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Structural organization and biological roles of the nuclear lamina

Dr. Yosef, Gruenbaum, et. al.

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Gene Ther Mol Biol Vol 1, 529-542. March, 1998.


Structural organization and biological roles of the nuclear lamina


Amnon Harel1, Michal Goldberg, Nirit Ulitzur2 and Yosef Gruenbaum

Department of Genetics, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.

Correspondence: Yosef Gruenbaum, Tel: +972-2-6585995, Fax: +972-2-5633066 or +972-2-6586975, E-mail: gru@vms.huji.ac.il

1. Present address: Department of Biology, University of California, San Diego, San Diego CA.

2. Present address: Department of Biochemistry, Stanford Medical School, Stanford CA.



The nuclear lamina is a protein meshwork that lies on the nucleoplasmic side of the nuclear envelope and is associated with the peripheral chromatin. It is involved in several biological activities including: the mitotic disassembly and reassembly of the nuclear envelope, determination of the size and shape of the nucleus, higher order chromatin organization, cell differentiation, and apoptosis. Lamins are the major proteins of the nuclear lamina. They are type V intermediate filaments and, like all intermediate filaments, they form filamentous structures. Lamins can interact in vitro with specific DNA sequences, with chromosomal proteins and with several proteins of the inner nuclear membrane, including otefin, LBR, LAP1 and LAP2. In this paper we show that Drosophila lamin Dm0 and otefin proteins are required for the assembly of the Drosophila nuclear envelope. We also demonstrate that the lack of lamin Dm0 activity causes the dissociation of peripheral chromatin from the nuclear envelope, accumulation of annulate lamellae and lethality. In addition, we show that the carboxy (tail) domain of lamin Dm0 can interact in vitro with chromosomes and the central (rod) domain of lamin Dm0 is essential and sufficient for the in vitro assembly of lamin Dm0 into filamentous structures. These results are discussed in relationship to the biological roles of the nuclear lamina.




I. Introduction

In eukaryotic cells, DNA replication and RNA processing occur in the nucleus, while protein synthesis occurs in the cytoplasm. These activities are physically separated by the nuclear envelope. The nuclear envelope is a complex structure composed of outer and inner lipid bilayer membranes. The two membranes are separated by a 20-40 nm perinuclear space and are connected at the nuclear pore complexes, which are passageways for transport of macromolecules between the nucleoplasm and the cytoplasm (reviewed in Davis, 1995;  Gorlich and Mattaj, 1996). Underlying the inner nuclear membrane there is a proteinaceous meshwork of intermediate filaments termed the nuclear lamina (Fig. 1A; reviewed in Hutchison et al., 1994;  Moir et al., 1995).


A. Proteins of the inner nuclear membrane and nuclear lamina

Several components of the inner nuclear membrane and the lamina have been identified. These include the integral membrane proteins (IMPs): LBR (Worman et al., 1990), LAP1, LAP2 (Furukawa et al., 1995;  Harris  et al., 1994;  Martin  et al., 1995), p34 (Simos and Georgatos, 1994) and p18 (Simos  et al., 1996), and the peripheral proteins: nuclear lamins (Fisher  et al., 1986;  McKeon, 1991), otefin (Harel  et al., 1989;  Padan  et al., 1990) and YA (Lopez  et al., 1994;  Lopez and Wolfner, 1997). The existing experimental data suggests that lamins can interact with LBR, LAP1, LAP2, otefin and YA (Foisner and Gerace, 1993;  Goldberg  et al., 1997;  Worman  et al., 1988) . p18 and p34 are associated with LBR and p18 is distributed equally between the inner and the outer nuclear membranes (Simos and Georgatos, 1994). The data on the peripheral proteins indicates that otefin is closely associated with the inner nuclear membrane, lamin can associate with both the inner nuclear membrane and the peripheral chromatin, and YA is associated with the peripheral chromatin (Fig. 1A; Goldberg et al., 1997). These proteins are present in the insoluble NMPCL (nu-


Figure 1. (A) Schematic view of the structural organization of nuclear envelope. OMN, outer nuclear membrane; INM, inner nuclear membrane; NPC, nuclear pore complex. (B) Schematic view and the putative roles of different regions in lamin Dm0 and otefin. The numbers are of amino acids positions in these proteins.

clear matrix-pore-complex-lamina) fraction, after salt and Triton X-100 extraction.

LBR (lamin B receptor) was isolated by its ability to bind in a saturable and specific fashion to lamin B. Binding of lamin B to LBR is affected by its phosphorylation. LBR is a 58 kDa protein containing a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments (Worman et al., 1990). Its sequence shows high homology to the yeast sterol C-14 reductase (Gerace and Foisner, 1994). Both the first transmembrane domain  (Smith and Blobel, 1993) and the amino-terminal domain of LBR (Soullam and Worman, 1993;  Soullam and Worman, 1995) mediate the targeting of LBR to the inner nuclear membrane. The highly charged amino-terminal domain of LBR can also direct cytosolic proteins to the nucleus and type II integral membrane proteins to the inner nuclear membrane in transfected COS-7 cells (Smith and Blobel, 1993). LBR is phosphorylated in a cell cycle-dependent manner on serine residues in interphase and on serine and threonine residues in mitosis. Its phosphorylation is mediated by p34cdc2-kinase and by an unidentified kinase that resides in the nuclear envelope and associates with LBR in vivo (Nikolakaki  et al., 1997;  Simos and Georgatos, 1992). LBR can interact with several proteins including p34 and p18 (Simos and Georgatos, 1994), lamin B (Worman et al., 1988) and with the human homologue of the Drosophila heterochromatin associated protein HP1 (Ye and Worman, 1996).

LAP1A-C - (Lamina-associated polypeptides 1A-C) is a group of three related integral membrane proteins of the inner nuclear membrane that are recognized by monoclonal antibody RL13. LAP1 proteins can bind both type A and B lamins (Foisner and Gerace, 1993). Cloning of LAP1C revealed that it is a type II integral membrane protein with a single membrane-spanning region and a hydrophilic amino terminal domain that is exposed to the nucleoplasm (Martin et al., 1995). The different LAP1 isotypes are differentially expressed during development and appear to bind lamins with different affinities (Martin et al., 1995).

LAP2 (Lamina-associated polypeptide 2 - also named thymopoietin) is a type II integral membrane protein of the inner nuclear membrane. The  LAP2 gene is alternatively spliced to give rise to at least 5 different products (Theodor  et al., 1997). The most abundant products: LAP2a, LAP2b, and LAP2g (75 kDa, 51 kDa and 39 kDa, respectively) are present in most cell types. LAP2a is present diffusely throughout the nucleus, while LAP2b and LAP2g are confined to the inner nuclear membrane (Harris  et al., 1995). LAP2b contains a large hydrophilic domain with several potential cdc2 kinase phosphorylation sites and a single putative membrane-spanning sequence close to its carboxy terminus. The amino-terminal domain of this protein is hydrophilic and is exposed to the nucleoplasm. LAP2 can bind directly to both lamin B and chromosomes and associates with chromosomes at the same time that lamins begin to reassemble around them (Foisner and Gerace, 1993;  Yang  et al., 1997). The phosphorylation of LAP2 during mitosis inhibits its binding to both lamin B and chromosomes. (Foisner and Gerace, 1993). The mechanism for inner membrane targeting and retention of LAP2 probably involves lateral diffusion in the interconnected membranes of the endoplasmatic reticulum and nuclear envelope, and interaction with components of the nuclear lamina and chromatin (Furukawa et al., 1995).

YA (Young Arrest) is an essential Drosophila gene for the transition from meiosis to the initiation of the rapid mitotic divisions by early embryos (Judd and Young, 1973;  Lin  et al., 1991;  Liu  et al., 1995). The chromosome condensation state is abnormal in nuclei in YA-deficient eggs and embryos (Liu et al., 1995). The YA protein is present during the first two hours of zygotic development, where it is localized to the nuclear lamina (Lin et al., 1991). Ectopically expressed YA associates with polytene chromosomes in vivo (Lopez and Wolfner, 1997), and YA can associate with both chromosomes and lamin Dm0 (Goldberg et al., 1997;  Lopez and Wolfner, 1997).

Otefin is a 45 kDa peripheral nuclear envelope protein with no apparent homology to other known proteins (Padan et al., 1990). It includes a large hydrophilic domain, a single carboxy terminal hydrophobic sequence of 17 amino acids and a high content of serine and threonine residues (Fig. 1B). With the exception of sperm cells, otefin is present in the nuclear envelope of all cells examined during the different stages of Drosophila development. In eggs and young embryos, otefin is also associated with the maternal fraction of membrane vesicles (Ashery-Padan  et al., 1997b). The COOH-terminal, 17-aa hydrophobic sequence of otefin is essential for the targeting of otefin to the nuclear periphery. Other sequences of otefin are required for its efficient targeting to the nuclear envelope and for further stabilizing otefin's interaction with the nuclear envelope (Ashery Padan  et al., 1997a). Otefin is a phosphoprotein in vivo and a substrate for in vitro phosphorylation by cdc2 kinase and cAMP-dependent protein kinase.

Lamins are the major proteins of the nuclear envelope. They are classified as type V intermediate filaments and, like all intermediate filaments, they contain an a helical rod domain flanked by amino (head) and carboxy (tail) domains (Fig. 1B).  Unlike the cytoplasmic intermediate filaments that are 10 nm wide, lamins can make up to 200 nm thick  fibers (Belmont  et al., 1993;  Paddy  et al., 1990). The rod domain of lamins is 52 nm long and contains three a helices, each composed of heptad repeats (reviewed in McKeon, 1987). These helices form coiled-coil interactions between lamin monomers. The lamin dimers associate longitudinally to form polar head-to-tail polymers. These polar head-to-tail polymers further associate laterally to form the 10 nm thick filaments (Heitlinger  et al., 1991). The 10 nm filaments further associate to form the 50-200 thick nuclear lamina (this study). The head-to-tail binding sites are at the ends of the rod domain that are highly conserved among all intermediate filament proteins. Point mutants that cause defects in binding were mapped to these conserved regions (Stuurman  et al., 1996;  Zhao  et al., 1996).

Lamins are divided into types A and B. Type A lamins are mainly expressed in differentiated cells, have a neutral isoelectric point and are soluble during mitosis. Type B lamins are expressed constitutively in all somatic cells, have an acidic isoelectric point and remain associated with membrane vesicles during mitosis (reviewed in  McKeon, 1991;  Nigg, 1992). Different eukaryotes possess between one to six lamin genes. Mammalian lamins A and C are the result of alternative splicing of the same gene. Lamins B1-B3 and C2 are coded by separate genes (Alsheimer and Benavente, 1996). The two major lamins in chicken are lamins A and B2 (Peter  et al., 1989). An additional minor species is termed lamin B1. Xenopus laevis has at least five different lamin genes (Stick, 1992;  Stick, 1994). Drosophila melanogaster has two lamin genes, termed lamin Dm0 and C (Bossie and Sanders, 1993;  Gruenbaum  et al., 1988). Caenorhabditis elegans probably has a single lamin gene, termed CeLam-1 (Riemer  et al., 1993).

Lamins undergo specific post translational modifications. All nuclear lamins except lamins C contain CaaX box at their carboxy terminus. The CaaX box undergoes proteolytic cleavage of the last three amino acids, farnesylation of the C-terminal cysteine, and carboxyl methylation. The isoprenylation is essential but not sufficient for the association of lamins with the nuclear envelope (Firmbach and Stick, 1995;  Firmbach-Kraft and Stick, 1993;  Hennekes and Nigg, 1994). Lamins are phosphorylated by several protein kinases in vivo and in vitro. These include: cdc2 kinase (Dessev  et al., 1991;  Heald and McKeon, 1990;  Peter  et al., 1990;  Ward and Kirschner, 1990), Casein kinase II (Li and Roux, 1992), PKA (Lamb  et al., 1991), bII PKC (Fields  et al., 1988;  Hennekes  et al., 1993;  Hocevar  et al., 1993;  Hocevar and Fields, 1991;  Kasahara  et al., 1991) and MAP kinase (Peter  et al., 1992). The phosphorylation state of lamins is cell-cycle regulated (Ottaviano and Gerace, 1985). It is involved in lamin polymerization and disassembly, and in importing lamin molecules into the nucleus. The Drosophila lamin Dm0 undergoes post translational modifications to give rise to at least three distinct isoforms termed, Dm1, Dm2 and Dmmit which differ in their phosphorylation pattern. Dm1 and Dm2 are present in most types of interphase nuclei as a random mixture of homo- and hetero-dimers (Smith  et al., 1987;  Stuurman  et al., 1995). Dmmit is present in the maternal pool and in mitotic cells (Smith and Fisher, 1989).

3-D in vivo studies in Drosophila and in mammalian cells revealed that lamin fibers are closely associated with chromatin fibers (Belmont et al., 1993;  Paddy et al., 1990). Studies in vitro have shown that lamins can specifically bind chromatin fragments and interphase chromatin (Hoger  et al., 1991;  Taniura  et al., 1995;  Yuan  et al., 1991), as well as condensed in vitro assembled chromatin (Ulitzur  et al., 1992) or mitotic chromosomes (Glass  et al., 1993;  Glass and Gerace, 1990). Lamins can also bind to specific DNA sequences (Baricheva  et al., 1996;  Luderus  et al., 1992;  Luderus  et al., 1994;  Shoeman and Traub, 1990;  Zhao et al., 1996) and to chromosomal proteins (Burke, 1990;  Glass et al., 1993;  Glass and Gerace, 1990;  Hoger et al., 1991;  Taniura et al., 1995;  Yuan et al., 1991). Binding of lamins to chromatin is specific and depends on the integrity of the chromosomes. Lamin A binds in vitro to poly-nucleosomes with a dissociation constant of about 1x10-9 M (Yuan et al., 1991). A binding site for mammalian lamins A and B was localized at the tail domain (Taniura et al., 1995). In the latter study, the dissociation constant of the tail domain binding to interphase chromatin was estimated to be in the range of 3x10-7 M and the binding was mediated by histones. Since lamins form large polymers in vivo, the actual association between the lamin filament and chromatin may be stronger. A specific binding site to mitotic chromosomes was also found in the rod domain. However, the in vivo relevance of this binding is not yet clear since the rod domain binding occurred only under acidic, non-physiological, conditions (Glass et al., 1993). Chicken lamin B and Drosophila lamin Dm0 polymers also bind specifically to M/SARs fragments (Luderus et al., 1992;  Luderus et al., 1994). These DNA sequences are several hundred base pairs long with several stretches of AT rich sequences and are likely to form an "open" form of chromatin. Indeed, the binding to these sequences could be competed to some extent with single strand DNA (Luderus et al., 1994). The binding of Drosophila lamin Dm0 to M/SARs is mediated by the rod domain and requires its polymerization (Zhao et al., 1996). Lamin-DNA interactions can occur, for example, in the centromeric regions since the l20p1.4 Drosophila centromeric sequence has DNA composition similar to M/SAR and it binds specifically to polymers of Drosophila lamin Dm0  (Baricheva et al., 1996). Lamin polymers can also bind strongly to telomeric sequences (Shoeman and Traub, 1990).


B. Biological roles of the nuclear lamina

Several functions have been ascribed to the nuclear lamina concerning nuclear organization and activity. These functions include: (i) regulating the size, shape and assembly of the nuclear envelope, (ii) a role in higher order chromatin organization by providing docking sites for chromatin , (iii) a role in DNA replication, (iv) a possible role in differentiation, as indicated by the change in lamina composition during development. In addition, the nuclear lamina is a major substrate for signals that control the cell cycle and lamins are specifically degraded in apoptosis (Nigg, 1992;  Oberhammer  et al., 1994).

(i) Nuclear envelope disassembly.

During mitosis, the nuclear envelope breaks down in prophase and starts to reassemble at late anaphase. Nuclear lamins and lamina associated proteins are likely to play a role both in the assembly and disassembly of the nuclear envelope. Disassembly of the nuclear lamina is controlled by phosphorylation of sites outside the rod domain of lamins that prevents the head-to tail association of the lamin molecules. For example, mutations in Ser-22 and Ser-392 of human lamin A in transfected COS cells prevented phosphorylation at these sites and blocked the disassembly of the nuclear lamina during mitosis (Heald and McKeon, 1990).

(ii) Nuclear envelope assembly depends on lamins and on lamin-associated proteins. Microinjection of lamin antibodies into cultured PtK2 cells resulted in daughter nuclei that remained arrested in a telophase-like configuration, and telophase-like chromatin that remained inactive (Benavente and Krohne, 1986). In mammalian cell-free extracts, antibodies directed against type A or B lamins blocked vesicles binding to chromatin, which is the first step of nuclear envelope assembly (Burke and Gerace, 1986). Similarly, anti-lamin Dm0 antibodies blocked the interaction between vesicles and chromatin in a Drosophila cell-free system that assembles nuclei from sperm chromatin (Ulitzur  et al., 1992;  Ulitzur et al., 1997). The role of lamin proteins in the association between nuclear vesicles and chromatin in Xenopus extracts has been the subject of debate; Depletion of lamin B3 from the assembly extract did not prevent the formation of nuclear envelopes consisting of membranes and nuclear pores. These lamin B3-depleted nuclei were small, fragile and failed to replicate their DNA (Jenkins  et al., 1995;  Meier  et al., 1991;  Newport  et al., 1990). In contrast, Dabauvalle et al. (Dabauvalle  et al., 1990) were able to block the formation of nuclear envelopes by using an antibody directed against both lamins B2 and B3. A major reason for the discrepancy between the above studies could be that Xenopus extracts contain lamins B2 and B1, in addition to lamin B3 (Lourim  et al., 1996;  Lourim and Krohne, 1993). In cell-free extracts of Xenopus eggs and Drosophila melanogaster it was shown that trypsinization of the membrane fraction abolished its ability to bind demembranated sperm chromatin and hence to support assembly of the nuclear envelope (Ulitzur et al., 1997;  Wilson and Newport, 1988). Possible target proteins for the Trypsin treatment are IMPs. Indeed, several studies suggest a role for LBR, LAP1 and LAP2 in nuclear assembly. LAP2 associates with chromosomes at the same time as lamins, which suggests a role for LAP2 in initial events of nuclear envelope reassembly (Foisner and Gerace, 1993). A recent study (Yang et al., 1997) shows that LAP1 and LAP2 become completely dispersed throughout ER membranes during mitosis and proposes that the reassembly of the nuclear envelope at the end of mitosis involves sorting of IMPs to chromosome surfaces by binding interactions with lamins and chromatin. Pyrpasopoulou et al. (Pyrpasopoulou  et al., 1996) analyzed the role of LBR in providing chromatin docking sites for nuclear vesicles by binding in vitro reconstituted vesicles of nuclear envelopes to chromatin. The results of this study suggest that LBR is involved in providing chromatin anchorage site at the nuclear envelope. It was also suggested that the homologue of LBR in sea urchin targets membranes to chromatin and later anchors the membrane to the lamina (Collas  et al., 1996). The essential role of otefin in the assembly of the nuclear envelope was recently demonstrated in a Drosophila cell-free system (Ashery-Padan et al., 1997b). The similar phenotype obtained when otefin or lamin Dm0 activities are inhibited (Ashery-Padan et al., 1997b) is probably due to the fact that otefin and lamin are part of the same protein complex in the vesicle fraction (Goldberg et al., 1997). In summary, the above data implies that the assembly of nuclear membranes following mitosis requires the function of protein complexes containing both peripheral and integral membrane proteins including: lamin, otefin, LAP2 and LBR.

Lamin genes are not present in significant homology in the yeast Saccharomyces cerevisiae (Gruenbaum, Y., unpublished observations) and in the protozoon Amoeba proteus (Schmidt  et al., 1995). In addition, the lamina-associated proteins LAP1, LAP2 and otefin are not present in significant homology in Saccharomyces cerevisiae (Gruenbaum, Y., unpublished observations), while LBR is the enzyme sterol C14 reductase (reviewed in Gerace and Foisner, 1994). One possible explanation for the appearance of lamins only in organisms with an open mitosis concerns their roles in nuclear envelope breakdown at the begining of mitosis and nuclear reassembly at the end of mitosis. These activities are not required in organisms with a closed mitosis. The involvement of the nuclear lamina in nuclear organization, development and DNA replication may have appeared later in evolution.


(iii). Nuclear and chromatin organization.

The nuclear lamina is a major component of the nuclear matrix. It was, therefore, suggested that a lamin filamentous meshwork is involved in nuclear and chromatin organization. An example for a direct involvement of a lamin protein in nuclear organization comes from an ectopic expression of the mouse sperm-specific lamin B3 in cultured somatic cells. This ectopic expression resulted in transformation of the nuclear morphology from spherical to hook-shaped (Furukawa and Hotta, 1993). Also, depletion of soluble lamin B3 from Xenopus nuclear assembly extracts gave in vitro assembled nuclei that were small and fragile (Meier et al., 1991;  Newport et al., 1990). Another evidence for the role of lamin in nuclear organization comes from the analysis of flies mutated in the Drosophila lamin Dm0 gene. Flies homozygous for a strong mutation in the lamin Dm0 gene had an aberrant nuclear structure and died following 9-16 hours of development. The dissociation of chromatin from the nuclear membrane was one of the first phenotypes observed in these flies (Osman, 1992). A weak mutation in the lamin Dm0 gene (<20% of lamin expression) resulted in a retarded development, reduced viability, sterility, and impaired locomotion. The nuclei in these mutant flies are enriched in nuclear pore complexes, in cytoplasmic annulate lamellae and contain defective nuclear envelopes (Lenz-Bohme  et al., 1997). In vitro studies support the role of nuclear lamin in chromatin organization. As discussed above, the nuclear lamina interacts in vivo with chromatin, and lamin proteins can bind histones and specific DNA sequences.

The Drosophila YA protein is needed to initiate embryonic cleavage divisions (Lopez et al., 1994). Ya is likely to be involved in mediating the association of chromosomes with the lamina (Goldberg et al., 1997), thus contributing to the organization of the nucleus in a developmental stage-specific manner (Lopez and Wolfner, 1997). Nuclei in YA-deficient eggs and embryos have abnormal chromosome condensation states (Liu et al., 1995), ectopically expressed YA associates with polytene chromosomes in vivo, and YA can associate with chromosomes in vitro (Lopez et al., 1994;  Lopez and Wolfner, 1997).


(iv). DNA replication requires nuclear lamins.

 Several reports demonstrated that, during interphase, lamin B molecules are present in foci in the nucleoplasm, in addition to their presence in the nuclear envelope. These foci coincide with sites of DNA replication (Goldman  et al., 1992;  Moir  et al., 1994;  Spann  et al., 1997). In addition, nuclei assembled in Xenopus egg extracts that were depleted of lamin B3 were unable to initiate DNA replication. These lamin B3-depleted nuclei had continuous nuclear envelopes and nuclear pores and were able to import proteins required for DNA synthesis such as PCNA, MCM3, ORC2 and DNA polymerase a (Goldberg  et al., 1995;  Meier et al., 1991;  Newport et al., 1990;  Spann et al., 1997). Addition of purified lamin B3 to the depleted extracts could rescue lamina assembly and DNA replication. Microinjection of a truncated human lamin, that was utilized as a dominant negative mutant to perturb lamin organization in mammalian cells, caused a dramatic reduction in DNA replication (Spann et al., 1997). Nuclear lamins are likely to be required for the elongation phase of DNA replication since the distribution of MCM3, ORC2, and DNA polymerase a that are required for the initiation stage of DNA replication was not affected by the depletion of lamin B3 activity (Spann et al., 1997).



II. Results and discussion

A. Mutations in the Drosophila lamin Dm0 gene reveal that it is an essential gene that is required for nuclear organization.

During egg chamber development, large amounts of lamin Dm0 are secreted by the nurse cells into the developing Drosophila oocyte (Ashery-Padan et al., 1997b;  Smith and Fisher, 1989;  Ulitzur et al., 1992). The amounts of lamin Dm0 RNA and protein that are maternally stored in the oocyte are sufficient for the assembly of many thousands of nuclei. In addition, lamin Dm0 is a very stable protein with an estimated half life of about 24 hr (Dr. Paul A. Fisher, personal communication). Therefore, flies mutated in their lamin Dm0 gene are expected to show a phenotype only following the consumption of the large maternal pool of lamin Dm0.

Drosophila melanogaster (canton S) males were mutagenized with ethyl methane sulphanate (ems) and offspring flies mutated in their second chromosome were crossed with flies containing the deletion Df(2L) gdh-A (Knipple  et al., 1991). This deletion is between 25D7-26A7 bands and contains the 25F1 locus of lamin Dm0 (Gruenbaum et al., 1988). One of the complementation groups was specific for a mutation in lamin Dm0 since it could be specifically rescued by a P-element mediated transformation with a CaspeR vector (Pirrotta, 1988) containing 1.2 kb upstream sequences of lamin Dm0 and either the complete genomic lamin Dm0 gene (EcoRI-EcoRI fragment; Osman  et al., 1990) or the two first exons and part  of the third exon of the genomic lamin gene (EcoRI-HindIII fragment; Osman et al., 1990) ligated to the HindIII-EcoRI fragment of lamin Dm0 cDNA (Gruenbaum et al., 1988). A second mutagenesis screen utilized a P-element targeted gene mutation, using the Birm-2/Birm-2; ry/ry line (Ballinger and Benzer, 1989;  Kaiser and Goodwin, 1990). Candidate lines for a mutation in lamin Dm0 were crossed with an ems-mutated line in lamin Dm0 (Osman, 1992). One of the isolated mutations, termed PM-15, was analyzed in more details. PM-15/PM-15 or PM-15/Df(2L)gdh-A flies showed an abnormal chromatin organization following 9-16 hr of development. The variability in the time of phenotype appearance is likely to be due to differences in the amounts of the maternal pool of lamin Dm0. Flies homozygous or trans-heterozygous for these mutations eventually die. Therefore, lamin Dm0 is an essential gene of the fruit fly (Osman, 1992). One of the first phenotypes in embryos homozygous for a mutation in lamin Dm0 gene is the detachment of the peripheral chromatin from the nuclear envelope. This detachment occurs in many regions of affected nuclei and is followed by condensation of chromatin (Fig. 2. compare panels C,D to panels A,B). The later phenotypes of these embryos include nuclei aggregation and formation of cytoplasmic annulate lamellae (Fig. 2E). A Drosophila line mutated in its lamin Dm0 gene (Lenz-Bohme et al., 1997), in which the amounts of lamin Dm0 protein are reduced to less than 20% of their normal levels, also revealed enrichment in annulate lamellae and in nuclear envelope clusters. These







Figure 2. A mutation in lamin Dm0 gene results in dissociation of chromatin from the nuclear envelope and accumulation of annulate lamellae (Osman et al., 1990). Embryos mutated in lamin Dm0 showed a visible phenotype following 9-16 hr of development. Electron microscope analysis of PM-15/PM-15 cells (C,D) revealed chromatin dissociation from the nuclear envelope as compared to normal cells (A,B). (E) Annulate lamellae in PM-15/Df(2L)gdh-A embryos following degradation of nuclei. The bars in panels A,C,E represent 1 µm. The bars in panels B,D represent 5 µm.


flies showed reduced viability, retardation in their development, sterility, and impaired locomotion. In some cells, defective nuclear envelopes were also observed (Lenz-Bohme et al., 1997). In summary, these studies demonstrate the essential role of lamins in nuclear and chromatin organization.


B. Filament assembly properties of lamin Dm0 and derivative proteins.

The assembly properties of lamin Dm0 were investigated in vitro using bacterially expressed and purifies lamin Dm0 and derivatives (Ulitzur et al., 1992). To test for the ability of filamentous protein to polymerize we used the sedimentation test (Heitlinger et al, 1991), which is based on the separation of pelletable polymers from soluble protein, following incubation under various chemicals and pH conditions. Reduction of salt concentration from 0.5 M NaCl to 50-150 mM NaCl in pH range of 5-9 was sufficient to induce 35-95% polymerization of lamin Dm0 protein. Electron microscope analysis of negative stained pellets confirmed the formation of filamentous structures (Fig. 3). The observed paracrystals were characterized by a distinct stain-excluding pattern with 25 nm axial repeat unit, which is half the size of the lamin rod domain (Fig. 3 A,B). Figure 3C shows a relatively rare case which reveals that these paracrystals are composed of separate lamin filaments. These filaments are 8-10 nm wide, which is the normal size of cytoplasmic intermediate filaments. Although there is no evidence for the existence of paracrystals in vivo, it is noteworthy that the width of these paracrystals fits is in the size range of lamin fibers that were visualized in Drosophila cells in vivo (Paddy et al., 1990).

The ability of the isolated rod domain of lamin Dm0 (amino acids 55-413) to polymerize was analyzed utilizing bacterially expressed protein that was purified to near ho-



Figure 3. Supramolecular structures formed by lamin Dm0 at low ionic strength. Lamin Dm0 protein at 2 mg/ml in buffer H (30 mM Tris-HCl pH 7.5, 1 mM DTT) containing 0.5 M NaCl was diluted 5 times in buffer H and incubated for 80 min on ice. Samples were placed on electron microscope grid and negatively stained with 1% uranyl acetate. Tightly packed paracrystals exhibit ~25 nm axial repeat unit and their thickness ranged between 40-200 nm (A,B). The thick paracrystalline arrays are composed of a large number of thin filaments (C).


mogeneity. Unlike the complete lamin Dm0 molecule, polymerization of the isolated rod domain was salt-independent. However, under acidic conditions (pH 5.5) and in the presence of 25 mM CaCl2, the isolated rod domain was organized in higher order structures, as judged by the sedimentation test (Fig. 4A) and by electron microscope analysis (Fig. 4B). The filamentous structure of the polymerized rod domain resembled that of the complete lamin protein, but lacked the 25 nm repeat unit. Under neutral and basic pH conditions the rod domain was organized into dimers which were 52 nm long and about 0.5 nm in diameter (not shown). In summary, these results demonstrate that the rod domain contains enough information to form the lamin filaments and that sequences outside the rod domain are required for the proper organization of the lamin filaments and for their assembly under physiological conditions.


C. Interaction between lamin Dm0 and chromatin.

Our previous analysis demonstrated that lamin Dm0 can interact specifically with sperm chromatin (Ulitzur et al., 1992). These experiments also showed that the addition of bacterially expressed lamin Dm0 to Drosophila embryonic extracts that can assemble nuclei from sperm chromatin resulted in increased amounts of lamin Dm0 around the peripheral chromatin (Ulitzur et al., 1992). The mitotic chromosome assay that measures the association between lamin and mitotic CHO chromosomes (Glass et al., 1993;  Glass and Gerace, 1990) was used to analyze domains in lamin Dm0 protein that are capable of interaction with chromatin. When lamin Dm0 protein was incubated for 30-60 min at 22oC with isolated mitotic chromosomes, in the presence of excess amounts of either 5% BSA or 10% FCS, a strong lamin staining was observed following immunofluorescence analysis with anti-lamin antibodies. The staining was mostly peripheral to the chromosomes and included aggregates of lamin (not shown). These aggregations are probably due to the organization of  lamin Dm0 into polymers since the aggregates were absent when mitotic chromosomes were incubated with lamin Dm0 containing the mutation R64>H (Zhao et al., 1996), which impairs the ability of lamin Dm0 to form filaments (Fig. 5B). The tail domain of lamin Dm0 (amino acids 425-622) contains specific binding site(s) to chromatin since it bound specifically to the mitotic chromosomes (Fig. 5A). The R64>H mutant protein was incubated with mitotic chromosomes in the presence of hundred fold molar excess of the isolated tail domain in order to find other possible domains in lamin that bind chromatin. As shown in Fig. 5C, staining with affinity purified polyclonal antibodies against the rod domain of lamin Dm0 gave intensity levels that were close to background levels. In conclusion, lamin Dm0 binds specifically to chromatin and its binding site(s) are localized to its tail domain. The specific lamin sequence that binds to chromatin, the affinity of its binding and the target chromosomal proteins are currently under investigation.

Figure 4. Polymerization properties of the isolated rod domain of lamin Dm0. (A) Isolated rod domain protein (2 mg/ml) in buffer H was diluted 5 folds in buffer H or in 50 mM sodium citrate pH 5.5, 25 mM CaCl2. Pellet (p) and supernatant (s) were separated by 30 min centrifugation at 15,000xg, boiled in sample loading buffer and subjected to SDS-10% PAGE stained with Comassie Brillant blue. The position of the size markers are shown on the left of the panel. The rod domain polymerized at pH 5.5 but not at pH 7.5. (B) The pellet fraction was placed on electron microscope grid and negatively stained with 0.75% uranyl acetate.

Figure 5. Binding of lamin Dm0 to chromosomes. Lamin Dm0 protein mutated in Arginine 64 (R64>H) (Zhao et al., 1996), which is impaired in its ability to form head-to-tail polymers (Stuurman et al., 1996;  Zhao et al., 1996), bound specifically to mitotic chromosomes (B). The tail domain of lamin Dm0 (amino acids 425-622) also bound specifically to mitotic chromosomes (A). The tail domain of lamin Dm0 could compete for the binding of the complete lamin Dm0 molecule to mitotic chromosomes since addition of a hundred fold molar excess of the tail domain could efficiently compete for the binding of R64>H (C). DAPI staining of DNA, left panels; antibody staining, right panels. Affinity purified polyclonal antibodies against the rod domain of lamin Dm0 B,C; monoclonal antibody 611A3A6 anti-lamin Dm0, A. This monoclonal antibody recognize an epitope in the tail domain. The bar represents  6µm and applies to all panels.



Figure 6. Inhibition of lamin Dm0 and otefin activity prevents the in vitro nuclear envelope assembly in Drosophila embryonic extracts. Twenty microliters of embryonic extracts were preincubated for 90 min with either 100 µg polyclonal anti-lamin Dm0 antibodies (C), 100 µg polyclonal anti-otefin antibodies (D), or 100 µg of preimmune serum antibodies (IgG fraction), (A,B). Sperm chromatin was added and the incubation proceeded for additional 90 min. Samples from the two experimental systems were viewed by standard transmission electron microscope. Decondensed chromatin was enveloped with nuclear membranes in preimmune antibodies-treated extracts (A,B) but not in anti-lamin Dm0 (C) or anti-otefin (D) antibodies-treated extracts. The bar  represents 1 µm.



D. Lamin and otefin are essential for nuclear envelope formation

To analyze lamin Dm0 and otefin function in nuclear envelope formation, 0-6 hr old Drosophila embryo extracts, in which interphase-like nuclei can be assembled from sperm chromatin (Berrios and Avilion, 1990;  Crevel and Cotterill, 1991;  Ulitzur and Gruenbaum, 1989), were incubated with either 100-300 µg polyclonal anti-Drosophila lamin Dm0 or anti-Drosophila otefin antibodies (IgG fractions). Incubation of the extract under the same conditions with 100-300 µg of preimmune rabbit sera (IgG fraction) or with normal rabbit IgG served as controls. No membrane assembly was observed when lamin Dm0 or otefin activities were inhibited (Ashery Padan et al., 1997;  Ulitzur et al., 1992;  Ulitzur et al., 1997). Electron microscope (Fig. 6), light, and fluorescent microscope analyses (not shown) revealed that while chromatin went through the characteristic decondensation process, membrane vesicles did not attach to its surface, and nuclear envelope did not assemble around it (Fig. 6C,D). Incubation of the extract with preimmune sera, (IgG fraction), or with commercially available normal rabbit IgG fraction had no effect on nuclear assembly, the presence of membranes around the chromatin was observed (Fig. 6A,B). Addition of 2 µg of interphase lamin isolated from Drosophila embryos to extracts that were preincubated with the anti-lamin Dm0 antibodies restored binding of vesicles to chromatin (Ulitzur et al., 1997).



The isolation of Drosophila lines with mutation in lamin Dm0 gene and the analysis of these lines was performed in collaboration with Dr. R. Falk and was part of the Ph.D. thesis of Dr. Midhat Osman. This study was supported by the US-Israel Binational Fund (BSF), and by the Israel Academy of Sciences.



Alsheimer, M., and Benavente, R.  (1996).  Change of karyoskeleton during mammalian spermatogenesis: expression pattern of nuclear lamin C2 and its regulation. Exp. Cell. Res. 228, 181-188.

Ashery Padan, R., Weiss, A. M., Feinstein, N., and Gruenbaum, Y.  (1997a).  Distinct regions specify the targeting of otefin to the nucleoplasmic side of the nuclear envelope. J. Biol. Chem. 272, 2493-2499.

Ashery-Padan, R., Ulitzur, N., Arbel, A., Goldberg, M., Weiss, A. M., Maus, N., Fisher, P. A., and Gruenbaum, Y.  (1997b).  Localization and posttranslational modifications of otefin, a protein required for vesicle attachment to chromatin, during Drosophila melanogaster development. Mol. Cell. Biol. 17, 4114-4123.

Ballinger, D. G. and Benzer, S.  (1989).  Targeted gene mutations in Drosophila. Proc. Natl. Acad. Sci. USA. 86, 9402-9406.

Baricheva, E. A., Berrrios, M., Bogachev, S. S., Borisevich, I. V., Lapik, E. R., Sharakhov, I. V., Stuurman, N., and Fisher, P. A.  (1996).  DNA from Drosophila b-heterochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ. Gene. 171, 171-176.

Belmont, A. S., Zhai, Y., and Thilenius, A.  (1993).  Lamin B distribution and association with peripheral chromatin revealed by optical sectioning and electron microscopy tomography. J. Cell Biol. 123, 1671-1685.

Benavente, R., and Krohne, G.  (1986).  Involvement of nuclear lamins in postmitotic reorganization of chromatin as demonstrated by microinjection of lamin antibodies. J. Cell Biol. 103, 1847-1854.

Berrios, M., and Avilion, A. A.  (1990).  Nuclear formation in Drosophila cell free system. Exp. Cell Res. 191, 64-70.

Bossie, C. A., and Sanders, M. M.  (1993).  A cDNA from Drosophila melanogaster encodes a lamin C-like intermediate filament protein. J. Cell Sci. 104, 1263-1272.

Burke, B.  (1990).  On the cell-free association of lamins A and C with metaphase chromosomes. Exp. Cell Res. 186, 169-176.

Burke, B., and Gerace, L.  (1986).  A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell 44, 639-652.

Collas, P., Courvalin, J. C., and Poccia, D.  (1996).  Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein. J. Cell. Biol. 135, 1715-1725.

Crevel, G., and Cotterill, S.  (1991).  DNA replication in cell-free extracts from Drosophila melanogaster. EMBO J. 10, 4361-4369.

Dabauvalle, M. C., Loos, K., and Scheer, U.  (1990).  Identification of a soluble precursor complex essential for nuclear pore assembly in vitro. Chromosoma 100, 56-66.

Davis, L. I.  (1995).  The nuclear pore complex. Annu. Rev. Biochem. 64, 865-896.

Dessev, G., Iovcheva, D. C., Bischoff, J. R., Beach, D., and Goldman, R.  (1991).  A complex containing p34cdc2 and cyclin B phosphorylates the nuclear lamin and disassembles nuclei of clam oocytes in vitro. J. Cell Biol. 112, 523-533.

Fields, A. P., Pettit, G. R., and May, W. S.  (1988).  Phosphorylation of lamin B at the nuclear membrane by activated protein kinase C. J. Biol. Chem. 263, 8253-8260.

Firmbach, K. I., and Stick, R.  (1995).  Analysis of nuclear lamin isoprenylation in Xenopus oocytes: isoprenylation of lamin B3 precedes its uptake into the nucleus. J. Cell Biol. 129, 17-24.

Firmbach-Kraft, I., and Stick, R.  (1993).  The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells. J. Cell Biol. 123, 1661-1670.

Fisher, D. Z., Chaudhary, N., and Blobel, G.  (1986).  cDNA sequencing of nuclear lamins A and C reveals primary and secondary structural homology to intermediate filament proteins. Proc Natl Acad Sci USA 83, 6450-6454.

Foisner, R., and Gerace, L.  (1993).  Integral membrane proteins of the nuclear envelope interact with lamins and chromosomes, and binding is modulated by mitotic phosphorylation. Cell 73, 1267-1279.

Furukawa, K., and Hotta, Y.  (1993).  cDNA cloning of a germ cell specific lamin B3 from mouse spermatocytes and analysis of its function by ectopic expression in somatic cells. EMBO J. 12, 97-106.

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

Gerace, L., and Foisner, R.  (1994).  Integral membrane proteins and dynamic organization of the nuclear envelope. Trends Cell Biol. 4, 127-131.

Glass, C. A., Glass, J. R., Taniura, H., Hasel, K. W., Blevitt, J. M., and Gerace, L.  (1993).  The alpha-helical rod domain of human lamins A and C contains a chromatin binding site. EMBO J. 12, 4413-4424.

Glass, J. R., and Gerace, L.  (1990).  Lamins A and C bind and assemble at the surface of mitotic chromosomes. J. Cell Biol. 111, 1047-1057.

Goldberg, M., Huihua, L., Ashery-Padan, R., Weiss, A. M., Fisher, P. A., Gruenbaum, Y., and Wolfner, M. F.  (1997).  Interactions between Drosophila  nuclear envelope proteins: lamin otefin and YA. submmited.

Goldberg, M., Jenkins, H., Allen, T., Whitfield, W. G., and Hutchison, C. J.  (1995).  Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts. J. Cell. Sci. 108, 3451-3461.

Goldman, A. E., Moir, R. D., Montag, L. M., Stewart, M., and Goldman, R. D.  (1992).  Pathway of incorporation of microinjected lamin A into the nuclear envelope. J. Cell Biol. 119, 725-735.

Gorlich, D., and Mattaj, I. W.  (1996).  Nucleocytoplasmic transport. Science. 271, 1513-1518.

Gruenbaum, Y., Landesman, Y., Drees, B., Bare, J. W., Saumweber, H., Paddy, M. R., Sedat, J. W., Smith, D. E., Benton, B. M., and Fisher, P. A.  (1988).  Drosophila nuclear lamin precursor Dm0 is translated from either of two developmentally regulated mRNA species apparently encoded by a single gene. J. Cell Biol. 106, 585-596.

Harel, A., Zlotkin, E., Nainudel, E. S., Feinstein, N., Fisher, P. A., and Gruenbaum, Y.  (1989).  Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos. J. Cell Sci. 94, 463-470.

Harris, C. A., Andryuk, P. J., Cline, S., Chan, H. K., Natarajan, A., Siekierka, J. J., and Goldstein, G.  (1994).  Three distinct human thymopoietins are derived from alternatively spliced mRNAs. Proc. Natl. Acad. Sci. USA 91, 6283-6287.

Harris, C. A., Andryuk, P. J., Cline, S. W., Mathew, S., Siekierka, J. J., and Goldstein, G.  (1995).  Structure and mapping of the human thymopoietin (TMPO) gene and relationship of human TMPO beta to rat lamin-associated polypeptide 2. Genomics 28, 198-205.

Heald, R., and McKeon, F. (1990).  Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis. Cell 61, 579-589.

Heitlinger, E., Peter, M., Haner, M., Lustig, A., Aebi, U., and Nigg, E. A.  (1991).  Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions. J. Cell Biol. 113, 485-95.

Hennekes, H., and Nigg, E. A.  (1994).  The role of isoprenylation in membrane attachment of nuclear lamins. A single point mutation prevents proteolytic cleavage of the lamin A precursor and confers membrane binding properties. J. Cell Sci. 107, 1019-1029.

Hennekes, H., Peter, M., Weber, K., and Nigg, E. A.  (1993).  Phosphorylation on protein kinase C sites inhibits nuclear import of lamin B2. J. Cell Biol. 120, 1293-1304.

Hocevar, B. A., Burns, D. J., and Fields, A. P.  (1993).  Identification of protein kinase C (PKC) phosphorylation sites on human lamin B. Potential role of PKC in nuclear lamina structural dynamics. J. Biol. Chem. 268, 7545-7552.

Hocevar, B. A., and Fields, A. P.  (1991).  Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells. J. Biol. Chem. 266, 28-33.

Hoger, T. H., Krohne, G., and Kleinschmidt, J. A.  (1991).  Interaction of Xenopus lamins A and LII with chromatin in vitro mediated by a sequence element in the carboxyterminal domain. Exp. Cell Res. 197, 280-289.

Hutchison, C. J., Bridger, J. M., Cox, L. S., and Kill, I. R.  (1994).  Weaving a pattern from disparate threads: lamin function in nuclear assembly and DNA replication. J. Cell Sci. 107, 3259-3269.

Jenkins, H., Whitfield, W. G., Goldberg, M. W., Allen, T. D., and Hutchison, C. J.  (1995).  Evidence for the direct involvement of lamins in the assembly of a replication competent nucleus. Acta Biochim. Pol. 42, 133-143.

Judd, B. H., and Young, M. W.  (1973).  An examination of the one cistron : one chromomere concept. Cold Spring Harbor Symp. Quant. Biol.  38, 573-579.

Kaiser, K. and Goodwin, S. F.  (1990).  "Site-selected" transposon mutagenesis of Drosophila. Proc. Natl. Acad. Sci. USA 87, 1686-1690.

Kasahara, K., Chida, K., Tsunenaga, M., Kohno, Y., Ikuta, T., and Kuroki, T.  (1991).  Identification of lamin B2 as a substrate of protein kinase C in BALB/MK-2 mouse keratinocytes. J. Biol Chem. 266, 20018-20023.

Knipple, D. C., Fuerst, T. R., and MacIntyre, R. J.  (1991).  Molecular cloning and analysis of the chromosomal region 26A of Drosophila melanogaster. Mol. Gen. Genet. 226, 241-249.

Lamb, N. J., Cavadore, J. C., Labbe, J. C., Maurer, R. A., and Fernandez, A.  (1991).  Inhibition of cAMP-dependent protein kinase plays a key role in the induction of mitosis and nuclear envelope breakdown in mammalian cells. EMBO J. 10, 1523-1533.

Lenz-Bohme, B., Wismar, J., Fuchs, S., Reifegerste, R., Buchner, E., Betz, H., and Schmitt, B.  (1997).  Insertional mutation of the Drosophila nuclear lamin dm(0) gene results in defective nuclear envelopes, clustering of nuclear pore complexes, and accumulation of annulate lamellae. J. Cell Biol. 137, 1001-1016.

Li, H., and Roux, S. J.  (1992).  Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei. Proc. Natl. Acad. Sci. USA 89, 8434-8348.

Lin, H., Song, K., Hutcheson, G. A., Goutte, C. E., and Wolfner, M. F.  (1991).  A maternally encoded nuclear envelope protein required for embryonic mitosis in Drosophila. Cold Spring Harbor Symp. Quant. Biol. 56, 719-727.

Liu, J., Song, K., and Wolfner, M. F.  (1995).  Mutational analyses of fs(1)Ya, an essential, developmentally regulated, nuclear envelope protein in Drosophila. Genetics 141, 1473-1481.

Lopez, J. M., Song, K., Hirshfeld, A. B., Lin, H., and Wolfner, M. F.  (1994).  The Drosophila fs(1)Ya protein, which is needed for the first mitotic division, is in the nuclear lamina and in the envelopes of cleavage nuclei, pronuclei, and nonmitotic nuclei. Dev. Biol. 163, 202-211.

Lopez, J. M., and Wolfner, M. F.  (1997).  The developmentally regulated drosophila embryonic nuclear lamina protein young arrest (fs(1)ya) is capable of associating with chromatin. J. Cell Sci. 5, 643-651.

Lourim, D., Kempf, A., and Krohne, G.  (1996).  Characterization and quantitation of three B-type lamins in Xenopus oocytes and eggs: increase of lamin LI protein synthesis during meiotic maturation. J. Cell Sci. 109, 1775-1785.

Lourim, D. and Krohne, G.  (1993).  Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations. J. Cell Biol. 123, 501-512.

Luderus, M. E., de Graaf, A., Mattia, E., den Blaauwen, J. L., Grande, M. A., de Jong, L., and van Driel, D. R.  (1992).  Binding of matrix attachment regions to lamin B1. Cell 70, 949-959.

Luderus, M. E., den Blaauwen, J. L., de Smit, Oj., Compton, D. A., and van Driel, D. R.  (1994).  Binding of matrix attachment regions to lamin polymers involves single-stranded regions and the minor groove. Mol. Cell. Biol. 14, 6297-6305.

Martin, L., Crimaudo, C., and Gerace, L.  (1995).  cDNA cloning and characterization of lamina-associated polypeptide 1C (LAP1C), an integral protein of the inner nuclear membrane. J. Biol. Chem. 270, 8822-8828.

McKeon, F. D.  (1987).  Nuclear lamin proteins and the structure of the nuclear envelope: where is the function? Bioessays. 7, 169-173.

McKeon, F. D. (1991).  Nuclear lamin proteins: domains required for nuclear targeting, assembly, and cell-cycle-regulated dynamics. Curr. Opin. Cell Biol. 3, 82-86.

Meier, J., Campbell, K. H., Ford, C. C., Stick, R., and Hutchison, C. J.  (1991).  The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. J. Cell Sci. 98, 271-279.

Moir, R. D., Montag, L. M., and Goldman, R. D.  (1994).  Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication. J. Cell Biol. 125, 1201-1212.

Moir, R. D., Spann, T. P., and Goldman, R. D.  (1995).  The dynamic properties and possible functions of nuclear lamins. Int. Rev. Cytol. 162b, 141-182.

Newport, J. W., Wilson, K. L., and Dunphy, W. G.  (1990).  A lamin-independent pathway for nuclear envelope assembly. J. Cell Biol. 111, 2247-2259.

Nigg, E. A.  (1992).  Assembly and cell cycle dynamics of the nuclear lamina. Semin. Cell Biol. 3, 245-253.

Nikolakaki, E., Meier, J., Simos, G., Georgatos, S. D., and Giannakouros, T.  (1997).  Mitotic phosphorylation of the lamin B receptor by a serine/arginine kinase and p34(cdc2). J. Biol. Chem. 272, 6208-6213.

Oberhammer, F. A., Hochegger, K., Froschl, G., Tiefenbacher, R., and Pavelka, M.  (1994).  Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase. J. Cell Biol. 126, 827-837.

Osman, M.  (1992).  Characterization of the lamin gene and search for biological roles of the lamin protein in Drosophila. Ph.D. thesis. The Hebrew University of Jerusalem.

Osman, M., Paz, M., Landesman, Y., Fainsod, A., and Gruenbaum, Y.  (1990).  Molecular analysis of the Drosophila nuclear lamin gene. Genomics 8, 217-224.

Ottaviano, Y., and Gerace, L.  (1985).  Phosphorylation of the nuclear lamins during interphase and mitosis. J. Biol. Chem. 260, 624-632.

Padan, R., Nainudel, E. S., Goitein, R., Fainsod, A., and Gruenbaum, Y.  (1990).  Isolation and characterization of the Drosophila nuclear envelope otefin cDNA. J. Biol. Chem. 265, 7808-7813.

Paddy, M. R., Belmont, A. S., Saumweber, H., Agard, D. A., and Sedat, J. W.  (1990).  Interphase nuclear envelope lamins form a discontinuous network that interacts with only a fraction of the chromatin in the nuclear periphery. Cell 62, 89-106.

Peter, M., Kitten, G. T., Lehner, C. F., Vorburger, K., Bailer, S. M., Maridor, G., and Nigg, E. A.  (1989).  Cloning and sequencing of cDNA clones encoding chicken lamins A and B1 and comparison of the primary structures of vertebrate A- and B-type lamins. J. Mol. Biol. 208, 393-404.

Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A.  (1990).  In vitro disassembly of the nuclear lamina and M phase-specific phosphorylation of lamins by cdc2 kinase. Cell 61, 591-602.

Peter, M., Sanghera, J. S., Pelech, S. L., and Nigg, E. A.  (1992).  Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2. Eur. J. Biochem. 205, 287-94.

Pirrotta, V.  (1988).  Vectors for P-mediated transformation in Drosophila. Biotechnology. 10, 437-456.

Pyrpasopoulou, A., Meier, J., Maison, C., Simos, G., and Georgatos, S. D.  (1996).  The lamin B receptor (LBR) provides essential chromatin docking sites at the nuclear envelope. EMBO J. 15, 7108-7119.

Riemer, D., Dodemont, H., and Weber, K.  (1993).  A nuclear lamin of the nematode Caenorhabditis elegans with unusual structural features; cDNA cloning and gene organization. Eur. J. Cell Biol. 62, 214-223.

Schmidt, M., Grossmann, U., and Krohne, G.  (1995).  The nuclear membrane-associated honeycomb structure of the unicellular organism Amoeba proteus: on the search for homologies with the nuclear lamina of metazoa. Eur. J. Cell Biol. 67, 199-208.

Shoeman, R. L., and Traub, P.  (1990).  The in vitro DNA-binding properties of purified nuclear lamin proteins and vimentin. J. Biol. Chem. 265, 9055-9061.

Simos, G., and Georgatos, S. D.  (1992).  The inner nuclear membrane protein p58 associates in vivo with a p58 kinase and the nuclear lamins. EMBO J. 11, 4027-4036.

Simos, G., and Georgatos, S. D.  (1994).  The lamin B receptor-associated protein p34 shares sequence homology and antigenic determinants with the splicing factor 2-associated protein p32. FEBS Lett. 346, 225-228.

Simos, G., Maison, C., and Georgatos, S. D.  (1996).  Characterization of p18, a component of the lamin B receptor complex and a new integral membrane protein of the avian erythrocyte nuclear envelope. J. Biol. Chem. 271, 12617-12625.

Smith, D. E. and Fisher, P. A.  (1989).  Interconversion of Drosophila nuclear lamin isoforms during oogenesis, early embryogenesis, and upon entry of cultured cells into mitosis. J. Cell Biol. 108, 255-265.

Smith, D. E., Gruenbaum, Y., Berrios, M., and Fisher, P. A.  (1987).  Biosynthesis and interconversion of Drosophila nuclear lamin isoforms during normal growth and in response to heat shock. J. Cell Biol. 105, 771-790.

Smith, S., and Blobel, G.  (1993).  The first membrane spanning region of the lamin B receptor is sufficient for sorting to the inner nuclear membrane. J. Cell Biol. 120, 631-637.

Soullam, B., and Worman, H. J.  (1993).  The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal. J. Cell Biol. 120, 1093-1100.

Soullam, B., and Worman, H. J.  (1995).  Signals and structural features involved in integral membrane protein targeting to the inner nuclear membrane. J. Cell Biol. 130, 15-27.

Spann, T. P., Moir, R. D., Goldman, A. E., Stick, R., and Goldman, R. D.  (1997).  Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis. J. Cell Biol. 136, 1201-1212.

Stick, R.  (1992).  The gene structure of Xenopus nuclear lamin A: a model for the evolution of A-type from B-type lamins by exon shuffling. Chromosoma 101, 566-574.

Stick, R.  (1994).  The gene structure of B-type nuclear lamins of Xenopus laevis: implications for the evolution of the vertebrate lamin family. Chromosome Res. 2, 376-382.

Stuurman, N., Maus, N., and Fisher, P. A.  (1995).  Interphase phosphorylation of the Drosophila nuclear lamin: site-mapping using a monoclonal antibody. J. Cell Sci. 108, 3137-3144.

Stuurman, N., Sasse, B., and Fisher, P. A.  (1996).  Intermediate filament protein polymerization: molecular analysis of Drosophila nuclear lamin head-to-tail binding. J. Struct. Biol. 117, 1-15.

Taniura, H., Glass, C., and Gerace, L.  (1995).  A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones. J. Cell Biol. 131, 33-44.

Theodor, L., Shoham, J., Berger, R., Gokkel, E., Trachtenbrot, L., Simon, A. J., Brok Simon, F., Nir, U., Ilan, E., Zevin Sonkin, D., Friedman, E., and Rechavi, G.  (1997).  Ubiquitous expression of a cloned murine thymopoietin cDNA. Acta Haematol. 97, 153-163.

Ulitzur, N., and Gruenbaum, Y.  (1989).  Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos. FEBS Lett. 259, 113-116.

Ulitzur, N., Harel, A., Feinstein, N., Goldberg, M., and Gruenbaum, Y.  (1997).  Nuclear membrane vesicle targeting to chromatin in a Drosophila embryo cell-free system. Mol. Biol. Cell. 8, 1439-1448.

Ulitzur, N., Harel, A., Feinstein, N., and Gruenbaum, Y.  (1992).  Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract. J. Cell Biol. 119, 17-25.

Ward, G. E., and Kirschner, M. W.  (1990).  Identification of cell cycle-regulated phosphorylation sites on nuclear lamin C. Cell 61, 561-577.

Wilson, K. L., and Newport, J.  (1988).  A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J. Cell Biol. 107, 57-68.

Worman, H. J., Evans, C. D., and Blobel, G.  (1990).  The lamin B receptor of the nuclear envelope inner membrane: a polytopic protein with eight potential transmembrane domains. J. Cell Biol. 111, 1535-1542.

Worman, H. J., Yuan, J., Blobel, G., and Georgatos, S. D.  (1988).  A lamin B receptor in the nuclear envelope. Proc. Natl. Acad. Sci. USA 85, 8531-8534.

Yang, L., Guan, T. L., and Gerace, L.  (1997).  Integral membrane proteins of the nuclear envelope are dispersed throughout the endoplasmic reticulum during mitosis. J. Cell Biol. 137, 1199-1210.

Ye, Q., and Worman, H. J.  (1996).  Interaction between an integral protein of the nuclear envelope inner membrane and humanchromodomain proteins homologous to Drosophila Hp1. J. Biol. Chem. 271, 14653-14656.

Yuan, J., Simos, G., Blobel, G., and Georgatos, S. D.  (1991).  Binding of lamin A to polynucleosomes. J. Biol. Chem. 266, 9211-9215.

Zhao, K., Harel, A., Stuurman, N., Guedalia, D., and Gruenbaum, Y.  (1996).  Binding of matrix attachment regions to nuclear lamin is mediated by the rod domain and depends on the lamin polymerization state. FEBS Lett. 380, 161-164.