A second lamin in Drosophila, Lamin C

A novel intermediate filament cDNA, pG-IF, has been isolated from a Drosophila melanogaster embryonic expression library screened with a polyclonal antiserum produced against a 46 kDa cytoskeletal protein isolated from Kc cells. This 46 kDa protein is known to be immunologically related to vertebrate intermediate filament proteins. The screen resulted in the isolation of four different cDNA groups. Of these, one has been identified as the previously characterized Drosophila nuclear lamin cDNA, Dm0, and a second, pG-IF, demonstrates homology to Dm0 by cross hybridization on Southern blots. DNA sequence analysis reveals that pG-IF encodes a newly identified intermediate filament protein in Drosophila. Its nucleotide sequence is highly homologous to nuclear lamins with lower homology to cytoplasmic intermediate filament proteins. pG-IF predicts a protein of 621 amino acids with a predicted molecular mass of 69,855 daltons. In vitro transcription and translation of pG-IF yields a protein with a SDS-PAGE estimated molecular weight of approximately 70 kDa. It contains sequence principles characteristic of class V intermediate filament proteins. Its near neutral pI (6.83) and the lack of a terminal CaaX motif suggests that it may represent a lamin C subtype in Drosophila. In situ hybridization to polytene chromosomes detects one band of hybridization on the right arm of chromosome 2 at or near 51A. This in conjunction with Southern blot analysis of various genomic digests suggests one or more closely placed genes while Northern blot analysis detects two messages in Kc cells (Bossie, 1993).

Invertebrates have long been thought to contain only a single lamin, which in Drosophila is the well-characterized Lamin. However, recently a Drosophila cDNA clone (pG-IF) has been identified that codes for an intermediate filament protein that harbors a nuclear localization signal but lacks a carboxy-terminal CaaX motif. Based on these data the putative protein encoded by pG-IF was tentatively called Drosophila Lamin C. To address whether the pG-IF encoded protein is expressed and whether it encodes a cytoplasmic intermediate filament protein or a nuclear lamin, antibodies were raised against the recombinant pG-IF protein. The antibodies decorate the nuclear envelope in Drosophila Kc tissue culture cells as well as in salivary and accessory glands, demonstrating that pG-IF encodes a nuclear lamin (Lamin C). Antibody decoration, in situ hybridization, western and northern blotting studies show that Lamin C is acquired late in embryogenesis. In contrast, Lamin is constitutively expressed. Lamin C is first detected in late stage 12 embryos in oenocytes, hindgut and posterior spiracles and subsequently also in other differentiated tissues. In third instar larvae Lamin C and Lamin are coexpressed in all tissues tested. Thus, Drosophila has two lamins: Lamin, containing a CaaX motif, is expressed throughout, while Lamin C, lacking a CaaX motif, is expressed only later in development. Expression of Drosophila Lamin C is similar to that of vertebrate lamin A, which loses its CaaX motif during incorporation into the lamina (Riemer, 1995).

To gain insight into the function of the developmentally regulated A-type lamins Drosophila was transformed with a construct containing the hsp70 promoter followed by the Drosophila lamin C (an analog of vertebrate A-type lamins) cDNA. Lamin C is expressed ectopically after heat shock of embryos and localizes to the nucleus. In embryos that normally do not contain lamin C, no phenotypic change is observed after lamin C expression. However, ectopic expression of lamin C during most larval (but not pupal) stages stalls growth, inhibits ecdysteroid signaling (in particular during the larval-prepupal transition), results in the development of melanotic tumors, and ultimately causes death. During pupation in control animals, when massive apoptosis of larval tissues takes place, lamin C is proteolyzed into a fragment with a size similar to that predicted by caspase cleavage. The ectopically expressed lamin C is identically cleaved, resulting in a large increase of the steady-state level of the lamin C fragment. A null mutation of the dcp-1 gene, one of the two known Drosophila caspase genes, also results in development of melanotic tumors and larval death, suggesting that the ectopically expressed lamin C inhibits apoptosis through competitive inhibition of caspase activity (Stuurman, 1999).

Lamin mutation

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of elbows and Achilles tendons, slowly progressive muscle wasting and weakness, and a life-threatening cardiomyopathy with conduction blocks. Two modes of inheritance exist: X-linked and autosomal dominant (EDMD-AD). EDMD-AD is clinically identical to the X-linked forms of the disease. Mutations in EMD, the gene encoding emerin, are responsible for the X-linked form. The locus for EDMD-AD has been mapped to an 8-cM interval on chromosome 1q11-q23 in a large French pedigree, and it was found that the EMD phenotype in four other small families is potentially linked to this locus. This region contains the lamin A/C gene (LMNA), a candidate gene encoding two proteins of the nuclear lamina, lamins A and C, produced by alternative splicing. Four mutations in LMNA co-segregate with the disease phenotype in the five families: one nonsense mutation and three missense mutations. These results are the first identification of mutations in a component of the nuclear lamina as a cause of inherited muscle disorder. Together with mutations in EMD, they underscore the potential importance of the nuclear envelope components in the pathogenesis of neuromuscular disorders (Bonne, 1999).

Numerous studies of the underlying causes of ageing have been attempted by examining diseases associated with premature ageing, such as Werner's syndrome and Hutchinson-Gilford progeria syndrome (HGPS). HGPS is a rare genetic disorder resulting in phenotypes suggestive of accelerated ageing, including shortened stature, craniofacial disproportion, very thin skin, alopecia and osteoporosis, with death in the early teens predominantly due to atherosclerosis1. However, recent reports suggest that developmental abnormalities may also be important in HGPS. Mice have been derived carrying an autosomal recessive mutation in the lamin A gene (Lmna) encoding A-type lamins, major components of the nuclear lamina. Homozygous mice display defects consistent with HGPS, including a marked reduction in growth rate and death by 4 weeks of age. Pathologies in bone, muscle and skin are also consistent with progeria. The Lmna mutation resulted in nuclear morphology defects and decreased lifespan of homozygous fibroblasts, suggesting premature cell death. A mouse model is presented for progeria that may elucidate mechanisms of ageing and development in certain tissue types, especially those developing from the mesenchymal cell lineage (Mounkes, 2003).

The Lmna gene encodes the A-type lamins, intermediate filament family members that make up the nuclear lamina, a fibrous network underlying the nuclear envelope. Alternative splicing of Lmna transcripts gives rise to four proteins: two major products called lamin A and lamin C, and two minor products, laminAdelta10 and lamin C2, the latter being specific to the testis. The A-type lamins interact with B-type lamins, which are encoded by the Lmnb1 and Lmnb2 genes, in a largely uncharacterized assembly process to form the nuclear lamina. The A-type lamins are developmentally regulated, whereas at least one B-type lamin is expressed in every cell type at all developmental stages. Embryonic stem cells, cells of the early embryo and hematopoietic stem cells do not express Lmna; however, derivatives of these cells do express Lmna during development, suggesting a role for Lmna in terminal differentiation. In addition to controlling interphase nuclear morphology, the nuclear lamina also functions in selective retention of proteins in the inner nuclear membrane, chromatin organization, DNA replication and gene expression. A role for A-type lamins in regulating gene expression is further supported by interactions with a growing number of transcription factors, for example, Rb7 and GCL indirectly through emerin (Mounkes, 2003).

The indispensable role of lamins in these diverse and fundamental processes may account for their association with a growing list of human diseases. Laminopathies can be divided loosely into two categories: those affecting striated muscle and those with phenotypes affecting adipose tissue and bone. Diseases of striated muscle caused by mutations in the Lmna gene are the autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD), limb girdle muscular dystrophy 1B (LGMD1B) and dilated cardiomyopathy with conduction system disease (DCM-CD). A single recessive mutation in Lmna has also been implicated in Charcot-Marie-Tooth syndrome type 2B1 (CMT2B1), a peripheral neuropathy with muscle weakness and wasting. Dunnigan's familial partial lipodystrophy (FPLD) and mandibuloacral dysplasia (MAD), disorders primarily resulting in loss and redistribution of white adipose tissue, are also associated with mutations in Lmna. Hyperlipidemia, insulin resistance and diabetes are common in FPLD and MAD patients, and bone defects in MAD patients include craniofacial abnormalities, osteolysis of terminal digits and hypoplasia of clavicles. Many clinical observations of MAD patients are also seen in progeria patients, suggesting that the two disorders may be allelic. Although it is not understood why mutations in Lmna cause such a wide array of phenotypes, the continued discovery of phenotypic overlap between some of these diseases indicates that they may represent a spectrum of related disorders rather than separate diseases. The present description of progeroid symptoms associated with Lmna^L530P/L530P mutant mice represents one of the most severe extremes in such a spectrum of laminopathies (Mounkes, 2003).

In an effort to create a mouse model for AD-EDMD, a nucleotide base change was introduced into the Lmna gene, directing a substitution of proline for leucine at residue 530 (L530P); this is a mutation that causes AD-EDMD in humans. Although mice heterozygous for this point mutation do not show signs of muscular dystrophy and have been overtly normal up to 6 months of age, mice homozygous for the mutation (Lmna^L530P/L530P mice) show phenotypes markedly reminiscent of symptoms observed in progeria patients. Homozygous Lmna^L530P/L530P mice are indistinguishable from their littermates at birth, but within 4-6 days develop severe growth retardation, dying within 4-5 weeks. Progeria patients are unremarkable at birth, but by 2 years of age develop severe growth retardation resulting in shortened stature, and the mean age of death is 12-15 years (Mounkes, 2003).

Homozygous Lmna^L530P/L530P mice show a slight waddling gait, suggesting immobility of joints. Similarly, progeria patients often adopt a 'horse-riding stance' with wide, shuffling gait due to joint stricture. Other progeroid features of Lmna^L530P/L530P mice include micrognathy and abnormal dentition -- in approximately half of the mutants a gap was observed between the lower two incisors, which also appeared yellowed. Such tooth gaps and discoloration are not observed in age-matched wild-type or heterozygous mice. Mutant mice continue to feed and defecate until death, and gonadal fat pads are present, suggesting that insufficient food consumption does not cause the decreased growth rate. On suspension by their tails, homozygous mutant mice did not clasp their hind legs, suggesting a normal peripheral motor response. No obvious abnormalities in social behavior or grooming were noted in the mutant mice. Mentation, IQ and personality are normal in progeria patients (Mounkes, 2003).

Pathology of the heart, skin, skeletal muscle and bone of 4-week old homozygous Lmna^L530P/L530P mice suggested developmental defects consistent with progeroid phenotypes. The most obvious sign of premature ageing occurred in the skin of mutant mice, which showed an epidermal layer thickened by regions of hyperkeratosis, thinning of the underlying dermis, atrophy of the accompanying muscle, and a complete absence of the subcutaneous fat layer. Increased deposits of collagen, similar to scleroderma, were observed in skin sections stained with Masson's trichrome. A decreased density of hair follicles observed in mutant skin sections may correlate to the alopecia observed in progeria patients; reduced numbers of eccrine and sebaceous glands were also observed in mutants. Mild to moderate degeneration mixed with hypoplasia and/or atrophy of both heart and skeletal muscle also occurs in mutant animals. Mutant mice had significantly smaller hearts with myocytes smaller than their wild-type counterparts. The amount of DNA in known weights of heart tissue was unchanged in mutants, suggesting that the smaller mutant hearts had the same number of cells as wild-type hearts. Counts of fibrocyte versus myocyte nuclei in mutant and wild-type hearts indicated that there were 2.6 fibrocytes per myocyte in wild-type hearts, but 4.1 fibrocytes per myocyte in mutant hearts. Although the total number of cells may be equivalent in mutant and wild-type hearts, there appeared to be fewer myocytes in mutant tissue with a compensatory increase in the fibrocyte population. Although not profound, a slight increase in extracellular collagen, indicative of degeneration, was noted in mutant hearts by Masson's trichrome staining. Hypoplasia and/or atrophy of esophageal muscles and myocardiocytes in homozygous Lmna^L530P/L530P mice suggested either incomplete development or loss of muscle mass in these tissues. Mild to moderate degeneration of other skeletal muscles, including pharyngeal, paravertebral, shoulder, hip, tibia, radius, femur, humerus and tongue, was also observed. However, dystrophic features such as centrally located nuclei and muscle fibers of varying diameter were not observed, ruling out muscular dystrophy, a prominent feature in Lmna-null mice (Mounkes, 2003).

Bone sections from 4-week-old mutant mice showed a decreased number and size of trabeculae, and a decrease in cortex width of vertebrae and femur, consistent with osteoporosis. In 50% of mutant mice surveyed, at least one scapula appeared to be smaller, thinner and misshapen compared with age-matched controls, suggesting incomplete development of the shoulder blade. Such defects in a quadruped where scapulae experience more perpendicular stress than the clavicle may correlate with clavicular malformation in MAD and progeria patients. The xiphisternum, formed of cartilage at the lower front midline of the rib cage, also appeared smaller in mutant animals in relation to overall ribcage size. No osteolysis of terminal digits was observed. Densitometry scanning of whole mice indicated a decrease in total bone mineral density of 25%-30% in mutant animals compared with wildtype or heterozygous littermates. Taken together these results suggest either a premature loss of bone mass or the incomplete development of the skeleton in homozygous mice, features prominent in patients with HGPS2. Consistent with several case histories of progeria subjects, juvenile testes and ovaries were observed in mutant mice, indicating incomplete gonadal development. Total necropsy of the animals revealed no incidence of tumors or gross organ deficiencies (Mounkes, 2003).

At the cellular level, nuclear morphology is disrupted in primary mouse embryo fibroblasts (MEFs) from Lmna^L530P/L530P homozygous embryos, consistent with phenotypes observed in fibroblast cell lines from AD-EDMD, FPLD and DCM patients. Discontinuities of the nuclear envelope were seen in 58% of mutant cells, whereas only 20% of heterozygous and 5.6% of wild-type cells showed herniations of the nucleus. Areas in which the inner nuclear membrane and outer nuclear membrane appeared to lose contact with each other, resulting in the expansion of the perinuclear space and ballooning of chromatin into a bleb-like structure, may reflect a weakened nuclear lamina in cells expressing Lmna^L530P/L530P. Consistent with a defective nuclear lamina, decreased levels of lamin A were incorporated into the lamina as deduced by indirect immunofluorescence. Lamin C is completely absent from the nuclear envelope in mutant primary MEFs, and there is an increase in lamin-C-reactive peptides in the cytoplasm, an observation not seen in wild-type cells when stained with the same antibody. These results suggest that the mutant L530P form of lamin A is competent to assemble at least partially or temporarily in the nuclear lamina, but lamin C is not assembled into the nuclear envelope in Lmna^L530P/L530P cells (Mounkes, 2003).

Similar defective lamin A and C incorporation into the lamina was observed in Lmna-null primary MEFs when a mutant complementary DNA encoding the L530P variant of Lmna was expressed. Western analysis indicates that any A-type lamins translated are unstable, since greatly diminished levels of mutant protein were detected in homozygous mutant cell lines. Cells heterozygous for Lmna^L530P/L530P had about half the wild-type amount of lamin A and C protein. Consistent with low levels of protein, decreased levels of Lmna transcripts were detected by Northern analysis in cells from homozygous embryos, suggesting instability of the mutant message, probably due to a splicing defect. Polymerase chain reaction with reverse transcription (RT-PCR) of the region surrounding exon 9, which encodes residue 530, suggests aberrant splicing between exons 9 and 10 in Lmna^L530P/L530P mice (Mounkes, 2003).

Thinning of the skin, hypoplasia and degeneration of cardiac and skeletal muscle, osteoporosis, and abnormal dentition in homozygous Lmna^L530P/L530P mice are all phenotypes consistent with those seen in progeria patients. Marked growth retardation and shortened lifespan are also hallmarks of progeria. The question of how faithfully progeria simulates natural ageing is, however, controversial, because cataracts, age-related cancers and cognitive decline are not observed in progeria patients. Lmna^L530P/L530P homozygous mutant mice also failed to show any signs of these age-induced pathologies. One possibility is that progeria is a developmental disease in which there is either accelerated or improper development of certain tissue types, while other tissues are relatively unaffected. The tissues most severely affected in progeria patients and the mutant mice arise from the mesenchymal stem cell lineage, making this a particularly interesting cell lineage for further study in Lmna^L530P/L530P mice. Accelerated development and premature death of terminally differentiated mesenchymal cells may account for phenotypes in progeric heart, muscle, bone and subcutaneous adipose tissues. Alternatively, terminally differentiated tissues with defects in the nuclear lamina may be unable to maintain the chromatin organization necessary to preserve a state of terminal differentiation, resulting in dedifferentiation and subsequent redifferentiation into other cell types with age in certain tissues. Preliminary data indicates that homozygous Lmna^L530P/L530P muscle myoblasts and fibroblasts readily differentiate into adipocytes, suggesting this latter hypothesis may be valid. Notably, muscles in older individuals show accumulation of fat compared with younger muscle, which could be a result of muscle tissue transdifferentiating into fat during old age. These questions are currently being studied in the Lmna^L530P/L530P mouse in expectation of learning more about the extent to which differentiation and the loss of cell identity contributes to the ageing process, especially with respect to the phenotypes seen in progeria (Mounkes, 2003).

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by symmetrical widespread myelin loss in the central nervous system, with a phenotype similar to chronic progressive multiple sclerosis. This study has identify a genomic duplication that causes ADLD. Affected individuals carry an extra copy of the gene for the nuclear laminar protein lamin B1, resulting in increased gene dosage in brain tissue from individuals with ADLD. Increased expression of lamin B1 in Drosophila melanogaster resulted in a degenerative phenotype. In addition, an abnormal nuclear morphology was apparent when cultured cells overexpressed this protein. This is the first human disease attributable to mutations in the gene encoding lamin B1. Antibodies to lamin B are found in individuals with autoimmune diseases, and it is also an antigen recognized by a monoclonal antibody raised against plaques from brains of individuals with multiple sclerosis. This raises the possibility that lamin B may be a link to the autoimmune attack that occurs in multiple sclerosis (Padiath, 2006).

Lamin subcellular distribution, assembly and phosphorylation

The nuclear lamina forms a protein mesh that underlies the nuclear membrane. In most mammalian cells it contains the intermediate filament proteins, lamins A, B and C. As their name indicates, lamins are generally thought to be confined to the nuclear periphery. They also form part of a diffuse skeleton that ramifies throughout the interior of the nucleus. Unlike their peripheral counterparts, these internal lamins are buried in dense chromatin and so are inaccessible to antibodies, but accessibility can be increased by removing chromatin. Knobs and nodes on an internal skeleton can then be immunolabelled using fluorescein- or gold-conjugated anti-lamin A antibodies. These results suggest that the lamins are misnamed as they are also found internally (Hozak, 1995).

The assembly kinetics of Xenopus wild type lamin A and 7 lamin A mutants were analyzed by the microinjection of fluorescein (5-IAF)-labeled protein into mouse 3T3 cells. The wild type protein and all mutants containing a nuclear localization signal are transported within 10 min into the nucleus. The wild type protein exhibits a strong lamina fluorescence 30 min after microinjection, whereas mutant molecules showed a delayed but complete, a delayed and incomplete, or no lamina assembly at all. The lamin A mutant lacking the carboxy-terminal cysteine of the CxxM-motif exhibits a delayed but complete assembly, whereas there is no significant interaction of this mutant with the lamina. An additional domain has been identified in the carboxy-terminal tail of lamin A that promotes its assembly into the lamina. In vitro this domain is required for the chromatin binding of lamin A. Lamin A molecules lacking the non-helical amino-terminal head domain show no significant lamina staining, whereas point mutations in conserved regions of the alpha helix result in an incomplete assembly (Schmidt, 1996).

Lamin A is synthesized in the cytoplasm as a precursor bearing a carboxyl-terminal CaaX box or isoprenylation signal. This precursor is post-translationally processed through multiple steps: isoprenylation with a farnesyl residue on the cysteine of the CaaX box, proteolytic removal of the last three amino acids, carboxymethylation of the cysteine residue and, finally, proteolytic removal of 15 amino acids from the carboxyl terminus. This last step gives rise to mature lamin A from which the isoprenylated terminus has been removed. Isoprenylation is a prerequisite for all other steps of processing. The subcellular location of these processing steps for lamin A is still a matter of debate. An antibody has been produced that is specific to the 18 amino acid carboxyl terminus of the lamin A precursor that does not recognize mature lamin A. This antibody detects intranuclear foci by immunofluorescence. Larger amounts of lamin A precursor are accumulated by treating cells with mevinolin (MVN), an inhibitor of isoprenoid synthesis. In MVN-treated cells, the lamin A precursor accumulates most strikingly in the peripheral nuclear lamina where it was assembled, while intranuclear foci are maintained. The addition of an excess of mevalonate (MVA), which restores isoprenylation activity, to MVN-treated cells leads to a progressive disappearance of the lamin A precursor from the peripheral lamina. This process is completed after 4 hours of MVA treatment, after which the lamin A precursor is restricted to intranuclear foci. It has been concluded that the non-isoprenylated lamin A precursor appears competent for assembly into the peripheral nuclear lamina, and that all the processing steps leading to mature lamin A can occur within the nuclear space (Sasseville, 1995).