Prelamin A-mediated nuclear envelope dynamics in normal and laminopathic cells

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1 1698 Biochemical Society Transactions (2011) Volume 39, part 6 Prelamin A-mediated nuclear envelope dynamics in normal and laminopathic cells Giovanna Lattanzi 1 National Research Council of Italy, Institute of Molecular Genetics, IGM-CNR, Unit of Bologna c/o IOR, Via di Barbiano 1/10, I Bologna, Italy Abstract Prelamin A is the precursor protein of lamin A, a major constituent of the nuclear lamina in higher eukaryotes. Increasing attention to prelamin A processing and function has been given after the discovery, from 2002 to 2004, of diseases caused by prelamin A accumulation. These diseases, belonging to the group of laminopathies and mostly featuring LMNA mutations, are characterized, at the clinical level, by different degrees of accelerated aging, and adipose tissue, skin and bone abnormalities. The outcome of studies conducted in the last few years consists of three major findings. First, prelamin A is processed at different rates under physiological conditions depending on the differentiation state of the cell. This means that, for instance, in muscle cells, prelamin A itself plays a biological role, besides production of mature lamin A. Secondly, prelamin A post-translational modifications give rise to different processing intermediates, which elicit different effects in the nucleus, mostly by modification of the chromatin arrangement. Thirdly, there is a threshold of toxicity, especially of the farnesylated form of prelamin A, whose accumulation is obviously linked to cell and organism senescence. The present review is focused on prelamin A-mediated nuclear envelope modifications that are upstream of chromatin dynamics and gene expression mechanisms regulated by the lamin A precursor. Introduction The LMNA gene (OMIM ), encoding the A-type lamins, was characterized in 1993 [1] and subsequently mapped to chromosome 1q21.2 q21.3 [2]. The discovery of mutations in emerin [3] and lamin A/C [4] in 1994 and 1999 respectively, as the cause of EDMD (Emery Dreifuss muscular dystrophy), brought to the attention of researchers the nuclear envelope as a structure implicated in fundamental mechanisms of tissue homoeostasis. A few years later, FPLD2 (Dunnigan-type familial partial lipodystrophy) [5] and a form of Charcot Marie Tooth neuropathy [6] were associated with lamin A/C mutations, widening the spectrum of LMNA-linked diseases, currently referred to as laminopathies. A key discovery occurred in 2002, when the homozygous R527H-coding mutation in LMNA was identified as the cause of MADA (mandibuloacral dysplasia with type A lipodystrophy), a disease featuring accelerated aging [7]. This finding suggested that lamin A/C may regulate cellular and organism senescence. In the Key words: emerin, Emery Dreifuss muscular dystrophy (EDMD), prelamin A, progeroid laminopathy, SUN1, SUN2. Abbreviations used: AFCMe, N-acetyl-S-farnesyl-l-cysteine methyl ester; BAF, barrier to autointegration factor; EDMD, Emery Dreifuss muscular dystrophy; EDMD1, X-linked EDMD; FPLD2, Dunnigan-type familial partial lipodystrophy; FTI, farnesyltransferase inhibitor; HGPS, Hutchinson Gilford progeria syndrome; HP1α, heterochromatin protein 1α; ICMT, isoprenylcysteine carboxymethyltransferase; LAP2α, lamina-associated polypeptide 2α; LINC, linker of nucleoskeleton and cytoskeleton; MADA, mandibuloacral dysplasia with type A lipodystrophy; MADB, mandibuloacral dysplasia with type B lipodystrophy; NARF, nuclear prelamin A recognition factor; PPARγ, peroxisome-proliferator-activated receptor γ ; RCE1, Rasconverting enzyme 1; RD, restrictive dermopathy; SREBP1, sterol-regulatory-element-binding protein 1; ZMPSTE24, zinc-dependent metalloproteinase Ste24 homologue. 1 giovanna.lattanzi@cnr.it following 2 years, MADB (mandibuloacral dysplasia with type B lipodystrophy) [8] and RD (restrictive dermopathy) [9] were linked to mutations in the prelamin A endoprotease ZMPSTE24 (zinc-dependent metalloproteinase Ste24 homologue), and HGPS (Hutchinson Gilford progeria syndrome) was associated with an aberrant splicing of the LMNA gene [10,11], giving rise to a truncated form of farnesylated prelamin A. Prelamin A accumulation was found in all of the progeroid laminopathies, and it is now established that diseases associated with prelamin A accumulation feature various degrees of adipose tissue, skin and bone defects, and accelerated aging. How prelamin A might act in these disorders, by modifying nuclear envelope dynamics, is discussed in the present review. The LMNA gene The LMNA gene encoding the A-type lamins is located on chromosome 1q21.2 q21.3 and spans 12 exons [1,2]. The mrna is alternatively spliced into four transcripts, which encode lamin A, lamin C, lamin A 10 and lamin C2 [12]. Most differentiated cells express lamin A and lamin C, whereas lamin C2 is mostly found in germinal cells and lamin A 10 expression has so far been elusive. Lamin A Lamin A is the most represented A-type lamin, obtained after post-translational processing of its precursor protein, called prelamin A [13]. Recent studies have shown that lamin A Biochem. Soc. Trans. (2011) 39, ; doi: /bst

2 Nuclear Envelope Disease and Chromatin Organization 1699 is not expressed in ipscs (induced pluripotent stem cells) and it is also absent from neural progenitors [14], a finding of utmost importance for the understanding of lamin-linked diseases. Lamin C Lamin C is expressed in most tissues expressing lamin A and co-polymerizes with lamin A at the nuclear lamina [15]. However, differences in the relative expression of lamin A compared with lamin C are observed by Western blot analysis, which could reflect either different stability of the alternatively spliced products, depending on the cell type or different solubility. Our recent data show that lamin C is much less prone to lysosomal degradation with respect to lamin A [16]. Both lamin A and lamin C can be also found in the nucleoplasm, probably in a different polymerization state. The function of lamin C is considered overlapping and redundant with lamin A [17], although a deep understanding of the role of lamin C is still lacking. Prelamin A Whereas lamin C is produced as mature protein, lamin A is translated as a 74 kda precursor, which undergoes four steps of post-translational modifications, including farnesylation, double endoprotease cleavage and carboxymethylation [18]. These modifications occur at the C-terminal CaaX motif (where a is an aliphatic residue), a sequence shared by farnesylated proteins, in which C is cysteine, the target of protein farnesyl transferase which catalyses prelamin A farnesylation. In human prelamin A, the aax sequence consists of a serine, an isoleucine and a methionine residue (SIM motif), and the methionine residue directs the addition of the C 15 farnesyl residue to cysteine. Following farnesylation, the aax tripeptide is cleaved by ZMPSTE24 or RCE1 (Ras-converting enzyme 1) and the C-terminal cysteine residue is carboxymethylated by ICMT (isoprenylcysteine carboxymethyltransferase). The second ZMPSTE24-mediated cleavage of 15 amino acids at the C- terminus of prelamin A leads to removal of the farnesyl residue and yields mature lamin A. The first cleavage step of prelamin A is zinc-dependent, whereas the second appears to be independent of zinc concentrations. This feature allows us to use different inhibitors to block prelamin A processing and obtain different processing intermediates. For instance, o-phenanthroline, a zinc chelator, can be used to accumulate full-length farnesylated prelamin A in cells, whereas AFCMe (N-acetyl-S-farnesyl-L-cysteine methyl ester) [19], a non-competitor peptide, can be used to accumulate carboxymethylated farnesylated prelamin A. FTIs (farnesyltransferase inhibitors) and statins, including mevinolin, can be used to accumulate unprocessed (nonfarnesylated) prelamin A [19]. Prelamin A processing is altered in laminopathies featuring premature aging and/or lipodystrophy, including HGPS, atypical Werner syndrome, RD, FPLD2 and MADA, as well as in MADB, associated with mutations of the ZMPSTE24 endoprotease gene [18]. Prelamin A partners Prelamin A partners can be split into two groups: enzymes that catalyse and regulate prelamin A maturation, and molecules that interact with prelamin A and modify nuclear envelope/chromatin dynamics. In the first group are the protein farnesyltransferases, which farnesylate the newly transcribed full-length protein, ZMPSTE24, which is required for the two cleavage steps of prelamin A processing, and ICMT, which catalyses prelamin A methylation, whose relevance for protein processing and intermolecular interactions is still not well understood [20]. A still open question in prelamin A studies, which deserves investigation, regards the mechanism(s) regulating expression and activity of prelamin A processing enzymes. Besides the zincdependent activity of ZMPSTE24, little is known about signalling pathways and biochemical factors capable of modulating prelamin A binding and catalytic activity by of those enzymes. Another open question is whether prelamin A copolymerizes with other lamins during its maturation process. Delbarre et al. [21] showed that accumulation of progerin, the truncated form of farnesylated prelamin A found in HGPS, affects the nuclear lamina composition, leading to formation of co-polymers of lamin B, lamin A and progerin itself. On the other hand, since the RCE1 endoprotease, which catalyses prelamin B cleavage, can also intervene in the first prelamin A-processing step, it is conceivable that altered prelamin A processing could interfere with prelamin B maturation. However, the polymerization status of prelamin A under physiological conditions is not yet obvious. The first prelamin A-binding partner was identified in 1999 by Barton and Worman [22]. It is a nuclear envelope protein called NARF (nuclear prelamin A recognition factor), which selectively interacts with farnesylated prelamin A [22]. This feature makes it particularly interesting to investigate the fate of NARF in laminopathic cells accumulating farnesylated prelamin A with deleterious effects. Another prelamin A-binding partner was identified in 2002 by the Shackleton group [23]: it is the transcription factor SREBP1 (sterol-regulatory-element-binding protein 1), which regulates adipocyte-specific gene transcription, including transactivation of PPARγ (peroxisome-proliferatoractivated receptor γ ) [23]. In 2005, we discovered that the cleaved active form of SREBP1 binds with high affinity to both farnesylated and non-farnesylated prelamin A and prelamin A influences its nuclear translocation [24]. Importantly, PPARγ levels are reduced when prelamin A sequesters SREBP1 at the nuclear envelope [24,25]. The regulation of PPARγ levels in adipocytes is also dependent on LAP2α (lamina-associated polypeptide 2α), another prelamin A-binding partner, which is localized in the nucleoplasm [26]. It is conceivable that a complex protein platform, including both prelamin A and LAP2α,

3 1700 Biochemical Society Transactions (2011) Volume 39, part 6 might regulate PPARγ activation. An interplay between prelamin A and LAP2α has been also demonstrated in murine myoblasts, where the interaction occurs at the early stages of myogenic differentiation, within a platform also including the heterochromatin constituent HP1α (heterochromatin protein 1α) [27]. Thus not only regulation of transcription factors, but also mechanisms implicated in chromatin remodelling, appear to be associated with prelamin A modulation. In this respect, it is noteworthy that prelamin A is able to bind BAF (barrier to autointegration factor), a DNA-binding protein located both in the cytoplasm and in the nucleus that is recruited to the nuclear membrane by prelamin A interaction [28]. These observations and others that will follow indicate a physiological role of prelamin A in cells that is not limited to localization or modulated production of mature lamin A. Prelamin A-dependent mechanisms The first function ascribed to prelamin A was to direct localization of lamin A to the nuclear periphery, beneath the nuclear envelope [29]. The farnesylated residue at the C-terminus of prelamin A should favour interactions with nuclear membrane constituents, thus allowing the mature lamin A to properly localize. ZMPSTE24 activity has been demonstrated in the nucleus [30], which should support the hypothesis that lamin A maturation occurs at the site where the protein is expected to localize. However, non-farnesylated prelamin A, either obtained by drug treatment of cells or by transfection of unprocessable prelamin A constructs, is able to properly reach the lamina [13], strongly suggesting that the role of the farnesylated prelamin A residue is mostly to mediate functional intermolecular interactions. The first mechanism dependent on prelamin A levels is the above-mentioned regulation of SREBP1 translocation into the nucleus [24]. Such a role in the modulation of transcription factor availability has been also described for lamin A, for instance in the regulation of c-fos levels within the nucleoplasm [31]. In this context, modulation of the amount of prelamin A, at either the transcriptional or the post-transcriptional level, or both, should in turn regulate gene expression. The relevance of prelamin A in the regulation of the transcription factor PPARγ is supported further by data showing that acquired partial lipodystrophy in patients undergoing highly active anti-retroviral therapy is caused by accumulation of farnesylated prelamin A, which in turn down-regulates PPARγ expression [25,32]. Balanced PPARγ levels are relevant not only for adipocyte differentiation, but also for the fate of stem cells that will differentiate into bone precursors or muscle progenitors [33]. Thus the study of PPARγ regulation in laminopathic cells deserves further study and will probably open new therapeutic perspectives, not just for adipose tissue and metabolic laminopathies. In muscle cells, prelamin A regulates expression of caveolin 3, a membrane protein induced in differentiated myoblasts and located at the sarcolemma of muscle fibres [27]. Other proteins, including troponin T, are also modulated in mouse muscle in response to prelamin A increase, thus suggesting that prelamin A could act indirectly through regulation of upstream genes or large-scale chromatin remodelling. In human myoblasts, prelamin A regulates the expression level of SUN1, a nuclear envelope protein, which is part of the so-called LINC (linker of nucleoskeleton and cytoskeleton) complex [34]. LINC proteins, also including lamins and nesprins, play a key role in nucleocytoplasmic interplay, mostly by mediating the nucleoskeleton connection to the cytoskeleton. SUN1 is required for nuclear positioning and, in Dictyostelium, has been shown to connect centrosomes to centromere clusters [35]. The role of prelamin A in the modulation of SUN1 is particularly relevant. The prelamin A form implicated in this function is the farnesylated and carboxymethylated protein [36]. Non-farnesylated prelamin A, in contrast, down-regulates SUN1 mrna and protein levels [34]. This mechanism is regulated not only at the transcriptional level, but also through stabilization of the protein at the nuclear envelope. The key role of the prelamin A SUN1 interaction in muscle cells resides in nuclear positioning within myotubes and muscle fibres. Lack of farnesylated prelamin A in muscle causes myonuclear clustering and misshapen nuclei, two events which could be related to each other, since both of them are likely to involve centrosome regulation [34]. Another key nuclear membrane protein whose localization and function appears to be dependent on prelamin A is emerin [37]. Emerin binds both non-farnesylated and farnesylated prelamin A, but is only required for the localization of the unprocessed form. Lack of interplay with emerin causes disorganization of the nuclear lamina, and prelamin A shows a honeycomb appearance when labelled by fluorescent antibodies [37]. Our results show that honeycomb structures correspond to sites where the lamina is deeply affected, whereas the whole arrangement of the nuclear membrane is substantially preserved. We suggest that prelamin A emerin interplay is required for the proper assemblage of the nuclear lamina in G 1 -phase cells. This is supported by the observation that the highest frequency of honeycomb structures in cells bearing LMNA or emerin mutations is found in G 1 -phase cells. Provided that emerin may form at least six distinct multiprotein complexes at the nuclear envelope [38], it remains to be established in which of them prelamin A takes part. The interplay between prelamin A and BAF [28], which clearly influences BAF localization, is particularly intriguing. Since BAF is a chromatin-binding protein, the main significance of its interplay with the lamin A precursor resides in the regulation of chromatin arrangement, which is probably the best recognized function of prelamin A. It is conceivable that different prelamin A levels could determine the amount of BAF recruited to the nuclear envelope, thus limiting its chromatin interaction [28]. We have shown that all of the prelamin A-processing intermediates may influence chromatin dynamics in a very specific and reproducible way [13]. Non-farnesylated prelamin A localizes at the nuclear periphery as well as in intranuclear aggregates, where

4 Nuclear Envelope Disease and Chromatin Organization 1701 it recruits heterochromatin, as demonstrated by staining HP1α and trimethylated histone H3 (Lys 9 ). Farnesylated carboxymethylated prelamin A does not bind HP1α, but causes loss of heterochromatin domains at the nuclear periphery. LAP2α is part of a complex that contains nonfarnesylated prelamin A and HP1α [13], which is consistent with the possibility that a protein platform including prelamin A and its nucleoplasmic binding partner might regulate chromatin dynamics in stem cells [39], with the aim to direct cell fate. In fact, modulation of chromatin remodelling by prelamin A could be a fine tool to modify expression of large groups of genes, as occurs during development, cell differentiation or aging. This hypothesis is strongly supported by data obtained in the study of syndromic laminopathies and lipodystrophies, which are characterized by prelamin A accumulation [18,40 42]. Prelamin A and disease Laminopathies featuring prelamin A accumulation Prelamin A accumulation is the hallmark of progeroid laminopathies. MADA and MADB and atypical Werner syndrome cells accumulate prelamin A bearing point mutations or wild-type prelamin A, as in the case of MADB [17]. In most cases of HGPS, a truncated prelamin A form, called progerin, is accumulated [43]. Moreover, wild-type prelamin A is recovered, instead of lamin A, in RD, a lethal developmental disorder, where null mutations of ZMPSTE24 impair lamin A maturation [44]. Prelamin A is also accumulated in lipodystrophies, either caused by LMNA missense mutations [24,32,40,45] or by drugs containing protease inhibitors, which alter ZMPSTE24 functionality [24,45]. Finally, statins, which are used worldwide to treat hypercholesterolaemia, bisphosphonates, widely used in osteoporosis treatment, and FTIs, which are used in chemotherapy of tumours, induce accumulation of unprocessed prelamin A in patients [18]. The toxicity of unprocessed prelamin A is obviously low, as suggested by the safety and efficacy of statins in the general population. Nevertheless, studies performed in animal models and clinical data show that accumulation of mutated non-farnesylated prelamin A may cause cardiomyopathy or lipodystrophy [20,46]. At the molecular level, things are a bit more complicated. Non-farnesylated (unprocessed) prelamin A accumulates in intranuclear aggregates and recruits heterochromatin [13]. However, our studies indicate that this prelamin A form is maintained at non-toxic levels in cells through scavenging mechanisms [47]. The toxicity of farnesylated prelamin A is well established. This means that, whereas proper levels of this protein isoform are required for normal activity of nuclei, as demonstrated in adipocytes and myoblasts [24,34], an increase in farnesylated prelamin A has deleterious effects. The reasons for such a toxicity are not completely understood. However, several aspects have been defined in recent years. The present review focuses on modifications of the nuclear envelope that occur in laminopathies featuring prelamin A accumulation. The first and best recognized defect caused by farnesylated prelamin A accumulation is nuclear envelope enlargement and misshaping, which can be detected using any antilamin antibodies or using antibodies directed against integral membrane proteins [13,19]. At the ultrastructural level, nuclear envelope invaginations can be observed both in cells treated with drugs that induce farnesylated prelamin A accumulation (AFCMe) [19] and in HGPS, MADA or RD fibroblasts [43,44,48]. Since enlargement of the nuclear membrane has been also reported in nuclei overexpressing B- type lamins [49], which are farnesylated, it is obvious that the farnesyl residue mediates such an effect. However, prelamin A partners involved in nuclear envelope modification have not been identified. The second effect of farnesylated prelamin A accumulation in laminopathic cells is nuclear lamina thickening. This aspect is particularly represented in MADA [48] and HGPS nuclei [43], whereas it is rarely observed in RD cells. We hypothesize that thickening of the lamina also requires mature lamin A, which is not produced in RD. Honeycomb appearance of the nuclear envelope is observed in MADA using antiemerin [48] (Figure 1) or anti-sun2 (D. Camozzi, M.R. D Apice, E. Schena, V. Cenni, M. Columbaro, C. Capanni, N.M. Maraldi, S. Squarzoni, M. Ortolani, G. Novelli and G. Lattanzi, unpublished work) antibodies. In nuclei showing emerin defects, prelamin A shows a distinct staining pattern [48], indicating an indirect effect of mutated prelamin A. Failure of lamin A and/or prelamin A to bind emerin could cause altered nuclear envelope/lamina organization, as shown in FPLD2 cells [50]. At the ultrastructural examination, the altered organization of the nuclear membrane proteins does not correspond to nuclear envelope holes [48]. It is conceivable that farnesylated prelamin A interferes with binding of emerin or SUN2 to nuclear lamina constituents, including mature lamin A, which are necessary for proper targeting of those nuclear membrane constituents. Emerin localization is HGPS cells overlaps with progerin, but honeycomb structures are not observed, although nuclear blebs are formed [37,43]. These aspects deserve further investigation. Importantly, SUN1, another nuclear envelope constituent, is increased in HGPS nuclei featuring accumulation of both progerin and wild-type prelamin A [38]. In fact, whereas accumulation of progerin lowers SUN1 levels, as seen in Western blot analysis, accumulation of wild-type farnesylated prelamin A, as occurs in a high percentage of late-passage HGPS fibroblast nuclei, up-regulates SUN1 [38], suggesting that the 50 amino acids lacking in the progerin sequence are necessary for prelamin A-dependent up-regulation and/or anchorage of SUN1. Emery Dreifuss muscular dystrophy On the other hand, prelamin A interplay with emerin is also important for laminopathies affecting skeletal muscle. In

5 1702 Biochemical Society Transactions (2011) Volume 39, part 6 Figure 1 Emerin organization is altered in MADA nuclei Prelamin A [detected by anti-(prelamin A) Sc-6214 antibody and FITC-conjugated secondary antibody, green] forms intranuclear aggregates. Emerin [detected by anti-emerin antibody from Monosan and TRITC (tetramethylrhodamine β-isothiocyanate)-conjugated secondary antibody, red] shows a honeycomb distribution in the nuclear envelope of the enlarged MADA nucleus (arrowhead). Nuclear blebs are observed in MADA cells. Control, control fibroblast nuclei, MADA, MADA fibroblast nuclei. DAPI (4,6-diamidino-2-phenylindole) was used to stain DNA. Scale bar, 10 μm. homoeostasis and aging. Understanding how prelamin A levels are regulated in normal cells, either through modulation of the processing rate or through degradation mechanisms, will represent an important advance in lamin A research and will open new perspectives for the therapy of laminopathies. Acknowledgements The scientific work of Cristina Capanni, Marta Columbaro, Elisabetta Mattioli, Vittoria Cenni and Stefano Squarzoni is gratefully acknowledged. Manfred Wehnert, Giuseppe Novelli, Nicolas Levy, Luciano Merlini, patients and their families are greatfully acknowledged for providing cell samples used in these studies. Funding This work was supported by grants from the Associazione Italiana Progeria Sammy Basso (A.I.Pro.Sa.B.), Italy, the Italian Ministero dell Istruzione, dell Università e della Ricerca Scientifica (MIUR) Progetti di Ricerca di Interesse Nazionale (PRIN) 2008 (to G.L.), Fondo Investimenti Ricerca di Base (FIRB) MIUR 2010 and Fondazione Carisbo, Italy. fact, the absence of emerin, as occurs in cells from EDMD1 (X-linked EDMD) causes mislocalization of unprocessable non-farnesylated prelamin A, a defect rescued by emerin expression [37]. Thus emerin is, in turn, necessary for fulllength prelamin A localization. How altered localization of full-length prelamin A, which is normally processed in EDMD1, affects the disease, remains to be established. We showed recently that SUN1 levels are up-regulated by farnesylated prelamin A, which accumulates in muscle cells, where prelamin A further anchors SUN1 to the nuclear envelope [34]. Recruitment of SUN1 in myotubes is necessary for proper positioning of myonuclei [51]. Thus reduced levels of farnesylated prelamin A, as observed in EDMD2 (autosomal dominant EDMD), fail to elicit SUN1 up-regulation and causes clustering of nuclei in differentiated muscle [34]. Clustering of muscle nuclei [52] could represent a pathogenetic factor in EDMD. Conclusions Expanding our knowledge of prelamin A function under normal or pathological conditions has brought to scientists attention a major role of prelamin A nuclear membrane protein interplay. Membrane proteins so far characterized for their interaction with prelamin A are NARF, emerin, SUN1 and BAF. 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7 1704 Biochemical Society Transactions (2011) Volume 39, part 6 49 Prufert, K., Vogel, A. and Krohne, G. (2004) The lamin CxxM motif promotes nuclear membrane growth. J. Cell Sci. 117, Capanni, C., Cenni, V., Mattioli, E., Sabatelli, P., Ognibene, A., Columbaro, M., Parnaik, V.K., Wehnert, M., Maraldi, N.M., Squarzoni, S. and Lattanzi, G. (2003) Failure of lamin A/C to functionally assemble in R482L mutated familial partial lipodystrophy fibroblasts: altered intermolecular interaction with emerin and implications for gene transcription. Exp. Cell Res. 291, Lei, K., Zhang, X., Ding, X., Guo, X., Chen, M., Zhu, B., Xu, T., Zhuang, Y., Xu, R. and Han, M. (2009) SUN1 and SUN2 play critical but partially redundant roles in anchoring nuclei in skeletal muscle cells in mice. Proc. Natl. Acad. Sci. U.S.A. 106, Mejat, A., Decostre, V., Li, J., Renou, L., Kesari, A., Hantai, D., Stewart, C.L., Xiao, X., Hoffman, E., Bonne, G. and Misteli, T. (2009) Lamin A/C-mediated neuromuscular junction defects in Emery Dreifuss muscular dystrophy. J. Cell Biol. 184, Puente, X.S., Quesada, V., Osorio, F.G., Cabanillas, R., Cadinanos, J., Fraile, J.M., Ordonez, G.R., Puente, D.A., Gutierrez-Fernandez, A., Fanjul-Fernandez, M. et al. (2011) Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am. J. Hum. Genet. 88, Received 14 July 2011 doi: /bst