Margalit A, Liu J, Fridkin A, Wilson KL, Gruenbaum Y. A lamin-dependent pathway that regulates nuclear organization, cell cycle progression and germ cell development. Novartis Found Symp. 2005;264 :231-40; discussion 240-5.Abstract
The C. elegans genome encodes a single lamin protein (Ce-lamin), three LEM domain proteins (Ce-emerin, Ce-MAN1 and LEM-3) and a single BAF protein (Ce-BAF). Down-regulation of Ce-lamin causes embryonic lethality. Abnormalities include rapid changes in nuclear morphology during interphase, inability of cells to complete mitosis, abnormal condensation of chromatin, clustering of nuclear pore complexes (NPCs), and missing or abnormal germ cells. Ce-emerin and Ce-MAN1 are both embedded in the inner nuclear membrane, and both bind Ce-lamin and Ce-BAF; in addition, both require Ce-lamin for their localization. Mutations in human emerin cause X-linked recessive Emery-Dreifuss muscular dystrophy. In C. elegans, loss of Ce-emerin alone has no detectable phenotype, while loss of 90% Ce-MAN1 causes approximately 15% embryonic lethality. However in worms that lack Ce-emerin, a approximately 90% reduction of Ce-MAN1 is lethal to all embryos by the 100-cell stage, with a phenotype involving chromatin condensation and repeated cycles of anaphase chromosome bridging and cytokinesis. The anaphase-bridged chromatin retained a mitosis-specific phosphohistone H3 epitope, and failed to recruit detectable Ce-lamin or Ce-BAF. Down-regulation of Ce-BAF showed similar phenotypes. These findings suggest that lamin, LEM-domain proteins and BAF are part of a lamina network essential for chromatin organization and cell division, and that Ce-emerin and Ce-MAN1 share at least one and possibly multiple overlapping functions, which may be relevant to Emery-Dreifuss muscular dystrophy.
Goldman RD, Shumaker DK, Erdos MR, Eriksson M, Goldman AE, Gordon LB, Gruenbaum Y, Khuon S, Mendez M, Varga R, et al. Accumulation of mutant lamin A causes progressive changes in nuclear architecture in Hutchinson-Gilford progeria syndrome. Proc Natl Acad Sci U S A. 2004;101 (24) :8963-8.Abstract
Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging disorder, commonly caused by a point mutation in the lamin A gene that results in a protein lacking 50 aa near the C terminus, denoted LADelta50. Here we show by light and electron microscopy that HGPS is associated with significant changes in nuclear shape, including lobulation of the nuclear envelope, thickening of the nuclear lamina, loss of peripheral heterochromatin, and clustering of nuclear pores. These structural defects worsen as HGPS cells age in culture, and their severity correlates with an apparent increase in LADelta50. Introduction of LADelta50 into normal cells by transfection or protein injection induces the same changes. We hypothesize that these alterations in nuclear structure are due to a concentration-dependent dominant-negative effect of LADelta50, leading to the disruption of lamin-related functions ranging from the maintenance of nuclear shape to regulation of gene expression and DNA replication.
Fridkin A, Karabinos A, Gruenbaum Y. Intermediate filaments in Caenorhabditis elegans. Methods Cell Biol. 2004;78 :703-18.
Fridkin A, Mills E, Margalit A, Neufeld E, Lee KK, Feinstein N, Cohen M, Wilson KL, Gruenbaum Y. Matefin, a Caenorhabditis elegans germ line-specific SUN-domain nuclear membrane protein, is essential for early embryonic and germ cell development. Proc Natl Acad Sci U S A. 2004;101 (18) :6987-92.Abstract
Caenorhabditis elegans mtf-1 encodes matefin, which has a predicted SUN domain, a coiled-coil region, an anti-erbB-2 IgG domain, and two hydrophobic regions. We show that matefin is a nuclear membrane protein that colocalizes in vivo with Ce-lamin, the single nuclear lamin protein in C. elegans, and binds Ce-lamin in vitro but does not require Ce-lamin for its localization. Matefin is detected in all embryonic cells until midembryogenesis and thereafter only in germ-line cells. Embryonic matefin is maternally deposited, and matefin is the first nuclear membrane protein known to have germ line-restricted expression. Animals homozygous for an mtf-1 deletion allele show that matefin is essential for germ line maturation and survival. However, matefin is also required for embryogenesis because mtf-1 (RNAi) embryos die around the approximately 300-cell stage with defects in nuclear structure, DNA content, and chromatin morphology. Down-regulating matefin in mes-3 animals only slightly enhances embryonic lethality, and elimination of UNC-84, the only other SUN-domain gene in C. elegans, has no affect on mtf-1 (RNAi) animals. Thus, mtf-1 mediates a previously uncharacterized pathway(s) required for embryogenesis as well as germ line proliferation or survival.
Mattout-Drubezki A, Gruenbaum Y. Dynamic interactions of nuclear lamina proteins with chromatin and transcriptional machinery. Cell Mol Life Sci. 2003;60 (10) :2053-63.Abstract
The nuclear lamina is a filamentous nuclear structure intimately connected to the inner nuclear membrane. It is composed of lamins, which are also present in the nuclear interior, and lamin-associated proteins. The nuclear lamina is involved directly or indirectly in many nuclear activities, including DNA replication and transcription, nuclear and chromatin organization, cell cycle regulation, cell development and differentiation, nuclear migration and apoptosis. Mutations in nuclear lamina genes cause a wide range of heritable human diseases, the molecular mechanisms for which are not well understood. This review describes our current knowledge of interactions between nuclear lamina proteins and chromatin, chromatin-remodeling factors, specific transcription factors and RNA polymerase II transcription machinery. Recent studies provide new insights into the nature and regulation of these interactions and suggest additional roles for the nuclear lamina.
Liu J, Lee KK, Segura-Totten M, Neufeld E, Wilson KL, Gruenbaum Y. MAN1 and emerin have overlapping function(s) essential for chromosome segregation and cell division in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2003;100 (8) :4598-603.Abstract
Emerin and MAN1 are LEM domain-containing integral membrane proteins of the vertebrate nuclear envelope. The function of MAN1 is unknown, whereas emerin is known to interact with nuclear lamins, barrier-to-autointegration factor (BAF), nesprin-1 alpha, and a transcription repressor. Mutations in emerin cause X-linked recessive Emery-Dreifuss muscular dystrophy. Emerin and MAN1 homologs are both conserved in Caenorhabditis elegans, but loss of Ce-emerin has no detectable phenotype. We therefore used C. elegans to test the hypothesis that Ce-MAN1 overlaps functionally with Ce-emerin. Supporting this model, Ce-MAN1 interacted directly with Ce-lamin and Ce-BAF in vitro and required Ce-lamin for its nuclear envelope localization. Interestingly, RNA interference-mediated removal of approximately 90% of Ce-MAN1 was lethal to approximately 15% of embryos. However, in the absence of Ce-emerin, approximately 90% reduction of Ce-MAN1 was lethal to all embryos by the 100-cell stage, with a phenotype involving repeated cycles of anaphase chromosome bridging and cytokinesis ["cell untimely torn" (cut) phenotype]. Immunostaining showed that the anaphase-bridged chromatin specifically retained a mitosis-specific phosphohistone H3 epitope and failed to recruit detectable Ce-lamin or Ce-BAF. These findings show that LEM domain proteins are essential for cell division and that Ce-emerin and Ce-MAN1 share at least one and possibly multiple overlapping functions, which may be relevant to Emery-Dreifuss muscular dystrophy.
Gruenbaum Y, Goldman RD, Meyuhas R, Mills E, Margalit A, Fridkin A, Dayani Y, Prokocimer M, Enosh A. The nuclear lamina and its functions in the nucleus. Int Rev Cytol. 2003;226 :1-62.Abstract
The nuclear lamina is a structure near the inner nuclear membrane and the peripheral chromatin. It is composed of lamins, which are also present in the nuclear interior, and lamin-associated proteins. The increasing number of proteins that interact with lamins and the compound interactions between these proteins and chromatin-associated proteins make the nuclear lamina a highly complex but also a very exciting structure. The nuclear lamina is an essential component of metazoan cells. It is involved in most nuclear activities including DNA replication, RNA transcription, nuclear and chromatin organization, cell cycle regulation, cell development and differentiation, nuclear migration, and apoptosis. Specific mutations in nuclear lamina genes cause a wide range of heritable human diseases. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Genetic analyses in Caenorhabditis elegans, Drosophila, and mice show new insights into the functions of the nuclear lamina, and recent structural analyses have begun to unravel the molecular structure and assembly of lamins and their associated proteins.
Cohen M, Feinstein N, Wilson KL, Gruenbaum Y. Nuclear pore protein gp210 is essential for viability in HeLa cells and Caenorhabditis elegans. Mol Biol Cell. 2003;14 (10) :4230-7.Abstract
Gp210 is an evolutionarily conserved membrane protein of the nuclear pore complex (NPC). We studied the phenotypes produced by RNAi-induced downregulation of gp210 in both human (HeLa) cells and Caenorhabditis elegans embryos. HeLa cell viability requires Gp210 activity. The dying cells accumulated clustered NPCs and aberrant membrane structures at the nuclear envelope, suggesting that gp210 is required directly or indirectly for nuclear pore formation and dilation as well as the anchoring or structural integrity of mature NPCs. Essential roles for gp210 were confirmed in C. elegans, where RNAi-induced reduction of gp210 caused embryonic lethality. The nuclear envelopes of embryos with reduced gp210 also had aberrant nuclear membrane structures and clustered NPCs, confirming that gp210 plays critical roles at the nuclear membrane through mechanisms that are conserved from nematodes to humans.
Laronne A, Rotkopf S, Hellman A, Gruenbaum Y, Porter ACG, Brandeis M. Synchronization of interphase events depends neither on mitosis nor on cdk1. Mol Biol Cell. 2003;14 (9) :3730-40.Abstract
Human HT2-19 cells with a conditional cdk1 mutation stop dividing upon cdk1 inactivation and undergo multiple rounds of endoreplication. We show herein that major cell cycle events remain synchronized in these endoreplicating cells. DNA replication alternates with gap phases and cell cycle-specific cyclin E expression is maintained. Centrosomes duplicate in synchrony with chromosome replication, giving rise to polyploid cells with multiple centrosomes. Centrosome migration, a typical prophase event, also takes place in endoreplicating cells. The timing of these events is unaffected by cdk1 inactivation compared with normally dividing cells. Nuclear lamina breakdown, in contrast, previously shown to be dependent on cdk1, does not take place in endoreplicating HT2-19 cells. Moreover, breakdown of all other major components of the nuclear lamina, like the inner nuclear membrane proteins and nuclear pore complexes, seems also to depend on cdk1. Interestingly, the APC/C ubiquitin ligase is activated in these endoreplicating cells by fzr but not by fzy. The oscillations of interphase events are thus independent of cdk1 and of mitosis but may depend on APC/Cfzr activity.
Gruenbaum Y, Lee KK, Liu J, Cohen M, Wilson KL. The expression, lamin-dependent localization and RNAi depletion phenotype for emerin in C. elegans. J Cell Sci. 2002;115 (Pt 5) :923-9.Abstract
Emerin belongs to the LEM-domain family of nuclear membrane proteins, which are conserved in metazoans from C. elegans to humans. Loss of emerin in humans causes the X-linked form of Emery-Dreifuss muscular dystrophy (EDMD), but the disease mechanism is not understood. We have begun to address the function of emerin in C. elegans, a genetically tractable nematode. The emerin gene (emr-1) is conserved in C. elegans. We detect Ce-emerin protein in the nuclear envelopes of all cell types except sperm, and find that Ce-emerin co-immunoprecipitates with Ce-lamin from embryo lysates. We show for the first time in any organism that nuclear lamins are essential for the nuclear envelope localization of emerin during early development. We further show that four other types of nuclear envelope proteins, including fellow LEM-domain protein Ce-MAN1, as well as Ce-lamin, UNC-84 and nucleoporins do not depend on Ce-emerin for their localization. This result suggests that emerin is not essential to organize or localize the only lamin (B-type) expressed in C. elegans. We also analyzed the RNAi phenotype resulting from the loss of emerin function in C. elegans under laboratory growth conditions, and found no detectable phenotype throughout development. We propose that C. elegans is an appropriate system in which to study the molecular mechanisms of emerin function in vivo.
Tzur YB, Hersh BM, Horvitz RH, Gruenbaum Y. Fate of the nuclear lamina during Caenorhabditis elegans apoptosis. J Struct Biol. 2002;137 (1-2) :146-53.Abstract
Invertebrates and in Drosophila, lamins and lamin-associated proteins are primary targets for cleavage by caspases. Eliminating mammalian lamins causes apoptosis, whereas expressing mutant lamins that cannot be cleaved by caspase-6 delay apoptosis. Caenorhabditis elegans has a single lamin protein, Ce-lamin, and a caspase, CED-3, that is responsible for most if not all somatic apoptosis. In this study we show that in C. elegans embryos induced to undergo apoptosis Ce-lamin is degraded surprisingly late. In such embryos CED-4 translocated to the nuclear envelope but the cytological localization of Ce-lamin remained similar to that in wild-type embryos. TUNEL labeling indicated that Ce-lamin was degraded only after DNA is fragmented. Ce-lamin, Ce-emerin, or Ce-MAN1 were not cleaved by recombinant CED-3, showing that these lamina proteins are not substrates for CED-3 cleavage. These results suggest that lamin cleavage probably is not essential for apoptosis in C. elegans.
Kakhlon O, Gruenbaum Y, Cabantchik IZ. Ferritin expression modulates cell cycle dynamics and cell responsiveness to H-ras-induced growth via expansion of the labile iron pool. Biochem J. 2002;363 (Pt 3) :431-6.Abstract
Repression or overexpression of ferritin accelerated or retarded cell cycling respectively, via changes in the cellular labile iron pool (LIP). A rise in LIP is caused by ferritin repression enhanced growth, induced by H-ras, and reverted growth arrest is induced by dominant negative H-ras. The studies indicate that repression of ferritin expression provides a mechanism by which certain oncogenes lead to cell growth stimulation.
Tour E, Pillemer G, Gruenbaum Y, Fainsod A. Gbx2 interacts with Otx2 and patterns the anterior-posterior axis during gastrulation in Xenopus. Mech Dev. 2002;112 (1-2) :141-51.Abstract
Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither, Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homologue, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, we demonstrate that, during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, we show that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. We therefore suggest that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as at midgastrulation.
Lee KK, Starr D, Cohen M, Liu J, Han M, Wilson KL, Gruenbaum Y. Lamin-dependent localization of UNC-84, a protein required for nuclear migration in Caenorhabditis elegans. Mol Biol Cell. 2002;13 (3) :892-901.Abstract
Mutations in the Caenorhabditis elegans unc-84 gene cause defects in nuclear migration and anchoring. We show that endogenous UNC-84 protein colocalizes with Ce-lamin at the nuclear envelope and that the envelope localization of UNC-84 requires Ce-lamin. We also show that during mitosis, UNC-84 remains at the nuclear periphery until late anaphase, similar to known inner nuclear membrane proteins. UNC-84 protein is first detected at the 26-cell stage and thereafter is present in most cells during development and in adults. UNC-84 is properly expressed in unc-83 and anc-1 lines, which have phenotypes similar to unc-84, suggesting that neither the expression nor nuclear envelope localization of UNC-84 depends on UNC-83 or ANC-1 proteins. The envelope localization of Ce-lamin, Ce-emerin, Ce-MAN1, and nucleoporins are unaffected by the loss of UNC-84. UNC-84 is not required for centrosome attachment to the nucleus because centrosomes are localized normally in unc-84 hyp7 cells despite a nuclear migration defect. Models for UNC-84 localization are discussed.
Goldman RD, Gruenbaum Y, Moir RD, Shumaker DK, Spann TP. Nuclear lamins: building blocks of nuclear architecture. Genes Dev. 2002;16 (5) :533-47.
Tour E, Pillemer G, Gruenbaum Y, Fainsod A. Otx2 can activate the isthmic organizer genetic network in the Xenopus embryo. Mech Dev. 2002;110 (1-2) :3-13.Abstract
Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.
Kakhlon O, Gruenbaum Y, Cabantchik ZI. Repression of ferritin expression modulates cell responsiveness to H-ras-induced growth. Biochem Soc Trans. 2002;30 (4) :777-80.Abstract
We assessed the role of the cell labile iron pool in mediating oncogene-induced cell proliferation via repression of ferritin expression. When HEK-293 cells, engineered to inducibly express either active (+) or dominant-negative (-) forms of the H-ras oncogene, were treated with antisense nucleotides to ferritin subunits they displayed (a) decreased ferritin levels, (b) increased labile iron pool and either (c) faster growth in cells induced to express H-Ras (+) or (d) recovery from growth retardation in dominant-negative H-Ras-induced cells. Our studies support the view that the role of down-modulation of ferritin expression by some oncogene-evoked proliferation proceeds via expansion of the cellular labile iron pool.
Cohen M, Tzur YB, Neufeld E, Feinstein N, Delannoy MR, Wilson KL, Gruenbaum Y. Transmission electron microscope studies of the nuclear envelope in Caenorhabditis elegans embryos. J Struct Biol. 2002;140 (1-3) :232-40.Abstract
Nuclear membranes and nuclear pore complexes (NPCs) are conserved in both animals and plants. However, the lamina composition and the dimensions of NPCs vary between plants, yeast, and vertebrates. In this study, we established a protocol that preserves the structure of Caenorhabditis elegans embryonic cells for high-resolution studies with thin-section transmission electron microscopy (TEM). We show that the NPCs are bigger in C. elegans embryos than in yeast, with dimensions similar to those in higher eukaryotes. We also localized the C. elegans nuclear envelope proteins Ce-lamin and Ce-emerin by pre-embedding gold labeling immunoelectron microscopy. Both proteins are present at or near the inner nuclear membrane. A fraction of Ce-lamin, but not Ce-emerin, is present in the nuclear interior. Removing the nuclear membranes leaves both Ce-lamin and Ce-emerin associated with the chromatin. Eliminating the single lamin protein caused cell death as visualized by characteristic changes in nuclear architecture including condensation of chromatin, clustering of NPCs, membrane blebbing, and the presence of vesicles inside the nucleus. Taken together, these results show evolutionarily conserved protein localization, interactions, and functions of the C. elegans nuclear envelope.
Wilson KL, Benavente R, Burke B, Craigie R, Foisner R, Furukawa K, Gerace L, Goldman RD, Gruenbaum Y, Harris C, et al. Problems with LAP nomenclature. Nat Cell Biol. 2001;3 (4) :E90.
Kakhlon O, Gruenbaum Y, Cabantchik ZI. Repression of ferritin expression increases the labile iron pool, oxidative stress, and short-term growth of human erythroleukemia cells. Blood. 2001;97 (9) :2863-71.Abstract
The role of ferritin expression on the labile iron pool of cells and its implications for the control of cell proliferation were assessed. Antisense oligodeoxynucleotides were used as tools for modulating the expression of heavy and light ferritin subunits of K562 cells. mRNA and protein levels of each subunit were markedly reduced by 2-day treatment with antisense probes against the respective subunit. Although the combined action of antisense probes against both subunits reduced their protein expression, antisense repression of one subunit led to an increased protein expression of the other. Antisense treatment led to a rise in the steady-state labile iron pool, a rise in the production of reactive oxygen species after pro-oxidative challenges and in protein oxidation, and the down-regulation of transferrin receptors. When compared to the repression of individual subunits, co-repression of each subunit evoked a more than additive increase in the labile iron pool and the extent of protein oxidation. These treatments had no detectable effects on the long-term growth of cells. However, repression of ferritin synthesis facilitated the renewal of growth and the proliferation of cells pre-arrested at the G(1)/S phase. Renewed cell growth was significantly less dependent on external iron supply when ferritin synthesis was repressed and its degradation inhibited by lysosomal antiproteases. This study provides experimental evidence that links the effect of ferritin repression on growth stimulation to the expansion of the labile iron pool.