MAN1 and emerin have overlapping function(s) essential for chromosome segregation and cell division in Caenorhabditis elegans Jun Liu*, Kenneth K. Lee†, Miriam Segura-Totten†, Ester Neufeld‡, Katherine L. Wilson†§, and Yosef Gruenbaum‡§¶ *Department of Molecular Biology and Genetics, 439 Biotechnology Building, Cornell University, Ithaca, NY 14853; †Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and ‡Department of Genetics, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Communicated by Roger D. Kornberg, Stanford University School of Medicine, Stanford, CA, February 11, 2003 (received for review December 11, 2002) Emerin and MAN1 are LEM domain-containing integral membrane family member, named SANE (16), has not yet been localized proteins of the vertebrate nuclear envelope. The function of MAN1 definitively. The LEM domains of LAP2 and emerin mediate is unknown, whereas emerin is known to interact with nuclear their direct binding to a chromatin protein named barrier-to- lamins, barrier-to-autointegration factor (BAF), nesprin-1␣, and a autointegration factor (BAF) (reviewed in ref. 17). All LEM transcription repressor. Mutations in emerin cause X-linked reces- proteins tested (LAP2␣,LAP2␤, and emerin) also have a sive Emery–Dreifuss muscular dystrophy. Emerin and MAN1 ho- separate domain that confers direct binding to A or B type mologs are both conserved in Caenorhabditis elegans, but loss of lamins (3, 18, 19). Ce-emerin has no detectable phenotype. We therefore used C. Only three LEM proteins are conserved in Caenorhabditis elegans to test the hypothesis that Ce-MAN1 overlaps functionally elegans: Ce-MAN1 and Ce-emerin, which have a transmembrane with Ce-emerin. Supporting this model, Ce-MAN1 interacted di- domain, and lem-3, which does not (2, 13). The small number of rectly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for LEM proteins in C. elegans and the presence of conserved genes its nuclear envelope localization. Interestingly, RNA interference- encoding BAF and one B type lamin facilitate the study of their mediated removal of Ϸ90% of Ce-MAN1 was lethal to Ϸ15% of functions and interactions in vivo. Reducing the level of either embryos. However, in the absence of Ce-emerin, Ϸ90% reduction lamin or BAF homologs in C. elegans causes abnormal nuclear of Ce-MAN1 was lethal to all embryos by the 100-cell stage, with structure, catastrophic exit from mitosis (chromosome misseg- a phenotype involving repeated cycles of anaphase chromosome regation and anaphase chromosome bridging), and early embry- bridging and cytokinesis [‘‘cell untimely torn’’ (cut) phenotype]. onic lethality (20, 21). In contrast, elimination of emerin has no Immunostaining showed that the anaphase-bridged chromatin detectable phenotype in C. elegans (22). In humans, emerin is specifically retained a mitosis-specific phosphohistone H3 epitope expressed in nearly all tissues, but the null phenotype is restricted and failed to recruit detectable Ce-lamin or Ce-BAF. These findings to skeletal muscles, cardiac function, and major tendons (23), show that LEM domain proteins are essential for cell division and suggesting that the unaffected tissues may express protein(s) that that Ce-emerin and Ce-MAN1 share at least one and possibly overlap functionally with emerin. multiple overlapping functions, which may be relevant to Emery– MAN1 and emerin are the only integral membrane LEM Dreifuss muscular dystrophy. proteins in C. elegans, with similar biochemical extraction prop- erties and cell cycle dynamics (13). We therefore used C. elegans amins form stable networks of filaments located near the to determine whether MAN1 has any functional overlap with Linner membrane of the nucleus and in the nuclear interior emerin. Our results strongly support the ‘‘overlap’’ hypothesis. (reviewed in ref. 1). Lamins interact with diverse partners, Like Ce-emerin, Ce-MAN1 binds directly to Ce-lamin and including membrane proteins emerin and LAP2␤ as well as Ce-BAF in vitro. Although Ce-MAN1 itself was revealed to be histones, transcription factors, and other proteins (reviewed in essential, we found that partial reduction of Ce-MAN1 was lethal ref. 1). This structural network of lamins and associated proteins in cells that completely lacked emerin, demonstrating that is collectively termed the nuclear ‘‘lamina.’’ In metazoan cells, emerin and MAN1 have overlapping functions essential for cell the nuclear lamina is essential to maintain the shape and division. integrity of the nucleus and for DNA replication, RNA tran- Materials and Methods scription, chromatin organization, cell cycle regulation, cell Antibodies and Indirect Immunofluorescence Staining of C. elegans. development and differentiation, nuclear migration, and apo- C. elegans (N2) embryos, larvae, and adults were fixed and ptosis (reviewed in refs. 1–4). prepared for indirect immunofluorescence staining as described Specific mutations in nuclear lamina genes cause a wide range (22) by using the following polyclonal antisera: rat anti-Ce- of heritable human diseases, termed laminopathies (reviewed in MAN1 serum 3597, rat anti-Ce-emerin serum 3598, mouse refs. 5–8). The best-studied laminopathy is Emery–Dreifuss anti-Ce-emerin serum 3272, and rat anti-UNC-84 serum 3595, muscular dystrophy (EDMD), characterized by early contrac- which were used at a 1:100 dilution (13, 22, 24). Affinity-purified tures of the Achilles, elbow, and neck tendons, progressive rabbit anti-Ce-lamin antibodies were used at a 1:400 dilution muscle wasting, and conduction defects in the heart (9, 10). The (20). Rat serum 3778 against Ce-BAF was generated through X linked form of EDMD is caused by loss of emerin (11), an Covance Research Products (Denver, PA) by using a keyhole integral protein of the nuclear inner membrane. The autosomal limpet hemocyanin-conjugated synthetic peptide (Boston Bio- dominant form of EDMD is caused by missense (and other) Molecules, Woburn, MA; see ref. 13) comprising Ce-BAF mutations in LMNA, the gene encoding A type lamins (7). Thus, EDMD can result from relatively subtle changes in lamin A filaments or from the loss of a specific protein (emerin) that Abbreviations: EDMD, Emery–Dreifuss muscular dystrophy; BAF, barrier-to-autointegra- binds lamin A. tion factor; dsRNA, double-stranded RNA; RNAi, RNA-mediated interference; DAPI, 4Ј,6- Emerin contains a LEM domain, the defining Ϸ40-residue diamidino-2-phenylindole. motif shared by a family of nuclear proteins that includes LAP2, §K.L.W. and Y.G. contributed equally to this work. emerin, MAN1, lem-3, and otefin (8, 12–15). A newly identified ¶To whom correspondence should be addressed. E-mail: [email protected]. 4598–4603 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0730821100 Downloaded by guest on September 25, 2021 residues 28–41 plus one cysteine for conjugation (PTYGTKLT- DAGFDKC). Rat anti-BAF was used at a 1:100 dilution. Anti- bodies specific for phosphohistone H3 were from Upstate Group (Waltham, MA). mAb mAb414, which recognizes a subset of nucleoporins in C. elegans (13), was purchased from Babco (Richmond, CA). All secondary antibodies were purchased from The Jackson Laboratory. Double labeling of Ce-MAN1 and Ce-lamin in C. elegans embryos by using immunogold TEM was performed exactly as described (25), with rat anti-Ce-MAN1 serum 3597 diluted 1:30 and rabbit polyclonal anti-Ce-lamin serum diluted 1:10. RNA-Mediated Interference (RNAi) Experiments. Double-stranded RNA (dsRNA) corresponding to Ce-emerin and Ce-lamin were synthesized as described (refs. 22 and 20, respectively). dsRNA corresponding to Ce-MAN1 residues 147–385 (see Fig. 1) was synthesized by using plasmid pJKL503.1. The dsRNA (0.1–1 ␮g͞␮l) was injected into both gonads of N2 hermaphrodites as described (20, 22, 24). From 12 to 60 h after injection, adults and embryos were either examined for viability as described (20) or fixed and prepared for indirect immunofluorescence staining as described (13). Ce-MAN1 Expression, Synthesis of [35S]Cysteine͞Methionine-Labeled Proteins, and Blot Overlay Assays. Blot overlay assays were done essentially as described (19). All constructs were verified by sequencing. Each Ce-MAN1 construct was transformed into Escherichia coli strain BL21 (DE3). Transformed cells contain- ing each plasmid were grown to an OD600 of 0.6, and Ce-MAN1 expression was induced by 0.4 mM isopropyl ␤-D-thiogalactoside for 4 h. Cells were pelleted 5 min at 14,000 ϫ g and resuspended in 2ϫ SDS sample buffer. Proteins from unfractionated bacterial lysates were separated on 10% SDS͞PAGE gels, transferred to nitrocellulose membranes (Schleicher & Schuell), and blocked for1hinPBScontaining0.1% Tween 20 5% nonfat dry milk. The expressed proteins had the expected mobility in SDS͞PAGE assays (data not shown). The T7 promoters on expression vectors pET7a (for Ce-lamin) and pET15b (for Ce-BAF) were used to Fig. 1. Localization of lamin and MAN1 in C. elegans and MAN1-binding 35 ͞ drive synthesis of [ S]cysteine methionine-labeled Ce-lamin interactions in vitro.(A) Indirect immunofluorescence double-staining of L1 and Ce-BAF proteins by using the TNT Quick Coupled Tran- larvae for endogenous lamin and MAN1; anterior is upwards. (Bar ϭ 10 ␮m.) scription͞Translation System (Promega), following the manu- (B) Immunogold TEM colocalization of lamin and MAN1 on chromatin in facturer’s protocol. Proteins were transcribed͞translated indi- detergent-extracted nuclei. C. elegans embryos were treated with 0.5% Triton vidually for 90 min at 30°C. Protein lysates from bacteria X-100 to remove nuclear membranes and then immunogold-labeled with expressing recombinant Ce-MAN1 polypeptides were resolved rabbit antibodies against the rod and tail domains of lamin, plus rat serum ͞ 3597 against the C terminus of MAN1. Secondary antibodies were conjugated by SDS PAGE, transferred to PVDF, and washed twice in blot ϭ rinse buffer (10 mM Tris⅐HCl, pH 7.4͞150 mM NaCl͞1mM with 12 nm gold (lamin) or 6 nm gold (MAN1), respectively.