Copyright C Munksgaard 2001Traffic 2001; 2: 675–683

Munksgaard International Publishers ISSN 1398-9219

Review

The Nuclear Envelope in Muscular Dystrophy andCardiovascular Diseases

Brian Burke1,*, Leslie C. Mounkes2 andColin L. Stewart2

1Department of Cell Biology and Anatomy, University of

Calgary, 3330 Hospital Drive NW, Calgary, Alberta T21

4N1, Canada2Laboratory of Cancer and Developmental Biology, NCI-

FCRDC, PO Box B, Frederick, MD 21701–1201, USA

*Corresponding author: Brian Burke, bburke@ucalgary.ca

Considerable interest has been focused on the nuclearenvelope in recent years following the realization thatseveral human diseases are linked to defects in genesencoding nuclear envelope specific proteins, most no-tably A-type lamins and emerin. These disorders, de-scribed as laminopathies or nuclear envelopathies, in-clude both X-linked and autosomal dominant forms ofEmery–Dreifuss muscular dystrophy, dilated cardio-myopathy with conduction system defects, limb girdlemuscular dystrophy 1B with atrioventricular conduc-tion disturbances, and Dunnigan-type familial partiallipodystrophy. Certain of these diseases are associatedwith nuclear structural abnormalities that can be seenin a variety of cells and tissues. These observationsclearly demonstrate that A-type lamins in particularplay a central role, not only in the maintenance of nu-clear envelope integrity but also in the large-scale or-ganization of nuclear architecture. What is not obvious,however, is why defects in nuclear envelope proteinsthat are found in most adult cell types should give riseto pathologies associated predominantly with skeletaland cardiac muscle and adipocytes. The recognition ofthese various disorders now raises the novel possibilitythat the nuclear envelope may have functions that gobeyond housekeeping and which impact upon cell-type specific nuclear processes.

Key words: cardiomyopathy, emerin, lamina-associ-ated polypeptides, lipodystrophy, muscular dystrophy,nuclear envelope, nuclear lamins, nuclear membranes.

Received 27 June 2001, revised and accepted for publi-cation 10 July 2001

Organization of the Nuclear Envelope

The nuclear envelope forms the interface between the nu-cleus and cytoplasm, and as such plays a central role in de-fining the biochemical identities of these two compartments.

675

The most prominent features of the nuclear envelope (Figure1) are a pair of biochemically distinct inner and outer nuclearmembranes (INM and ONM), between which is the peri-nuclear space (PNS) (1,2). The ONM features numerousbound ribosomes and displays frequent connections with theperipheral rough ER, to which it is functionally related. TheINM, in contrast, contains a unique spectrum of membraneproteins, is ribosome-free and maintains close associationswith the underlying chromatin (3). Despite these composi-tional and functional differences, the INM and ONM exhibitcontinuities of their lipid bilayers where they are spanned bynuclear pore complexes (NPCs), the channels that mediatemacromolecular trafficking between the nucleus and cyto-plasm. In this way, the INM, ONM and ER form a singlecontinuous membrane system, with the PNS representing anextension of the ER lumen. Metazoans also contain an ad-ditional NE structural feature, known as the nuclear lamina(2,4). This is a thin (20nm) protein meshwork that lines thenuclear face of the INM and which maintains interactionswith both chromatin and INM-specific integral membraneproteins. Recent work from several laboratories has demon-strated that the lamina is of fundamental importance, not onlyin maintaining the structural integrity of the NE but also inorganizing chromatin within the nucleus (5–7).

The Nuclear Lamina

The nuclear lamina is composed primarily of members of thenuclear lamin family of intermediate filament proteins (4).These are the products of three genes: LMNA, LMNB1 andLMNB2. Like other intermediate filament proteins, the nu-clear lamins feature a central alpha helical rod domain flank-ed by N- and C-terminal globular domains (Figure 2). Laminmonomers readily self-associate to form parallel coiled-coilhomodimers, which in turn can form ‘head-to-tail’ polymers.Lateral interactions may then give rise to higher-order struc-tures that include 10nm intermediate-like filaments [review-ed in (4)]. At least in amphibian oocytes, such lamin fila-ments are arranged within the nuclear lamina as an orthog-onal meshwork with a crossover spacing of about 50nm,approximately the length of a single lamin homodimer (8).This filament meshwork is intimately associated with the INMas well as with the nucleoplasmic face of NPCs.

The nuclear lamins fall into two broad sequence classes: A-type and B-type (9). The major mammalian B-type lamins,

Burke et al.

Figure1: Overview of nuclear envelope organization. (A) The outer nuclear membrane (ONM) is coated with ribosomes and is adomain of the rough endoplasmic reticulum (ER). The inner nuclear membrane (INM) is connected to the ONM at the periphery of eachnuclear pore complex (NPC). The nuclear lamina lines the nuclear face of the INM and is closely associated with peripheral heterochromatin.Localization of emerin to the INM is dependent upon LMNA expression (LMNAπ/π) in mouse embryo fibroblasts. (B) In cells lacking afunctional A-type lamin gene (LMNA–/–) emerin is localized largely to the peripheral ER.

B1 and B2, are encoded by two separate genes (LMNB1 andLMNB2, respectively), at least one of which is expressed inall cells at any given time or stage of the life cycle (10–12).A third minor B type lamin, lamin B3, is a splice variant oflamin B2 and is expressed only in male germ cells (13). TheA-type lamins, of which there are at least four, are all derivedfrom a single primary transcript encoded by the LMNA gene.Alternative splicing produces the two major A-type lamins,lamin A and lamin C, as well as minor products that includelamin AD10 (14) and lamin C2 (15), the latter being specificto testis. Lamins A and C are identical for the first 566 aminoacids and diverge only at their C-termini (Figure 2). Human

Figure2: Domain organization of lamins A and C and localization of disease-linked mutations. These proteins feature a centralcoiled-coil domain (coils 1a, 1b and 2) flanked by nonhelical N- and C-terminal domains. The two proteins are identical in sequence toresidue 566, after which they diverge (shaded areas). The position of the nuclear localization sequence is represented by a black strip.Various mutations are indicated by color-coded bars for dilated cardiomyopathy (DCM) Emery–Dreifuss muscular dystrophy (EDMD), familialpartial lipodystrophy (FPLD) and limb-girdle muscular dystrophy (LGMD1B). Most of these mutations are missense. Nonsense (Z), singleamino acid deletion (D) and frameshifts (F) are indicated. The asterisk represents a single frameshift mutation (within codon 520) which hasbeen found to cause DCM, EDMD or LGMD1B in different members of the same family. Only a single mutation, which is linked to DCM,has been found within the unique lamin C sequence.

676 Traffic 2001; 2: 675–683

lamin A contains a unique C-terminal extension of 98 aminoacids, the last four of which form a so-called CaaX sequence.This motif, which is also observed at the C-termini of B-typelamins, represents a site of farnesylation and is essential forcorrect targeting to the nuclear envelope (16–18). However,while B-type lamins are constitutively farnesylated, this modi-fication is lost from lamin A as a result of proteolytic cleavagewithin the lamin A C-terminal domain soon after its incorpor-ation into the nuclear lamina (19). Lamin C also exhibits aunique C-terminal extension. However, in this case it consistsof only 6 amino acids and does not contain a CaaX motif.Perhaps because lamin C cannot be farnesylated, efficient

Nuclear Laminopathies

assembly of newly synthesized lamin C into the nuclear lam-ina is dependent upon the presence of lamin A (W.H. Rahar-jo, P. Enarson, T. Sullivan, C. Stewart, and B. Burke, submittedfor publication).

Developmental Regulation of LaminExpression

In contrast to B-type lamins, A-type lamin expression is de-velopmentally regulated. As a general rule, early embryoniccells including embryonic stem cells do not express A-typelamins (10,12). During mouse embryogenesis, lamins A andC only appear about midway through gestation, initially ingiant cells of the trophoblast and in cells of the visceral endo-derm. This is followed by asynchronous expression of theLmna gene in a variety of tissues. In certain cell types, Lmna

expression does not commence until after birth. Even in adultanimals, stem cells of the immune and hematopoietic sys-tems (11), as well as epithelial stem cells in the villus cryptsof the gut, do not express A-type lamins. It is only after differ-entiation that lamins A and C may be detected in derivativesof these cells. The appearance of A-type lamins in differenti-ating tissues has been postulated to signal an important func-tion for the lamin A and/or C products in initiating thechromatin organization necessary to determine cell fates andmaintain a differentiated state (10). However, ablation of theLmna gene in mice has little effect on embryonic develop-ment (5). Indeed neonatal Lmna –/– mice are virtually indis-tinguishable from their wild-type and heterozygous siblings.Evidently, A-type lamins are not essential in terms of normalcell growth and differentiation (at least for the majority ofcells). On the other hand, neither are they entirely dispens-able since, as will be described below, mice lacking a func-tional Lmna gene exhibit profoundly retarded postnatalgrowth, associated with the rapid onset of muscular dys-trophy leading to death by 6ª8weeks of age (5).

Inner Nuclear Membrane Proteins

Eleven mammalian inner nuclear membrane proteins havebeen described to date. The properties of these are summar-ized in Table1. With the exception of nurim (20), all of thesehave large nucleoplasmic domains which can interact withlamins and/or chromatin. In the case of lamin B receptor(LBR), chromatin interaction is mediated by Hp1 (21), achromatin-associated protein that plays an essential role inthe maintenance of chromatin structure and which is involvedin the repression of gene expression. A second chromatin-associated protein, HA95, which is related to A-kinase-an-choring protein 95 (AKAP95), also interacts with LBR as wellas with lamina-associated protein 2b (LAP2b below) (22).LBR itself displays sterol reductase activity and may thereforecontribute to sterol metabolism. Indeed, LBR can functionallyreplace a yeast C14 sterol reductase, to which it bears sig-nificant sequence similarity (23). LAP2b,g,d,e (24–26), emer-in (27,28) and MAN1 (29) belong to a family of proteins de-

677Traffic 2001; 2: 675–683

fined by the presence of a ‘LEM domain’. The LEM domainis composed of a ,43 amino acid motif that is exposed tothe nucleoplasm (29). In the case of LAP2, the LEM domainhas been shown to bind BAF (barrier to autointegration fac-tor), an abundant chromatin-associated protein (30). In thisway BAF may provide a link between certain INM proteinsand chromatin. Although the function of BAF is still poorlyunderstood, it is clearly an essential protein, since repressionof BAF expression in C. elegans embryos is lethal (31). Emer-in, LAP1 and 2 family members and LBR also interact withlamins. Since lamins themselves have a chromatin-bindingfunction, they may therefore provide an additional link be-tween chromatin and INM components (32).

INM protein localization

How do integral proteins such as LBR, emerin and LAP1/2become localized to the INM? The consensus that has nowemerged is that this occurs via a mechanism of selective re-tention (33,34). In this scheme, proteins that are mobilewithin the ER membranes may gain access to the INM viathe membrane continuities at the periphery of each nuclearpore complex (NPC), a process that does not require a target-ing signal. However, only those proteins that can specificallyinteract with nuclear components, e.g. lamins or chromatin,will be retained and concentrated. Evidence in favor of thismodel stems from several sources. Torrisi, Bonnatti and col-leagues have demonstrated that in cells infected with Sindbisvirus, the newly synthesized envelope glycoprotein could bedetected not only within the ER and ONM, but also withinthe INM (35,36). However, since the viral glycoprotein couldbe ‘chased’ from both the peripheral ER and the INM withsimilar kinetics, it was concluded that these two pools couldfreely exchange. In related studies, newly synthesized vesicu-lar stomatitis virus envelope glycoprotein (G protein) was alsodetected in the INM of infected cells (37). The selective re-tention model predicts that integral proteins of the INMshould exhibit a reduction in translational mobility upon local-ization to the NE. This prediction has been borne out in im-aging studies of live cells expressing green fluorescent pro-tein (GFP)-tagged INM proteins such as emerin and LBR(33,34). FRAP measurements suggest a minimum three-foldreduction in mobility of tagged proteins localized to the INMvs. those still in the peripheral ER. Finally, in fibroblasts de-rived from Lmna –/– mice, emerin, which interacts with thelamin A C-terminal domain (W.H. Raharjo, P. Enarson, T. Sulli-van, C. Stewart and B. Burke, submitted for publication(38,39)), is mislocalized to the peripheral ER (Figure 1) (5).Correct emerin localization can be restored in these cells,however, by the introduction of heterologous lamin A (5). Evi-dently, appropriate emerin localization in fibroblasts is contin-gent upon the presence of A-type lamins.

Laminopathies

Cardiac and skeletal myopathies

Emery–Dreifuss muscular dystrophy (EDMD) (40,41) was thefirst human disease found to result from a nuclear envelope-

Burke et al.

Table1: Properties of mammalian inner nuclear membrane proteins

INM Polypeptide Lamin Chromatin Commentsprotein mass binding binding

LAP1A 75kDa A/B No (73)LAP1B 68kDa A/B No (73)LAP1C 57kDa A/B? No Type ll membrane protein.

173 residue lumenal domain,311 residue nucleoplasmicdomain (74)

LAP2b 50kDa B Yes Type ll membrane protein.Large 410 residue nucleoplasmicdomain. LEM domain (24,75)

LAP2e,d,g 38–46kDa Most likely B Probable Type ll membrane proteins.Splice variants of LAP2b.Two other splice variants,LAP2a,z are solubleproteins (26,76).

LBR 70kDa B Yes Multi-spanning protein.Sterol C-14 reductase activity(23,77)

Emerin 29kDa A – Defects in emerin linked toEmery–Dreifuss musculardystrophy. LEM domain (27)

Nurim 29kDa – – Multi-spanning membraneprotein (20)

MAN1 82kDa Unknown Probable LEM domain (29)

specific defect (27,28). Although quite rare, this disorder isone of the three major X-linked muscular dystrophies andis characterized by childhood onset with progressive musclewasting and weakening. This is usually accompanied by con-tractures of the Achilles tendons, as well as of tendons of theelbows and neck, often resulting in severe postural changes.Late-onset defects of EDMD include abnormal cardiacrhythms, conduction block, and cardiomyopathy that canlead to sudden cardiac arrest (42). These cardiac abnormali-ties invariably demand implantation of a pacemaker. Muta-tions causing EDMD were originally mapped to a gene en-coding a 29-kDa integral membrane protein, termed emerin(27). This protein, which is found in the majority of cell types,was subsequently shown to be a component of the nuclearenvelope and a resident of the INM (28,43).

Most of the EDMD-linked emerin defects described to dateare nonsense mutations (44,45). Since emerin is a type 2membrane protein with a C-terminal membrane anchor do-main, most such mutations would be predicted to results insynthesis of soluble forms of emerin. In reality, the majorityof EDMD mutations result in the almost complete loss ofthe protein, suggesting that C-terminal truncation destabilizesemerin in vivo (45,46). A few EDMD mutations, includingsome missense and in-frame deletions, do give rise to de-tectable protein. However, these mutant forms of emerin tendto be mislocalized to the cytoplasm (47–50). A consistentfeature of X-linked EDMD therefore is a loss of emerin fromthe nuclear periphery.

A clinically identical autosomal dominant form of EDMD

678 Traffic 2001; 2: 675–683

(EDMD-AD) has been mapped to the LMNA gene, andat least 22 distinct disease-causing mutations have beenidentified (51,52). The majority of these are missensemutations and can be found throughout most exonswhich encode the common portions of the lamin A and Cproteins (exons 1–9). A single nonsense mutation at co-don 6 has also been described and this will functionallyeliminate LMNA expression (52). Patients carrying thismutation are effectively haploinsufficient. Consistent withthese observations is the finding that mice which arehomozygous for a targeted disruption of the Lmna genedevelop a syndrome that bears a striking resemblance tohuman EDMD and features both cardiac and skeletalmyopathy (5). In addition to these gross pathologicalchanges, cells derived from the Lmna null mice exhibit anarray of defects related to changes in nuclear envelopeintegrity (5). In particular, nuclei in a variety of Lmna –/–cells and tissues exhibit grossly altered morphologies,often displaying nuclear membrane herniations which aredevoid of the normal complement of NE components,such as B-type lamins, LAP2 and NPCs (5). At the ultra-structural level these herniations appear as regions of thenuclear periphery where the ONM and perinuclear spaceare massively distended (Figure 3). An additional featureof Lmna –/– nuclei is loss of peripheral heterochromatinas well as mislocalization of emerin to the ER (Figure 1).A similar range of nuclear structural defects has been ob-served in both skin and muscle cells obtained from X-linked EDMD patients (53,54). The inference here is thatemerin and A-type lamins cooperate in maintenance ofnuclear architecture.

Nuclear Laminopathies

Figure3: Cells from LMNA–/– mice exhibit nuclear envelope‘herniations’ in which regions of the outer nuclear membrane(small arrows) become massively dilated and are separatedfrom the inner nuclear membrane (large arrows) by an ex-panded perinuclear space (asterisk). Nuclear pore complexesare excluded from these areas. The two electron micrographs repre-sent low (upper panel) and high (lower panel) magnification viewsof the same cell. By immunofluorescence microscopy, herniated re-gions of the nuclear membranes (arrowhead, inset) appear to lackmany nuclear envelope components such as LAP2 (red). Chroma-tin, revealed by staining with Höchst dye, is shown in blue.

Linkage studies have connected two other human myo-pathies to mutations in the LMNA gene (51). These arelimb-girdle muscular dystrophy with atrio-ventricular con-duction disturbances (LGMD-1B) (55) and dilated cardio-myopathy with conduction system disease (DCM-CD)(56). Like EDMD, LGMD-1B features skeletal myopathyand cardiac conduction defects (55). The main distin-guishing features of LGMD-1B with respect to EDMD arethe absence of early tendon contractures and the pre-dominance of proximal limb weakness. DCM-CD is pri-marily characterized by ventricular dilation and impairedsystolic contraction. In some cases of inherited autosomal

679Traffic 2001; 2: 675–683

dominant cardiomyopathy, skeletal muscle defects remi-niscent of EDMD-AD and LGMD may be seen (51,57).Such variability in phenotypes can be observed evenwithin family members carrying the same mutant allele ofLMNA. For instance, Brodsky et al. (57) have described afamily in which several members are heterozygous for aLMNA allele in which a single nucleotide deletion in exon6 results in a frameshift at codon 320 (within the laminA/C coiled coil domain). One family member was foundto display a pure DCM-CD phenotype. A second display-ed symptoms more consistent with a diagnosis of LGMD-1B, while a third had symptoms characteristic of EDMD.The implication is that these three disorders, DCM-CD,AD-EDMD and LGMD-1B, may represent points on asingle clinical continuum that may itself be influenced bythe effects of modifying genes in varying genetic back-grounds, or perhaps environmental factors such as stressand diet.

Lipodystrophy

A further very different tissue-specific disease associatedwith mutations in the lamin A/C gene is Dunnigan-type fam-ilial partial lipodystrophy (FPLD) (58,59). FPLD usually be-comes evident at puberty and is characterized by selectiveloss of subcutaneous fat from the limbs and trunk, with ac-cumulations mainly in the upper body, particularly the faceand neck (60,61). These changes in adipose distribution areassociated with hypertriglyceridemia and hyperinsulinemia,often culminating in insulin-resistant diabetes. With few ex-ceptions, muscular weakness and tendon contractures arenot evident, but early coronary heart disease has been ob-served (60). However, this most likely results from hyperlipid-emia, a common metabolic complication of FPLD (62), anddoes not appear to be related to the type of cardiomyopathyobserved in DCM-CD, EDMD and LGMD-1B.

Two exons encoding regions of the lamin A/C C-terminal do-main contain hotspots for missense mutations that result inFPLD (58,59,62–65). Within exon 8, single nucleotidechanges in codons 465, 482, 486 have been documented,as have substitutions in codons 582 and 584 within the lam-in A-specific exon 11. While only FPLD mutations have beenmapped within the latter exon, this is not true of exon 8.An I469T substitution (66) and a single nucleotide deletion1397delA (67) are both associated with EDMD without thecharacteristic redistribution of subcutaneous fat found inFPLD. Structural studies indicate that residues 482 and 486lie on the outer surface of the C-terminal globular domain (S.Shoelson, personal communication), suggesting thatchanges in these amino acids could specifically modify thecapacity of the LMNA products for a number of potentialbinding partners, such as INM proteins, nucleoplasmic pro-teins, chromatin or indeed other A- or B-type lamin mol-ecules. Conversely, several EDMD mutations that map toexon 8 as well as to other 3ƒ exons are predicted to alterresidues that lie within the interior of the C-terminal domain,perhaps interfering with the 3D organization and folding ofthis region of the lamin A/C molecule.

Burke et al.

Assembly and Targeting of Defective Lamins

The effects of disease-linked A-type lamin point mutants onlamina assembly and nuclear organization have been investi-gated by introducing mutated human lamin A/C cDNAs intoa variety of cell types, including HeLa cells and lamin-de-ficient primary mouse embryo fibroblasts (PMEFs). In thisway it has been possible to mimic the alleles associated withvarious phenotypic presentations of EDMD, DCM-CD, orFPLD (W.H. Raharjo, P. Enarson, T. Sullivan, C. Stewart, andB. Burke, submitted for publication). When introduced intoHeLa cells, which express wild-type lamins A and C, anEDMD L530P mutant lamin A cDNA was found to cause asignificant loss of emerin from the nuclear envelope. Despitethe fact that this mutant is capable of associating with thenuclear periphery, the L530P substitution clearly has a domi-nant negative effect on emerin retention in the INM. Consist-ent with this finding, co-immunoprecipitation experimentsusing in vitro translated emerin and lamin A demonstrate thatthe L530P mutation effectively abolishes the ability of laminA to interact with emerin (W.H. Raharjo, P. Enarson, T. Sulliv-an, C. Stewart, and B. Burke, submitted for publication). Simi-lar experiments involving mutants associated with DCM-CD(N195K, L85R) reveal a broad spectrum of effects on nuclearmorphology and targeting to the nuclear envelope but withonly a weak impact on emerin localization. In contrast to wild-type lamin A, none of these mutant lamins (N195K, L85R,L530P) is capable of rescuing the aberrant distribution of em-erin that is observed in Lmna-deficient fibroblasts (Figure 1).Östlund et al. have described similar results in terms of thetargeting and assembly of 15 A-type lamin mutants (C.Östlund, G. Bonne, K. Schwartz and H. J. Worman, submittedfor publication). The consensus that has emerged from all ofthese studies is that at least 50% of EDMD, LGMD-1B andDCM-CD A-type lamin mutants have easily observable de-fects in terms of assembly into the lamina and disruption ofwild-type lamins. Interestingly, however, the R482W-LMNA

FPLD mutant (either lamin A or C) is indistinguishable in allrespects from wild-type lamin A/C, having no immediate ad-verse effects on either nuclear structure or emerin distri-bution. Like wild-type lamin A the R482W mutant is fully cap-able of recruiting emerin to the INM in Lmna-deficient fibro-blasts and binds efficiently to emerin in vitro. These findingsare consistent with the suggestion that the FPLD mutationsmight modify some cell-type specific function of lamin Arather than causing a generalized defect in lamina organiza-tion (68).

A surprising finding that has emerged from these studies onA-type lamin mutants is that distinct phenotypes can be ob-served for both EDMD and DCM-CD mutants, dependingupon whether it is lamin A or lamin C which actually carriesthe amino acid substitutions. For instance, while the L85Rand L530P lamin A mutants associate with the NE, thesesame mutations render lamin C completely assembly incom-petent. This finding reveals another level of complexity inunderstanding how lamins A and C interact either with eachother or with other NE proteins to form the nuclear lamina,

680 Traffic 2001; 2: 675–683

and indicates that these two proteins are not functionally in-terchangeable.

Disease Mechanisms

Given that the LMNA gene is expressed in the majority ofadult cell types, how is it that different mutant alleles can giverise to such diverse tissue-restricted phenotypes? Severalmodels have been proposed to account for this paradox.However, these are not mutually exclusive, and it is possiblethat multiple mechanisms will be found to lie at the heart ofthese various pathologies. It has been suggested that nucleicontaining defective lamin or emerin proteins may be mech-anically more fragile than their wild-type counterparts andthat this fragility ultimately leads to nuclear damage and celldeath (69,70). In fact, purified hepatocyte nuclear envelopesfrom Lmna –/– mice do indeed exhibit increased fragility, asevidenced by a marked tendency to vesiculate (5). Wild-typeNEs in contrast remain largely intact and present the appear-ance of nuclear ‘ghosts’. Clearly a priority will be to confirmthese observations using NEs derived from mice harboringdisease-linked Lmna point mutations. This notion of in-creased nuclear fragility is particularly attractive in terms ofcardiac and skeletal muscle pathologies, since the forcesgenerated during muscle contraction might potentially leadto preferential breakage of nuclei containing a defective nu-clear lamina. It could also account for the commonality ofcardiac defects in EDMD, DCM-CD and LGMD-1B with vari-ability in skeletal muscle involvement (69). Since skeletalmuscle is a syncitium, damage to single nuclei may not beparticularly deleterious so long as each muscle cell containssufficient undamaged nuclei to remain functional. Further-more, if cell death does eventually occur, proliferation of myo-blasts will lead to replacement of compromised musclefibers. In the heart, which is not a syncitium, damage to asingle nucleus will inevitably lead to cell death, and with obvi-ous consequences in terms of cardiac function. The mechan-ical stress model is far less attractive with respect to the etiol-ogy of FPLD, since it seems highly unlikely that adipocytenuclei would ever be subjected to forces comparable tothose encountered in muscle. Furthermore, since there is nomuscle involvement in FPLD and no adipocyte involvementin any of the LMNA-linked myopathies, mechanical damageto nuclei cannot provide a universal foundation for all of theLMNA-associated disorders.

A second model that has been proposed relates to the rolethat the NE may play in the regulation of global patterns ofgene expression. Transcriptional regulators such as theretinoblastoma susceptibility gene product, p110Rb, areknown to interact with A-type lamins (71) and at the sametime it is clear that the lamina exerts a profound influence onthe organization of heterochromatin within the nucleus (5).Since heterochromatin is generally silent in terms of tran-scriptional activity, changes in chromatin organization, whichhas been documented in both Lmna null mice and EDMDpatients, could lead to changes in gene expression programs

Nuclear Laminopathies

with potentially deleterious effects. A more detailed dis-cussion of this proposal is provided in two recent reviews byWilson and colleagues (3,68).

A final novel possibility is that LMNA mutations resulting inchanges in nuclear membrane composition could haveknock-on effects on the composition and function of the pe-ripheral ER. The INM, ONM, and ER together form a singlecontinuous membrane system containing functionally distinctdomains into which certain membrane proteins may be seg-regated. We know that loss of one component of this system,lamin A/C, causes the mislocalization of emerin to the ER (5).The implication is that the nuclear lamina plays an essentialrole in this domain organization by maintaining the segre-gation of at least some INM proteins from the ONM andperipheral ER. Thus, the tissue specificity associated with dis-eases caused by LMNA mutations might be ascribed to im-paired function in particularly sensitive processes enacted bythe ER. For example, the ER is the major site of cholesteroland fatty acid synthesis. Abnormal accumulation of proteinsin the ER could possibly alter lipidogenesis or lipogenic sig-naling in LMNA-deficient cells, resulting in aberrant adipocy-te development and lipodystrophic disease. In the case ofskeletal and cardiac muscle, Ca2π release during contractioncycles could be compromised due to alterations in the sarco-plasmic reticulum. Alternatively, generalized ER stress due toinappropriate accumulation of nuclear envelope proteins,such as emerin, in the ER could potentially promote aberrantintracellular signaling pathways with downstream effects ongene expression and cell viability (72).

Perspectives

Whatever the mechanisms underlying the various lamin-opathies discussed here, the phenotypic heterogeneity in pa-tients from the same family underscores the importance ofgenetic backgrounds as well as possible environmental fac-tors in determining the breadth and severity of EDMD,LGMD-B1, DCM-CD and FPLD defects. Since it has beenpossible to mimic features of EDMD in lamin-deficient mice,this provides us with a unique opportunity to identify candi-dates for modifying genes in inbred mouse strains and tounderstand further how A-type lamin function relates to nu-clear architectural organization and muscular and cardio-vascular diseases. In the future, the introduction of specificpoint mutations into the Lmna gene in mice as well as themodification of other genes encoding nuclear envelope andchromatin-associated proteins should shed new light on theetiology of laminopathies and pave the way for the develop-ment of novel treatment strategies.

Acknowledgments

Brian Burke is supported by grants from the Canadian Institutes of HealthResearch and the Alberta Heritage Foundation for Medical Research. We

681Traffic 2001; 2: 675–683

would like to thank Davide Salina for the electron micrographs presentedin Figure 3.

References

1. Gant TM, Wilson KL. Nuclear assembly. Annu Rev Cell Dev Biol1997;13:669–695.

2. Gerace L, Burke B. Functional organization of the nuclear envelope.Ann Rev Cell Biol 1988;4:335–374.

3. Wilson KL. The nuclear envelope, muscular dystrophy and gene ex-pression. Trends Cell Biol 2000;10:125–129.

4. Stuurman N, Heins S, Aebi U. Nuclear lamins. their structure, as-sembly, and interactions. J Struct Biol 1998;122:42–66.

5. Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, NagashimaK, Stewart CL, Burke B. Loss of A-type lamin expression compromisesnuclear envelope integrity leading to muscular dystrophy. J Cell Biol1999;147:913–920.

6. Liu J, Ben-Shahar TR, Riemer D, Treinin M, Spann P, Weber K, Fire A,Gruenbaum Y. Essential roles for Caenorhabditis elegans lamin genein nuclear organization, cell cycle progression, and spatial organizationof nuclear pore complexes. Mol Biol Cell 2000;11:3937–3947.

7. Lenz B,̂ hme B, Wismar J, Fuchs S, Reifegerste R, Buchner E, Betz H,Schmitt B. Insertional mutation of the Drosophila nuclear lamin Dm0

gene results in defective nuclear envelopes, clustering of nuclear porecomplexes, and accumulation of annulate lamellae. J Cell Biol1997;137:1001–1016.

8. Aebi U, Cohn JB, Buhle L, Gerace L. The nuclear lamina is a meshworkof intermediate type filaments. Nature 1986;323:560–564.

9. Moir RD, Spann TP, Lopez-Soler RI, Yoon M, Goldman AE, Khuon S,Goldman RD. Review. the dynamics of the nuclear lamins during thecell cycle – relationship between structure and function. J Struct Biol2000;129:324–334.

10. Roeber R-A, Weber K, Osborn M. Differential timing of lamin A/C ex-pression in the various organs of the mouse embryo and the younganimal: a developmental study. Development 1989;105:365–378.

11. Roeber R-A, Sauter H, Weber K, Osborn M. Cells of the cellular im-mune and hemopoietic system of the mouse lack lamins A/C: distinc-tion versus other somatic cells. J Cell Sci 1990;95:587–598.

12. Stewart C, Burke B. Teratocarcinoma stem cells and early mouse em-bryos contain only a single major lamin polypeptide closely resem-bling lamin B. Cell 1987;51:383–392.

13. Furukawa K, Hotta Y. cDNA cloning of a germ cell-specific lamin B3from mouse spermatocytes and analysis of its ectopic expression insomatic cells. EMBO J 1993;12:97–106.

14. Machiels BM, Zorenc AH, Endert JM, Kuijpers HJ, van Eys GJ, Ramae-kers FC, Broers JL. An alternative splicing product of the lamin A/Cgene lacks exon 10. J Biol Chem 1996;271:9249–9253.

15. Alsheimer M, Benavente R. Change of karyoskeleton during mam-malian spermatogenesis: expression pattern of nuclear lamin C2 andits regulation. Exp Cell Res 1996;228:181–188.

16. Holtz D, Tanaka RA, Hartwig J, McKeon F. The CaaX motif of lamin Afunctions in conjunction with the nuclear localization signal to targetassembly to the nuclear envelope. Cell 1989;59:969–977.

17. Krohne G, Waizenegger I, Hoeger TH. The conserved carboxy-terminalcysteine of nuclear lamins is essential for association with the nuclearenvelope. J Cell Biol 1989;109:2003–2011.

18. Kitten GT, Nigg EA. The CaaX motif is required for isoprenylation, car-boxy methylation and nuclear membrane association of lamin B2. JCell Biol 1991;113:13–24.

19. Weber K, Plessmann U, Traub P. Maturation of nuclear lamin A in-volves a specific carboxy-terminal trimming, which removes the polyi-soprenylation site from the precursor; implications for the structure ofthe nuclear lamina. FEBS Lett 1989;257:411–414.

Burke et al.

20. Rolls MM, Stein PA, Taylor SS, Ha E, McKeon F, Rapoport TA. A visualscreen of a GFP-fusion library identifies a new type of nuclear envel-ope membrane protein. J Cell Biol 1999;146:29–44.

21. YeQ, Callebaut I, Pezhman A, Courvalin JC, Worman HJ. Domain-spe-cific interactions of human HP1-type chromodomain proteins and in-ner nuclear membrane protein LBR. J Biol Chem 1997;272:14983–14989.

22. Martins SB, Eide T, Steen RL, Jahnsen T, Skalhegg BS, Collas P. HA95is a protein of the chromatin and nuclear matrix regulating nuclearenvelope dynamics. J Cell Sci 2000;113 Part 21:3703–3713.

23. Silve S, Dupuy PH, Ferrara P, Loison G. Human lamin B receptor ex-hibits sterol C14-reductase activity in Saccharomyces cerevisiae. Bio-chim Biophys Acta 1998;1392:233–244.

24. Foisner R, Gerace L. Integral membrane proteins of the nuclear envel-ope interact with lamins and chromosomes, and binding is modulatedby mitotic phosphorylation. Cell 1993;73:1267–1279.

25. Harris CA, Andryuk PJ, Cline S, Chan HK, Natarajan A, Siekierka JJ,Goldstein G. Three distinct human thymopoietins are derived from al-ternatively spliced mRNAs. Proc Natl Acad Sci USA 1994;91:6283–6287.

26. Berger R, Theodor L, Shoham J, Gokkel E, Brok-Simoni F, AvrahamKB, Copeland NG, Jenkins NA, Rechavi G, Simon AJ. The character-ization and localization of the mouse thymopoietin/lamina-associatedpolypeptide 2 gene and its alternatively spliced products. GenomeRes 1996;6:361–370.

27. Bione S, Maestrini E, Rivella S, Mancini M, Regis S, Romeo G, TonioloD. Identification of a novel X-linked gene responsible for Emery–Drei-fuss muscular dystrophy. Nature Genet 1994;8:323–327.

28. Manilal S, Nguyen TM, Sewry CA, Morris GE. The Emery–Dreifussmuscular dystrophy protein, emerin, is a nuclear membrane protein.Hum Mol Genet 1996;5:801–808.

29. Lin F, Blake DL, Callebaut I, Skerjanc IS, Holmer L, McBurney MW,Paulin-Levasseur M, Worman HJ. MAN1, an inner nuclear membraneprotein that shares the LEM domain with lamina-associated polypep-tide 2 and emerin. J Biol Chem 2000;275:4840–4847.

30. Shumaker DK, Lee KK, Tanhehco YC, Craigie R, Wilson KL. LAP2 bindsto BAF.DNA complexes: requirement for the LEM domain and modu-lation by variable regions. EMBO J 2001;20:1754–64.

31. Zheng R, Ghirlando R, Lee MS, Mizuuchi K, Krause M, Craigie R. Bar-rier-to-autointegration factor (BAF) bridges DNA in a discrete, higher-order nucleoprotein complex. Proc Natl Acad Sci USA 2000;97:8997–9002.

32. Glass CA, Glass JR, Taniura H, Hasel KW, Blevitt JM, Gerace L. Thealpha-helical rod domain of human lamins A and C contains achromatin binding site. EMBO J 1993;12:4413–4424.

33. Ostlund C, Ellenberg J, Hallberg E, Lippincott-Schwartz J, WormanHJ. Intracellular trafficking of emerin, the Emery–Dreifuss musculardystrophy protein. J Cell Sci 1999;112:1709–1719.

34. Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley JF, Worman HJ,Lippincott-Schwartz J. Nuclear membrane dynamics and reassemblyin living cells: targeting of an inner nuclear membrane protein in inter-phase and mitosis. J Cell Biol 1997;138:1193–1206.

35. Torrisi MR, Lotti LV, Pavan A, Migliaccio G, Bonatti S. Free diffusion toand from the inner nuclear membrane of newly synthesized plasmamembrane glycoproteins. J Cell Biol 1987;104:733–737.

36. Torrisi MR, Bonatti S. Immunocytochemical study of the partition anddistribution of Sindbis virus glycoproteins in freeze-fractured mem-branes of infected baby hamster kidney cells. J Cell Biol 1985;101:1300–1306.

37. Bergmann JE, Singer SJ. Immunoelectron microscopic studies of theintracellular transport of the membrane glycoprotein (G) of vesicularstomatitis virus in infected Chinese hamster ovary cells. J Cell Biol1983;97:1777–1787.

38. Clements L, Manilal S, Love DR, Morris GE. Direct interaction between

682 Traffic 2001; 2: 675–683

emerin and lamin A. Biochem Biophys Res Commun 2000;267:709–714.

39. Sakaki M, Koike H, Takahashi N, Sasagawa N, Tomioka S, Arahata K,Ishiura S. Interaction between emerin and nuclear lamins. J Biochem(Tokyo) 2001;129:321–327.

40. Emery AE, Dreifuss FE. Unusual type of benign x-linked muscular dys-trophy. J Neurol Neurosurg Psychiatry 1966;29:338–342.

41. Emery AE. Emery–Dreifuss muscular dystrophy – a 40 year retrospec-tive. Neuromuscul Disord 2000;10:228–232.

42. Emery AE. Emery–Dreifuss muscular dystrophy and other related dis-orders. Br Med Bull 1989;45:772–787.

43. Nagano A, Koga R, Ogawa M, Kurano Y, Kawada J, Okada R, HayashiYK, Tsukahara T, Arahata K. Emerin deficiency at the nuclear mem-brane in patients with Emery–Dreifuss muscular dystrophy. Nat Genet1996;12:254–259.

44. Yates JR, Wehnert M. The Emery–Dreifuss muscular dystrophy muta-tion database. Neuromuscul Disord 1999;9:199.

45. Manilal S, Recan D, Sewry CA, Hoeltzenbein M, Llense S, Leturcq F,Deburgrave N, Barbot J, Man N, Muntoni F, Wehnert M, Kaplan J,Morris GE. Mutations in Emery–Dreifuss muscular dystrophy and theireffects on emerin protein expression. Hum Mol Genet 1998;7:855–864.

46. Yates JR, Bagshaw J, Aksmanovic VM, Coomber E, McMahon R,Whittaker JL, Morrison PJ, Kendrick-Jones J, Ellis JA. Genotype-phenotype analysis in X-linked Emery–Dreifuss muscular dystrophyand identification of a missense mutation associated with a milderphenotype. Neuromuscul Disord 1999;9:159–165.

47. Di Blasi C, Morandi L, Raffaele di Barletta M, Bione S, Bernasconi P,Cerletti M, Bono R, Blasevich F, Toniolo D, Mora M. Unusual expres-sion of emerin in a patient with X-linked Emery–Dreifuss musculardystrophy. Neuromuscul Disord 2000;10:567–571.

48. Ellis JA, Craxton M, Yates JR, Kendrick-Jones J. Aberrant intracellulartargeting and cell cycle-dependent phosphorylation of emerin contrib-ute to the Emery–Dreifuss muscular dystrophy phenotype. J Cell Sci1998;111:781–792.

49. Fairley EA, Kendrick-Jones J, Ellis JA. The Emery–Dreifuss musculardystrophy phenotype arises from aberrant targeting and binding ofemerin at the inner nuclear membrane. J Cell Sci 1999;112: 2571–2582.

50. Ellis JA, Brown CA, Tilley LD, Kendrick-Jones J, Spence JE, Yates JR.Two distal mutations in the gene encoding emerin have profoundlydifferent effects on emerin protein expression. Neuromuscul Disord2000;10:24–30.

51. Bonne G, Mercuri E, Muchir A, Urtizberea A, Becane HM, Recan D,Merlini L, Wehnert M, Boor R, Reuner U, Vorgerd M, Wicklein EM,Eymard B, Duboc D, Penisson-Besnier I et al. Clinical and moleculargenetic spectrum of autosomal dominant Emery–Dreifuss musculardystrophy due to mutations of the lamin A/C gene. Ann Neurol2000;48:170–180.

52. Bonne G, Di Barletta MR, Varnous S, Becane HM, Hammouda EH,Merlini L, Muntoni F, Greenberg CR, Gary F, Urtizberea JA, Duboc D,Fardeau M, Toniolo D, Schwartz K. Mutations in the gene encodinglamin A/C cause autosomal dominant Emery- Dreifuss muscular dys-trophy. Nat Genet 1999;21:285–288.

53. Ognibene A, Sabatelli P, Petrini S, Squarzoni S, Riccio M, Santi S,Villanova M, Palmeri S, Merlini L, Maraldi NM. Nuclear changes in acase of X-linked Emery–Dreifuss muscular dystrophy. Muscle Nerve1999;22:864–869.

54. Fidzianska A, Toniolo D, Hausmanowa-Petrusewicz I. Ultrastructuralabnormality of sarcolemmal nuclei in Emery–Dreifuss muscular dys-trophy (EDMD). J Neurol Sci 1998;159:88–93.

55. Muchir A, Bonne G, van der Kooi AJ, van Meegen M, Baas F, BolhuisPA, de Visser M, Schwartz K. Identification of mutations in the geneencoding lamins A/C in autosomal dominant limb girdle muscular

Nuclear Laminopathies

dystrophy with atrioventricular conduction disturbances (LGMD1B).Hum Mol Genet 2000;9:1453–1459.

56. Fatkin D, MacRae C, Sasaki T, Wolff MR, Porcu M, Frenneaux M,Atherton J, Vidaillet HJ Jr, Spudich S, De Girolami U, Seidman JG,Seidman CE, Muntoni F, Muehle G, Johnson W et al. Missense muta-tions in the rod domain of the lamin A/C gene as causes of dilatedcardiomyopathy and conduction-system disease. N Engl J Med1999;341:1715–1724.

57. Brodsky GL, Muntoni F, Miocic S, Sinagra G, Sewry C, Mestroni L.Lamin A/C gene mutation associated with dilated cardiomyopathywith variable skeletal muscle involvement. Circulation 2000;101:473–476.

58. Cao H, Hegele RA. Nuclear lamin A/C R482Q mutation in Canadiankindreds with Dunnigan-type familial partial lipodystrophy. Hum MolGenet 2000;9:109–112.

59. Shackleton S, Lloyd DJ, Jackson SN, Evans R, Niermeijer MF, SinghBM, Schmidt H, Brabant G, Kumar S, Durrington PN, Gregory S, O’R-ahilly S, Trembath RC. LMNA, encoding lamin A/C, is mutated in par-tial lipodystrophy. Nat Genet 2000;24:153–156.

60. Burn J, Baraitser M. Partial lipoatrophy with insulin resistant diabetesand hyperlipidaemia (Dunnigan syndrome). J Med Genet 1986;23:128–130.

61. Garg A, Peshock RM, Fleckenstein JL. Adipose tissue distribution pat-tern in patients with familial partial lipodystrophy (Dunnigan variety).J Clin Endocrinol Metab 1999;84:170–174.

62. Vigouroux C, Magre J, Vantyghem MC, Bourut C, Lascols O,Shackleton S, Lloyd DJ, Guerci B, Padova G, Valensi P, Grimaldi A,Piquemal R, Touraine P, Trembath RC, Capeau J. Lamin A/C gene. sex-determined expression of mutations in Dunnigan-type familial partiallipodystrophy and absence of coding mutations in congenital and ac-quired generalized lipoatrophy. Diabetes 2000;49:1958–1962.

63. Speckman RA, Du Garg AF, Bennett L, Veile R, Arioglu E, Taylor SI,Lovett M, Bowcock AM. Mutational and haplotype analyses of familieswith familial partial lipodystrophy (Dunnigan variety) reveal recurrentmissense mutations in the globular C-terminal domain of lamin A/C.Am J Hum Genet 2000;66:1192–1198.

64. Garg A, Vinaitheerthan M, Weatherall PT, Bowcock AM. Phenotypicheterogeneity in patients with familial partial lipodystrophy (Dunniganvariety) related to the site of missense mutations in lamin A/C gene.J Clin Endocrinol Metab 2001;86:59–65.

683Traffic 2001; 2: 675–683

65. Hegele RA, Cao H, Anderson CM, Hramiak IM. Heterogeneity of nu-clear lamin A mutations in Dunnigan-type familial partial lipodys-trophy. J Clin Endocrinol Metab 2000;85:3431–3435.

66. Raffaele Di Barletta M, Ricci E, Galluzzi G, Tonali P, Mora M, MorandiL, Romorini A, Voit T, Orstavik KH, Merlini L, Trevisan C, Biancalana V,Housmanowa-Petrusewicz I, Bione S, Ricotti R et al. Different muta-tions in the LMNA gene cause autosomal dominant and autosomalrecessive Emery–Dreifuss muscular dystrophy. Am J Hum Genet2000;66:1407–1412.

67. Genschel J, Schmidt HH. Mutations in the LMNA gene encoding lam-in A/C. Hum Mutat 2000;16:451–459.

68. Wilson KL, Zastrow MS, Lee KK. Lamins and disease: insights intonuclear infrastructure. Cell 2001;104:647–650.

69. Hutchison CJ, Alvarez-Reyes M, Vaughan OA. Lamins in disease. Whydo ubiquitously expressed nuclear envelope proteins give rise totissue-specific disease phenotypes? J Cell Sci 2001;114:9–19.

70. Morris GE, Manilal S. Heart to heart: from nuclear proteins to Emery–Dreifuss muscular dystrophy. Hum Mol Genet 1999;8:1847–1851.

71. Ozaki T, Saijo M, Murakami K, Enomoto H, Taya Y, Sakiyama S. Com-plex formation between lamin A and the retinoblastoma gene product:identification of the domain on lamin A required for its interaction.Oncogene 1994;9:2649–2653.

72. Pahl HL, Baeuerle PA. The ER-overload response: activation of NF-kappa B. Trends Biochem Sci 1997;22:63–67.

73. Senior A, Gerace L. Integral membrane proteins specific to the innernuclear membrane and associated with the nuclear lamina. J Cell Biol1988;107:2029–2036.

74. Martin L, Crimaudo C, Gerace L. cDNA cloning and characterizationof lamina-associated polypeptide 1C (LAP1C), an integral protein ofthe inner nuclear membrane. J Biol Chem 1995;270:8822–8828.

75. Furukawa K, Pante N, Aebi U, Gerace L. Cloning of a cDNA for lamina-associated polypeptide 2 (LAP2) and identification of regions that spe-cify targeting to the nuclear envelope. EMBO J 1995;14:1626–1636.

76. Harris CA, Andryuk PJ, Cline SW, Mathew S, Siekierka JJ, GoldsteinG. Structure and mapping of the human thymopoietin (TMPO) geneand relationship of human TMPO beta to rat lamin-associated poly-peptide 2. Genomics 1995;28:198–205.

77. Worman HJ, Evans CD, Blobel G. The lamin B receptor of the nuclearenvelope inner membrane: a polytopic protein with eight potentialtransmembrane domains. J Cell Biol 1990;111:1535–1542.