing with the DAPI Here, we report independent measures of the structural staining), reflecting the differential X-chromosome inactivation in the het- erozygous cell population. 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 even in mice as old as 12–15 mo (unpublished data). Hereafter and gene regulation. We show that, in contrast to A-type we have focused our studies on analyzing EmdϪ/y fibroblasts lamin-deficient cells, emerin-deficient fibroblasts have appar- derived from male embryos to avoid possible heterogeneity ently normal nuclear mechanics but display similar, although due to X-chromosome–associated gene dosage compensation less profound, deficiencies in strain-induced gene regulation, effects in cell lines of female origin. leading to an increased rate of apoptosis in response to me- chanical strain. These data suggest that emerin-associated Nuclear shape laminopathies are predominantly caused by an impaired sig- Fibroblasts derived from EDMD patients often have irregularly naling response and not through direct strain-induced injury to shaped nuclei and show blebbing of the nuclear membrane the nuclear membrane. (Fidzianska et al., 1998; Fidzianska and Hausmanowa-Petru- sewicz, 2003). Emerin-deficient mouse embryo fibroblasts Results had a significantly increased fraction of abnormally shaped nuclei (Fig. 2, a and b) and nuclei with membrane and Emerin null fibroblasts chromatin protrusions (nuclear blebs) when compared with Emerin-deficient mouse embryo fibroblasts were derived from wild-type fibroblasts. These nuclear shape abnormalities were male emerin hemizygous mice (EmdϪ/y) and have been shown less pronounced compared with those of A-type lamin-deficient to lack emerin expression (unpublished data). Emerin-deficient cells (Fig. 2 b). To assess the degree of irregular nuclear shape mice were derived by deletion of the entire coding region of the more quantitatively, we measured nuclear cross-sectional area Emd gene, which resulted in the complete absence of emerin and perimeter of Hoechst 33342 stained nuclei and computed protein as shown by Western and immunohistochemical analy- the nuclear contour ratio (4␲ ϫ area/perimeter2), which yields sis of muscle fibroblasts isolated from the null mice (Fig. 1). a quantitative measure of nuclear roundness. For a circular