Increased solubility of lamins and redistribution of lamin C in X-linked Emery–Dreifuss muscular dystrophy fibroblasts Ewa Markiewicz, a Rachel Venables, a Mauricio-Alvarez-Reyes, a Roy Quinlan, a Margareth Dorobek, b Irena Hausmanowa-Petrucewicz, b and Christopher Hutchison a, * a SchoolofBiologicalandBiomedicalSciences,UniversityofDurham,SouthRoad,DurhamDH13LE,UK b NeuromuscularUnit,MedicalResearchCentre,PolishAcademyofSciences,1aBanachaStr.,Warsaw02-097,Poland Received 18 June 2002, and in revised form 3 October 2002 Abstract Emery–Dreifussmusculardystrophy(EDMD)iscausedbymutationsinthegeneencodingthenuclearmembraneproteinemerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fi- broblastsfromX-EDMDpatientscomparedtonormalindividuals.Laminandemerinsolubilitywasmildlyincreasedinfibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopystudiesusinglaminA-andlaminC-specificantibodiesconfirmedthatlaminCbutnotlaminAbecameredistributedto the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2a was also mislocalized in X-EDMD fibroblasts. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Emery–Driefuss muscular dystrophy; Lamin A/C; Emerin; Lamina-associated polypeptides; Nuclear envelope; Nuclear lamina 1. Introduction Laminopathy refers to a group of diseases caused by underlying mutations in the genes encoding lamins A and C or in the nuclear membrane protein emerin (re- viewed by Cohen et al., 2001; Hutchison et al., 2001; MorrisandManilal,1999).Positionalcloningfirstledto the realization that mutations in the gene encoding la- minA/C(LMNA)orinthegeneencodingemerin(STA) give rise to Emery–Dreifuss muscular dystrophy (EDMD) (Bione et al., 1994; Bonne et al., 1999). The three forms of this disease autosomal dominant or re- cessive (AD-EDMD and AR-EDMD, both caused by mutations in LMNA) and X-linked (X-EDMD caused by mutations in STA) display similar phenotypes that are atypical of other muscular dystrophies (e.g., DuchenneandBeckerreviewedbyEmery,2000;Morris, 2000). The disease starts with stiffness of joints in the neck, elbows, and Achilles tendons. Muscle weakness may progress only very slowly and the life-threatening condition associated with this disease is a conduction block (which can lead to sudden death) and progressive cardiomyopathy (Emery, 1989). Three other dominant diseases are caused by mutations in LMNA. These in- clude dilated cardiomyopathy with conduction system disease (DCM-CD) (Brodsky et al., 2000; Fatkin et al., 2000), Dunnigan-type familial partial lipodystrophy (FPLD)(CaoandHegele,2000;Shackletonetal.,2000), and a subset of limb girdle muscular dystrophy type 1B (LGMD1B;Muchiretal.,2000).Inaddition,autosomal recessive diseases with mutations in LMNA have now been reported including Charcot–Marie–Tooth disorder type2(CMT-2;DeSandra-Giovannolietal.,2002)and mandibuloacral dysplasia (MAD; Novelli et al., 2002). For a number of patients with EDMD-like phenotypes, Journal of Structural Biology 140 (2002) 241–253 www.academicpress.com Journal of Structural Biology * Corresponding author. Fax: +44-191-374-4127. E-mailaddress: c.j.hutchison@durham.ac.uk (C. Hutchison). 1047-8477/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII:S1047-8477(02)00573-7