Farnesylation of lamin B1 is important for retention of nuclear chromatin during neuronal migration

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left “naked” (free from the nuclear lamina). Thus, farnesylation of lamin B1—but not lamin B2—is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.The nuclear lamina is an intermediate filament meshwork that lies beneath the inner nuclear membrane. This lamina provides structural support for the nucleus and also interacts with nuclear proteins and chromatin, thereby affecting many functions within the cell nucleus (1, 2). In mammals, the main protein components of the nuclear lamina are lamins A and C (A-type lamins) and lamins B1 and B2 (B-type lamins).Both B-type lamins and prelamin A (the precursor of lamin A) terminate with a CaaX motif, which triggers three posttranslational modifications (3–5): farnesylation of the carboxyl-terminal cysteine (the “C” in the CaaX motif) (6), endoproteolytic cleavage of the last three amino acids (the -aaX) (7, 8), and carboxyl methylation of the newly exposed farnesylcysteine (9, 10). Prelamin A undergoes a second endoproteolytic cleavage event, mediated by zinc metalloproteinase STE24 (ZMPSTE24), which removes 15 additional amino acids from the carboxyl terminus, including the farnesylcysteine methyl ester (11–14). Lamins B1 and B2 do not undergo the second cleavage step and therefore retain their farnesyl lipid anchor.The discovery that Hutchinson-Gilford progeria syndrome (HGPS) is caused by a LMNA mutation yielding an internally truncated farnesyl–prelamin A (15) has focused interest in the farnesylation of nuclear lamins. This interest has been fueled by the finding that disease phenotypes in mouse models of HGPS could be ameliorated by blocking protein farnesylation with a protein farnesyltransferase inhibitor (FTI) (16–19). Most recently, children with HGPS seemed to show a positive response to FTI treatment (20).The prospect of using an FTI to treat children with HGPS naturally raises the issue of the importance of protein farnesylation for lamin B1 and lamin B2. The B-type lamins are expressed in all mammalian cells and have been highly conserved during vertebrate evolution. The B-type lamins have been reported to participate in many functions within the cell nucleus, including DNA replication (21) and the formation of the mitotic spindle (22). Recently, both lamin B1 and lamin B2 have been shown to be important for neuronal migration within the developing brain (23–26). A deficiency of either protein causes abnormal layering of cortical neurons (23, 24). Coffinier et al. (24) proposed that the neuronal migration defect might be the consequence of impaired integrity of the nuclear lamina. Whether the farnesylation of lamin B1 or lamin B2 is important for neuronal migration is not known.Over the past few years, it has become increasingly clear that mouse models are important for elucidating the in vivo relevance of lamin posttranslational processing. In the case of prelamin A, cell culture studies suggested that protein farnesylation plays a vital role in the targeting of prelamin A to the nuclear rim (27–29), but recent studies with gene-targeted mice have raised questions about the in vivo relevance of those findings. For example, knock-in mice that produce mature lamin A directly (bypassing prelamin A synthesis and protein farnesylation) are free of disease, and the nuclear rim positioning of lamin A in the tissues of mice is quite normal (30).To assess the importance of protein farnesylation for B-type lamins, we reasoned that mouse models would be even more important because these lamins are important for neuronal migration in the developing brain, a complex process that requires the use of animal models. In the present study, we investigated the in vivo functional relevance of protein farnesylation in B-type lamins by creating knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2.     

Abnormal development of the cerebral cortex and cerebellum in the setting of lamin B2 deficiency

Nuclear lamins are components of the nuclear lamina, a structural scaffolding for the cell nucleus. Defects in lamins A and C cause an array of human diseases, including muscular dystrophy, lipodystrophy, and progeria, but no diseases have been linked to the loss of lamins B1 or B2. To explore the functional relevance of lamin B2, we generated lamin B2-deficient mice and found that they have severe brain abnormalities resembling lissencephaly, with abnormal layering of neurons in the cerebral cortex and cerebellum. This neuronal layering abnormality is due to defective neuronal migration, a process that is dependent on the organized movement of the nucleus within the cell. These studies establish an essential function for lamin B2 in neuronal migration and brain development.     

Biogenesis of the nuclear lamina: in vivo synthesis and processing of nuclear protein precursors.

Utilizing antibodies against lamins A, B1, and B2, we have studied the biogenesis of the nuclear lamina in chicken embryo fibroblasts. (Lamins B1 and B2 have been identified recently as structurally distinct "lamin B" proteins.) We demonstrate that, unique among the nuclear proteins studied to date, lamin A is synthesized as a higher molecular mass precursor. A short-lived higher molecular mass variant (t 1/2 approximately equal to 3 min) accompanying the mature-size protein was also detected in the case of lamin B2 biosynthesis, but no precursor was found for lamin B1. By combining pulse-chase experiments with subcellular fractionation, we provide evidence that synthesis of lamin proteins occurs on free polysomes; subsequently, the newly synthesized proteins become rapidly associated with a crude nuclear fraction. The lamin A precursor is processed within the nucleus with a half-time of about 30 min. Concomitantly, lamin proteins acquire a characteristic resistance to detergent extraction, suggesting their insertion into a submembraneous protein network. The described biogenetic pathway involving precursor synthesis and processing is very unusual for nuclear proteins; it may have interesting implications for the mechanisms of transport and assembly of poorly soluble nuclear proteins.     

Nuclear lamins and peripheral nuclear antigens during fertilization and embryogenesis in mice and sea urchins.

Nuclear structural changes during fertilization and embryogenesis in mice and in sea urchins have been followed by using antibodies against the nuclear lamins A/C and B and against antigens at the periphery of nuclei and chromosomes. Lamins are found on all pronuclei and nuclei during mouse fertilization, but with a diminished intensity on the second polar body nucleus. On sperm in both systems, lamins are reduced and detected only at the acrosomal and centriolar fossae. In sea urchin eggs, lamins are found on both pronuclei. Unlike in other dividing cells, the mitotic chromosomes of sea urchin eggs and embryos retain an association with lamins. The peripheral antibodies delineate each chromosome and nucleus except the mature mouse sperm nucleus. A dramatic change from the expected lamin distribution occurs during early development. In mouse morulae or blastocysts, lamins A/C are no longer recognized, although lamin B remains. In sea urchins both lamins A/C and lamin B, as detected with polyclonal antibodies, are lost after the blastula stage, although a different lamin A/C epitope emerges as recognized by a monoclonal antibody. These results demonstrate that pronucleus formation in both systems involves a new association or exposure of lamins, that the polar body nucleus is largely restricted from the cytoplasmic pool of lamins, and that mitotic chromosomes in the rapidly proliferating sea urchin egg retain associated lamins. They also suggest that changes in the expression or exposure of different lamins are a common feature of embryogenesis.     

Change of karyoskeleton during spermatogenesis of Xenopus: expression of lamin LIV, a nuclear lamina protein specific for the male germ line.

Lamins are the major constituent proteins of the nuclear lamina. In the frog, Xenopus laevis, they are the products of a multigene family whose expression can be correlated to certain routes of cell differentiation. For example, lamins LI (Mr, 72,000) and LII (Mr, 68,000) is expressed, together with LI/LII, in certain highly differentiated cell types such as neurons and muscle cells and is the only lamin present in diplotene oocytes. Here we report the identification by means of two monoclonal antibodies of a fourth lamin (LIV) of Mr 75,000, which is expressed specifically during the later stages of spermatogenesis. In the seminiferous tubules, Sertoli cells contain LI/LII and LIII whereas, among the spermatogenic cells, spermatogonia contain only LI and LII. In contrast, in spermatids and sperm cells these lamins are completely replaced by lamin LIV. Primary spermatocytes are negative with both antibodies, indicating that a switch in the expression of lamins occurs early in spermatogenesis. Lamin LIV is distributed in patches along the nuclear envelopes of elongated spermatids and sperm cells rather than in the characteristic continuous lamina pattern found in most other cells. We hypothesize that the specific expression of lamin LIV is related to the conspicuous changes of nuclear architecture and chronmatin composition that are known to take place during the late stages of sperm development.     

The A-type lamins: nuclear structural proteins as a focus for muscular dystrophy and cardiovascular diseases.

Mutations in the lamin A (LMNA) gene are associated with the tissue-specific diseases Emery-Dreifuss muscular dystrophy (EDMD), limb girdle muscular dystrophy (LGMD-1B), dilated cardiomyopathy with conduction system disease (DCM-CD), and Dunnigan's familial partial lipodystrophy (FPLD). Lamins A and C, the products of the LMNA gene, are nuclear intermediate filament proteins and are the major structural components of the lamina network that underlies and supports the nuclear envelope. Nuclear fragility and mislocalization of the nuclear envelope protein emerin are two defects induced by a lack of the A-type lamins. These observations reveal that organization and structural integrity of the nucleus are critical factors in the origins of certain dystrophic and cardiovascular diseases.     

Lamin expression in human adipose cells in relation to anatomical site and differentiation state

Familial partial lipodystrophy-Dunnigan variety (FPLD) is an autosomal dominant form of lipodystrophy resulting in a loss of sc fat from the trunk and limbs with retention of fat in the visceral depots, face, and neck. Specific point mutations in the gene encoding the nuclear lamina proteins, lamins A and C, have been established to cause this syndrome. We undertook studies to determine which members of the lamin family were expressed in human fat cells, to examine the effect of differentiation state on lamin A and C expression in human preadipocytes, and to test the hypothesis that regional variation in lamin A/C expression might underlie the stereotyped anatomical pattern of FPLD. Lamins A, C, and B1, but not B2, were expressed in sc mature human adipocytes. Subcutaneous preadipocytes expressed all four lamins, with lamin A and C expression increasing with ex vivo differentiation. Consistent with previously reported resistance to ex vivo differentiation, omental preadipocytes did not show an increase in lamin A or C mRNA under these conditions. Lamin A/C mRNA levels were similar in isolated mature adipocytes and preadipocytes from omental, sc, and neck sites. However, lamin C was consistently lower, and the ratio of lamin A/C mRNA was higher in sc mature adipocytes compared with omental mature adipocytes. We conclude that the depot-specific pattern of lamin A/C expression does not provide clues to the mechanism of FPLD. Nonetheless, these studies provide new information regarding the expression of lamin isoforms in normal human adipose cells, which will inform future studies of the molecular pathogenesis of FPLD.     

Heterotypic and homotypic associations between the nuclear lamins: site-specificity and control by phosphorylation.

Using purified components in affinity chromatography and blot binding assays, we have found that rat liver lamins A, B, and C can associate in homotypic and heterotypic fashions. Heterotypic A-B and C-B complexes are unusually stable and involve the common amino-terminal domain of lamins A and C, but not their helical "rod" domain. A synthetic peptide, comprising the first 32 amino acid residues of lamins A and C, is able to fully compete with the intact molecules for binding to lamin B. Conversely, heterotypic A-C associations and homotypic A-A and C-C interactions appear significantly weaker than A/C-B binding and do not involve the lamin A and C amino-terminal domain. Homotypic B-B complexes are not formed to any considerable extent unless isolated lamin B subunits are "superphosphorylated" in vitro with protein kinase A. However, when lamins A and C are similarly modified, no changes in their binding specificity can be detected. These data suggest that the nuclear lamina, unlike other multicomponent intermediate filaments, constitutes a nonobligatory heteropolymer. They also indicate that cAMP-dependent phosphorylation of interphase lamin B could cause remodeling of the lamina and establishment of homopolymeric domains.     

The tail domain of lamin Dm0 binds histones H2A and H2B

In multicellular organisms, the higher order organization of chromatin during interphase and the reassembly of the nuclear envelope during mitosis are thought to involve an interaction between the nuclear lamina and chromatin. The nuclear distribution of lamins and of peripheral chromatin is highly correlated in vivo, and lamins bind specifically to chromatin in vitro. Deletion mutants of Drosophila lamin Dm0 were expressed to map regions of the protein that are required for its binding to chromosomes. The binding activity requires two regions in the lamin Dm0 tail domain. The apparent Kd of binding of the lamin Dm0 tail domain was found to be approximately 1 microM. Chromatin subfractions were examined to search for possible target molecules for the binding of lamin Dm0. Isolated polynucleosomes, nucleosomes, histone octamer, histone H2A/H2B dimer, and histones H2A or H2B displaced the binding of lamin Dm0 tail to chromosomes. This displacement was specific, because polyamines or proteins such as histones H1, H3, or H4 did not displace the binding of the lamin Dm0 tail to chromosomes. In addition, DNA sequences, including M/SARs, did not interfere with the binding of lamin Dm0 tail domain to chromosomes. Taken together, these results suggest that the interaction between the tail domain of lamin Dm0 and histones H2A and H2B may mediate the attachment of the nuclear lamina to chromosomes in vivo.     

A progeria mutation reveals functions for lamin A in nuclear assembly,architecture, and chromosome organization

Numerous mutations in the human A-type lamin gene (LMNA) cause the premature aging disease, progeria. Some of these are located in the α-helical central rod domain required for the polymerization of the nuclear lamins into higher order structures. Patient cells with a mutation in this domain, 433G>A (E145K) show severely lobulated nuclei, a separation of the A- and B-type lamins, alterations in pericentric heterochromatin, abnormally clustered centromeres, and mislocalized telomeres. The induction of lobulations and the clustering of centromeres originate during postmitotic nuclear assembly in daughter cells and this early G1 configuration of chromosomes is retained throughout interphase. In vitro analyses of E145K-lamin A show severe defects in the assembly of protofilaments into higher order lamin structures. The results show that this central rod domain mutation affects nuclear architecture in a fashion distinctly different from the changes found in the most common form of progeria caused by the expression of LAΔ50/progerin. The study also emphasizes the importance of lamins in nuclear assembly and chromatin organization.     

Mechanical model of blebbing in nuclear lamin meshworks

Much of the structural stability of the nucleus comes from meshworks of intermediate filament proteins known as lamins forming the inner layer of the nuclear envelope called the nuclear lamina. These lamin meshworks additionally play a role in gene expression. Abnormalities in nuclear shape are associated with a variety of pathologies, including some forms of cancer and Hutchinson–Gilford Progeria Syndrome, and often include protruding structures termed nuclear blebs. These nuclear blebs are thought to be related to pathological gene expression; however, little is known about how and why blebs form. We have developed a minimal continuum elastic model of a lamin meshwork that we use to investigate which aspects of the meshwork could be responsible for bleb formation. Mammalian lamin meshworks consist of two types of lamin proteins, A type and B type, and it has been reported that nuclear blebs are enriched in A-type lamins. Our model treats each lamin type separately and thus, can assign them different properties. Nuclear blebs have been reported to be located in regions where the fibers in the lamin meshwork have a greater separation, and we find that this greater separation of fibers is an essential characteristic for generating nuclear blebs. The model produces structures with comparable morphologies and distributions of lamin types as real pathological nuclei. Thus, preventing this opening of the meshwork could be a route to prevent bleb formation, which could be used as a potential therapy for the pathologies associated with nuclear blebs.     

Decreased and aberrant nuclear lamin expression in gastrointestinal tract neoplasms

BACKGROUND: Altered expression of lamins A/C and B1, constituent proteins of the nuclear lamina, may occur during differentiation and has also been reported in primary lung cancer. AIMS: To examine the expression of these proteins in gastrointestinal neoplasms. PATIENTS: Archival human paraffin wax blocks and frozen tissue from patients undergoing surgical resection or endoscopic biopsy. METHODS: Immunohistochemistry and western blotting using polyclonal antisera against A type lamins and lamin B1. RESULTS: The expression of lamin A/C was reduced and was frequently undetectable by immunohistochemistry in all primary colon carcinomas and adenomas, and in 7/8 primary gastric cancers. Lamin B1 expression was reduced in all colon cancers, 16/18 colonic adenomas, and 6/8 gastric cancers. Aberrant, cytoplasmic labelling with both antibodies occurred in some colonic cancers and around one third of colonic adenomas. Cytoplasmic lamin A/C expression was detected in 3/8 gastric cancers. Lamin expression was reduced in gastric dysplasia, but not intestinal metaplasia, atrophy, or chronic gastritis. Lamin expression was low in carcinomas of oesophagus, prostate, breast, and uterus, but not pancreas. CONCLUSIONS: Reduced expression of nuclear lamins, sometimes together with aberrant, cytoplasmic immunoreactivity is common in gastrointestinal neoplasms. Altered lamin expression may be a biomarker of malignancy in the gastrointestinal tract.     

Antibodies to nuclear lamin C in chronic hepatitis delta virus infection

Sera of patients with chronic hepatitis delta virus infection stained the nuclear periphery in indirect immunofluorescence. Using proteins of isolated nuclei, isolated nuclear matrices, the nuclear pore complex-lamina fraction and purified lamins A and C as antigen source in immunoblotting experiments, nuclear lamin C was identified as the reactive antigen. Most sera tested (8 of 10) recognized nuclear lamin C exclusively, but not the nuclear lamins A and B. Antibodies reacting with both nuclear lamins A and C, which share extensive sequence homologies, have been reported to occur in autoimmune hepatitis and primary biliary cirrhosis. The present findings suggest that the novel autoantibody associated with chronic hepatitis delta virus infection recognizes an epitope localized in the short carboxyterminal region of nuclear lamin C.     

Nesprin-1 mutations in human and murine cardiomyopathy

Mutations in LMNA, the gene encoding the nuclear membrane proteins, lamins A and C, produce cardiac and muscle disease. In the heart, these autosomal dominant LMNA mutations lead to cardiomyopathy frequently associated with cardiac conduction system disease. Herein, we describe a patient with the R374H missense variant in nesprin-1α, a protein that binds lamin A/C. This individual developed dilated cardiomyopathy requiring cardiac transplantation. Fibroblasts from this individual had increased expression of nesprin-1α and lamins A and C, indicating changes in the nuclear membrane complex. We characterized mice lacking the carboxy-terminus of nesprin-1 since this model expresses nesprin-1 without its carboxy-terminal KASH domain. These Δ/ΔKASH mice have a normally assembled but dysfunctional nuclear membrane complex and provide a model for nesprin-1 mutations. We found that Δ/ΔKASH mice develop cardiomyopathy with associated cardiac conduction system disease. Older mutant animals were found to have elongated P wave duration, elevated atrial and ventricular effective refractory periods indicating conduction defects in the myocardium, and reduced fractional shortening. Cardiomyocyte nuclei were found to be elongated with reduced heterochromatin in the Δ/ΔKASH hearts. These findings mirror what has been described from lamin A/C gene mutations and reinforce the importance of an intact nuclear membrane complex for a normally functioning heart.