Lamin Gene Research Articles

Hippocampal LMNA Gene Expression is Increased in Late-Stage Alzheimer's Disease.

Lamins are fibrillary proteins that are crucial in maintaining nuclear shape and function. Recently, B-type lamin dysfunction has been linked to tauopathies. However, the role of A-type lamin in neurodegeneration is still obscure. Here, we examined A-type and B-type lamin expression levels by RT-qPCR in Alzheimer’s disease (AD) patients and controls in the hippocampus, the core of tau pathology in the brain. LMNA, LMNB1, and LMNB2 genes showed moderate mRNA levels in the human hippocampus with highest expression for the LMNA gene. Moreover, LMNA mRNA levels were increased at the late stage of AD (1.8-fold increase; p-value < 0.05). In addition, a moderate positive correlation was found between age and LMNA mRNA levels (Pearson’s r = 0.581, p-value = 0.018) within the control hippocampal samples that was not present in the hippocampal samples affected by AD. A-type and B-type lamin genes are expressed in the human hippocampus at the transcript level. LMNA mRNA levels are up-regulated in the hippocampal tissue in late stages of AD. The effect of age on increasing LMNA expression levels in control samples seems to be disrupted by the development of AD pathology.

High-throughput microfluidic micropipette aspiration device to probe time-scale dependent nuclear mechanics in intact cells.

The mechanical properties of the cell nucleus are increasingly recognized as critical in many biological processes. The deformability of the nucleus determines the ability of immune and cancer cells to migrate through tissues and across endothelial cell layers, and changes to the mechanical properties of the nucleus can serve as novel biomarkers in processes such as cancer progression and stem cell differentiation. However, current techniques to measure the viscoelastic nuclear mechanical properties are often time consuming, limited to probing one cell at a time, or require expensive, highly specialized equipment. Furthermore, many current assays do not measure time-dependent properties, which are characteristic of viscoelastic materials. Here, we present an easy-to-use microfluidic device that applies the well-established approach of micropipette aspiration, adapted to measure many cells in parallel. The device design allows rapid loading and purging of cells for measurements, and minimizes clogging by large particles or clusters of cells. Combined with a semi-automated image analysis pipeline, the microfluidic device approach enables significantly increased experimental throughput. We validated the experimental platform by comparing computational models of the fluid mechanics in the device with experimental measurements of fluid flow. In addition, we conducted experiments on cells lacking the nuclear envelope protein lamin A/C and wild-type controls, which have well-characterized nuclear mechanical properties. Fitting time-dependent nuclear deformation data to power law and different viscoelastic models revealed that loss of lamin A/C significantly altered the elastic and viscous properties of the nucleus, resulting in substantially increased nuclear deformability. Lastly, to demonstrate the versatility of the devices, we characterized the viscoelastic nuclear mechanical properties in a variety of cell lines and experimental model systems, including human skin fibroblasts from an individual with a mutation in the lamin gene associated with dilated cardiomyopathy, healthy control fibroblasts, induced pluripotent stem cells (iPSCs), and human tumor cells. Taken together, these experiments demonstrate the ability of the microfluidic device and automated image analysis platform to provide robust, high throughput measurements of nuclear mechanical properties, including time-dependent elastic and viscous behavior, in a broad range of applications.

Differential expression of nuclear lamin subtypes in the neural cells of the adult rat cerebral cortex

Lamins are type V intermediate filament proteins that are located beneath the inner nuclear membrane. In mammalian somatic cells, LMNB1 and LMNB2 encode somatic lamins B1 and B2, respectively, and the LMNA gene is alternatively spliced to generate somatic lamins A and C.Mutations in lamin genes have been linked to many human hereditary diseases, including neurodegenerative disorders. Knowledge about lamins in the nervous system has been accumulated recently, but a precise analysis of lamin subtypes in glial cells has not yet been reported. In this study we investigated the composition of lamin subtypes in neurons, astrocytes, oligodendrocyte-lineage cells, and microglia in the adult rat cerebral cortex using an immunohistochemical staining method. Lamin A was not observed in neurons and glial cells. Lamin C was observed in astrocytes, mature oligodendrocytes and neurons, but not observed in oligodendrocyte progenitor cells. Microglia also did not stain positive for lamin C which differed from macrophages, with lamin C positive. Lamin B1 and B2 were observed in all glial cells and neurons. Lamin B1 was intensely positive in oligodendrocyte progenitor cells compared with other glial cells and neurons. Lamin B2 was weakly positive in all glial cells compared to neurons. Our current study might provide useful information to reveal how the onset mechanisms of human neurodegenerative diseases are associated with mutations in genes for nuclear lamin proteins.

Neuronal Lamin regulates motor circuit integrity and controls motor function and lifespan.

Neuronal aging involves a progressive decline in cognitive abilities and loss of motor function. Mutations in human Lamin genes (LMNA, LMNB1, LMNB2) lead to a wide-range of diseases including muscular dystrophy, peripheral neuropathy and progeria. Here we investigate the role of neuronal Lamin in regulating age-related phenotypes. Neuronal targeting of Lamin led to shortened lifespan, progressive impairment of motor function and loss of dopaminergic (DA) neurons within the protocerebral anterior medial (PAM) cluster in the Drosophila melanogaster brain. Loss of neuronal Lamin caused an age-related decline in neural physiology, with slower neurotransmission and increased chance of motor circuit failure with age. Unexpectedly, Lamin-dependent decline in motor function was specific for the chemical synapses of the dorsal longitudinal muscle (DLM). Together these findings highlight a central role for Lamin dysfunction in regulating neuronal survival and motor circuit physiology during aging.

N-acetyl cysteine alleviates oxidative stress and protects mice from dilated cardiomyopathy caused by mutations in nuclear A-type lamins gene.

Cardiomyopathy caused by lamin A/C gene (LMNA) mutations (hereafter referred as LMNA cardiomyopathy) is an anatomic and pathologic condition associated with muscular and electrical dysfunction of the heart, often leading to heart failure-related disability. There is currently no specific therapy available for patients that target the molecular pathophysiology of LMNA cardiomyopathy. We showed here an increase in oxidative stress levels in the hearts of mice carrying LMNA mutation, associated with a decrease of the key cellular antioxidant glutathione (GHS). Oral administration of N-acetyl cysteine, a GHS precursor, led to a marked improvement of GHS content, a decrease in oxidative stress markers including protein carbonyls and an improvement of left ventricular structure and function in a model of LMNA cardiomyopathy. Collectively, our novel results provide therapeutic insights into LMNA cardiomyopathy.

Microtubule cytoskeleton regulates Connexin 43 localization and cardiac conduction in cardiomyopathy caused by mutation in A-type lamins gene.

Mutations in the lamin A/C gene (LMNA) cause an autosomal dominant inherited form of dilated cardiomyopathy associated with cardiac conduction disease (hereafter referred to as LMNA cardiomyopathy). Compared with other forms of dilated cardiomyopathy, mutations in LMNA are responsible for a more aggressive clinical course owing to a high rate of malignant ventricular arrhythmias. Gap junctions are intercellular channels that allow direct communication between neighboring cells, which are involved in electrical impulse propagation and coordinated contraction of the heart. For gap junctions to properly control electrical synchronization in the heart, connexin-based hemichannels must be correctly targeted to intercalated discs, Cx43 being the major connexin in the working myocytes. We here showed an altered distribution of Cx43 in a mouse model of LMNA cardiomyopathy. However, little is known on the molecular mechanisms of Cx43 remodeling in pathological context. We now show that microtubule cytoskeleton alteration and decreased acetylation of α-tubulin lead to remodeling of Cx43 in LMNA cardiomyopathy, which alters the correct communication between cardiomyocytes, ultimately leading to electrical conduction disturbances. Preventing or reversing this process could offer a strategy to repair damaged heart. Stabilization of microtubule cytoskeleton using Paclitaxel improved intraventricular conduction defects. These results indicate that microtubule cytoskeleton contributes to the pathogenesis of LMNA cardiomyopathy and that drugs stabilizing the microtubule may be beneficial for patients.

Potential association of LMNA-associated generalized lipodystrophy with juvenile dermatomyositis

BackgroundJuvenile dermatomyositis (JDM) is an auto-immune muscle disease which presents with skin manifestations and muscle weakness. At least 10% of the patients with JDM present with acquired lipodystrophy. Laminopathies are caused by mutations in the lamin genes and cover a wide spectrum of diseases including muscular dystrophies and lipodystrophy. The p.T10I LMNA variant is associated with a phenotype of generalized lipodystrophy that has also been called atypical progeroid syndrome.Case presentationA previously healthy female presented with bilateral proximal lower extremity muscle weakness at age 4. She was diagnosed with JDM based on her clinical presentation, laboratory tests and magnetic resonance imaging (MRI). She had subcutaneous fat loss which started in her extremities and progressed to her whole body. At age 7, she had diabetes, hypertriglyceridemia, low leptin levels and low body fat on dual energy X-ray absorptiometry (DEXA) scan, and was diagnosed with acquired generalized lipodystrophy (AGL). Whole exome sequencing (WES) revealed a heterozygous c.29C > T; p.T10I missense pathogenic variant in LMNA, which encodes lamins A and C. Muscle biopsy confirmed JDM rather than muscular dystrophy, showing perifascicular atrophy and perivascular mononuclear cell infiltration. Immunofluroscence of skin fibroblasts confirmed nuclear atypia and fragmentation.ConclusionsThis is a unique case with p.T10I LMNA variant displaying concurrent JDM and AGL. This co-occurrence raises the intriguing possibility that LMNA, and possibly p.T10I, may have a pathogenic role in not only the occurrence of generalized lipodystrophy, but also juvenile dermatomyositis. Careful phenotypic characterization of additional patients with laminopathies as well as individuals with JDM is warranted.

Nuclear Lamins: Thin Filaments with Major Functions.

The nuclear lamina is a nuclear peripheral meshwork that is mainly composed of nuclear lamins, although a small fraction of lamins also localizes throughout the nucleoplasm. Lamins are classified as type V intermediate filament (IF) proteins. Mutations in lamin genes cause at least 15 distinct human diseases, collectively termed laminopathies, including muscle, metabolic, and neuronal diseases, and can cause accelerated aging. Most of these mutations are in the LMNA gene encoding A-type lamins. A growing number of nuclear proteins are known to bind lamins and are implicated in both nuclear and cytoskeletal organization, mechanical stability, chromatin organization, signaling, gene regulation, genome stability, and cell differentiation. Recent studies reveal the organization of the lamin filament meshwork in somatic cells where they assemble as tetramers in cross-section of the filaments.

Evolutionary changes in lamin expression in the vertebrate lineage

ABSTRACT The nuclear lamina is involved in fundamental nuclear functions and provides mechanical stability to the nucleus. Lamin filaments form a meshwork closely apposed to the inner nuclear membrane and a small fraction of lamins exist in the nuclear interior. Mutations in lamin genes cause severe hereditary diseases, the laminopathies. During vertebrate evolution the lamin protein family has expanded. While most vertebrate genomes contain 4 lamin genes, encoding the lamins A, B1, B2, and LIII, the majority of non-vertebrate genomes harbor only a single lamin gene. We have collected lamin gene and cDNA sequence information for representatives of the major vertebrate lineages. With the help of RNA-seq data we have determined relative lamin expression levels for representative tissues for species of 9 different gnathostome lineages. Here we report that the level of lamin A expression is low in cartilaginous fishes and ancient fishes and increases toward the mammals. Lamin B1 expression shows an inverse tendency to that of lamin A. Possible implications for the change in the lamin A to B ratio is discussed in the light of its role in nuclear mechanics.

Proteome Analysis of Drosophila Mutants Identifies a Regulatory Role for 14-3-3ε in Metabolic Pathways.

The evolutionary conserved family of 14-3-3 proteins appears to have a role in integrating numerous intracellular pathways, including signal transduction, intracellular trafficking, and metabolism. However, little is known about how this interactive network might be affected by the direct abrogation of 14-3-3 function. The loss of Drosophila 14-3-3ε resulted in reduced survival of mutants during larval-to-adult transition, which is known to depend on an energy supply coming from the histolysis of fat body tissue. Here we report a differential proteomic analysis of larval fat body tissue at the onset of larval-to-adult transition, with the loss of 14-3-3ε resulting in the altered abundance of 16 proteins. These included proteins linked to protein biosynthesis, glycolysis, tricarboxylic acid cycle, and lipid metabolic pathways. The ecdysone receptor (EcR), which is responsible for initiating the larval-to-adult transition, colocalized with 14-3-3ε in wild-type fat body tissues. The altered protein abundance in 14-3-3ε mutant fat body tissue was associated with transcriptional deregulation of alcohol dehydrogenase, fat body protein 1, and lamin genes, which are known targets of the EcR. This study indicates that 14-3-3ε has a critical role in cellular metabolism involving either molecular crosstalk with the EcR or direct interaction with metabolic proteins.

A Case Report of a Chinese Familial Partial Lipodystrophic Patient with Lamin A/C Gene R482Q Mutation and Polycystic Ovary Syndrome

Individuals with Familial partial lipodystrophy (FPLD), Dunnigan variety is a rare autosomal dominant disorder caused by missense mutations in Lamin gene are predisposed to insulin resistance and its complications including features of polycystic ovarian syndrome. We present a single case report about a 26-year-old Chinese woman consulted for infertility. On physical examination acanthosis nigricans and central distribution of fat were found. Her masculine type morphology, muscular appearance of the limbs and excess fat deposits in the face and neck promote us to suspect the existence of partial lipodystrophy. Biochemistry testing confirmed glucose intolerance associated with a severe insulin resistance, hypertriglyceridemia, and polycystic ovary syndrome. The detection of a heterozygous missense mutation in LAMIN A/C gene at position 482 confirmed the diagnosis of FPLD2. In conclusion, characteristic features of FPLD and mutation screening allow early diagnosis of this disorder, and facilitate appropriate clinical treatment.

Lamin A and lamin-associated polypeptide 2 (LAP-2) in human skin fibroblasts in the process of aging

Mutations of lamin genes lead to diseases, one of which is progeria. This disease is caused by violation of splicing of lamin A gene and accumulation of farnesylated prelamin A (progerin) in the nucleus. LAP-2 is an important factor that regulates and stabilizes the nucleoplasmic pool of lamin A. However, roles of lamin A and LAP-2 in renewal regulation of the population of fibroblasts in the human dermis in different age periods still have not been investigated. The purpose of this investigation was to define expression of lamin A and LAP-2 in human dermal fibroblasts at different ages. Lamin A and LAP-2 were detected in skin sections by indirect immunohistochemistry. It was found that the number of fibroblasts containing lamin A gradually decreased from 90.4 to 76.9% from 20 weeks of pregnancy to 85 years. It was revealed that 32% of dermal fibroblasts were positively stained for LAP-2 in the period from 20 weeks of pregnancy to 20 years, 37.8% in the period of 21–40 years, and 49–51% in the period of 41–85 years. Lamin A expression levels in fibroblast nuclei were essentially constant from 20 weeks of pregnancy to 85 years, while LAP-2 content in fibroblast nuclei was reducing from 0 to 20 years as compared with the embryonic period and was gradually increasing after 21 year. The total number of fibroblasts and PCNA-positive fibroblasts in the dermis decreased with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of pregnancy to 20 years. The results suggest involvement of lamin A and LAP-2 in the age-related decrease in the number of fibroblasts in the human dermis.

Lamins and metabolism.

Lamins are nuclear intermediate filaments (IFs) with important roles in most nuclear activities, including nuclear organization and cell-cycle progression. Mutations in human lamins cause over 17 different diseases, termed laminopathies. Most of these diseases are autosomal dominant and can be roughly divided into four major groups: muscle diseases, peripheral neuronal diseases, accelerated aging disorders and metabolic diseases including Dunnigan typefamilial partial lipodystrophy (FLPD), acquired partial lipodystrophy (APL) and autosomal dominant leucodystrophy. Mutations in lamins are also associated with the metabolic syndrome (MS). Cells derived from patients suffering from metabolic laminopathies, as well as cells derived from the corresponding animal models, show a disruption of the mechanistic target of rapamycin (mTOR) pathway, abnormal autophagy, altered proliferative rate and down-regulation of genes that regulate adipogenesis. In addition, treating Hutchinson-Gilford progeria syndrome (HGPS) cells with the mTOR inhibitor rapamycin improves their fate. In this review, we will discuss the ways by which lamin genes are involved in the regulation of cell metabolism.

Aging Human Hematopoietic Stem Cells Manifest Massive Epigenetic Reprogramming and Altered Gene Splicing of Key Hematopoietic Gene Sets

Aging leads to impairment of hematopoietic stem cell (HSC) function with decreased self-renewal, imbalanced differentiation potential and an increased risk to develop myeloid malignancies. These malignancies are associated with epigenetic deregulation, which contributes to pathogenesis. Notably, studies in murine models have revealed epigenetic changes in aged HSC. However, it is unknown if this occurs in normal human HSC aging and whether it may contribute to HSC dysfunction. Therefore, we performed comprehensive epigenomic and transcriptional profiling in primary human HSC (Lin-, CD34+, CD38-) isolated from young (18-30 yo), mid (45-55 yo) and old (65-75 yo) healthy donors. Using a micro-ChIP-seq protocol we profiled H3K4me1, H3K4me3, H3K27me3 and H3K27ac in 4-7 donors per age group, as well as genome-wide DNA methylation (5mC), hydroxymethylation (5hmC) and RNA-seq.Analysis of enhancer-associated marks revealed that with age there is marked reduction in both H3K4me1 and H3K27ac (20,783 and 15,625 peaks lost, respectively; log10likelihood ratio >3). Gene ontology analysis of these lost peaks revealed their association with genes involved in hematopoiesis and, RNA splicing and chromatin organization, respectively (ChIPenrich, FDR<0.05). In addition, regions depleted in H3K4me1 are enriched for PU.1, FLI1, ETS, and CTCF binding sites (Homer, q<0.00001). We next asked if aging results in specific remodeling of poised (H3K4me1>H3K4me3, H3K27ac-) and active (H3K4me1>H3K4me3, H3K27ac+) enhancers. We found age-related loss of H3K4me1 enrichment at 10,696 poised enhancers, which are associated with hematopoiesis and T- and B-cell receptor signaling (FDR<0.05). We also identified 17,242 active enhancers in young HSC, 7,057 of which are depleted in old HSC. This loss of active enhancers targets genes associated with hematopoiesis, immune signaling and myeloid malignancies (FDR<0.05). Next we analyzed the impact of aging on promoter-associated marks, H3K4me3 and H3K27me3. Remarkably, while aging leads to loss of 22,689 H3K4me3 peaks, only 1,339 H3K27me3 peaks are lost. Loss of H3K4me3 targets genes involved in inflammatory response, development and WNT signaling (FDR<0.05). Given this uneven change in H3K4me3/H3K27me3 with aging, we hypothesized this may correlate with changes in bivalently marked promoters, which regulate key developmental genes. Out of 3,947 bivalent promoters in young HSC, 842 are lost in aged HSC. This loss of bivalency affects genes involved in WNT, Cadherin and Hedgehog signaling pathways (FDR<0.05). Next we analyzed changes in cytosine modifications. We observe widespread gain of 5hmC (n=14,554 differentially hydroxymethylated regions [DHMR]; FDR <0.005), with specific enrichment at introns and exons (p<2.2e-16), as well as enrichment for GATA and KLF binding sites (Homer, q<0.00001). These DHMR target genes involved in hematopoiesis, proteins regulated by alternative splicing, and pathways associated with cancer (FDR<0.05). In contrast, much more subtle changes are found in 5mC with HSC aging, with only 529 differentially methylated regions (q-value <0.05, meth.diff >20%). However, these subtle changes also target genes associated with cadherin and WNT signaling. Finally, RNA-seq analysis revealed that this age-associated epigenetic reprogramming is accompanied by an overall trend to gene downregulation. Amongst the genes most affected are the nuclear lamin gene LMNA (mutated in progeria syndrome), splicing factors SRSF7 and U2AF1 and, the transcription factors KLF3/6 and HIF1α (FDR <0.05, fold change >1.5). Notably, changes in expression also include significant differential exon usage, which may be mediated by DHMR at intron-exon boundaries: 575 genes show altered exon usage (FDR <0.05, fold change>1.5) including LMNA and the epigenetic modifiers BRD9, CITED2, KDM6A and SETD6.In summary, we have completed the first comprehensive epigenomic profiling of aging in human HSC. Our findings show massive epigenetic remodeling in aged HSC, consisting of loss of activating histone marks primarily targeting enhancers and bivalent promoters at genes involved in hematopoiesis and developmental pathways. Cytosine modifications show widespread changes in 5hmC, targeting intron-exon boundaries. Globally, this epigenetic reprogramming results in overall gene downregulation and altered splicing of genes important for HSC regulation. DisclosuresNo relevant conflicts of interest to declare.

Comparing lamin proteins post-translational relative stability using a 2A peptide-based system reveals elevated resistance of progerin to cellular degradation

ABSTRACTNuclear lamins are the major components of the nuclear lamina at the periphery of the nucleus, supporting the nuclear envelope and participating in many nuclear processes, including DNA replication, transcription and chromatin organization. A group of diseases, the laminopathies, is associated with mutations in lamin genes. One of the most striking cases is Hutchinson-Gilford progeria syndrome (HGPS) which is the consequence of a lamin A dominant negative mutant named progerin. Due to the abnormal presence of a permanent C-terminal farnesyl tail, progerin gradually accumulates on the nuclear membrane, perturbing a diversity of signalings and transcriptional events. The accumulation of progerin has led to the speculation that progerin possesses higher stability than the wild type lamin A protein. However, the low solubility of lamin proteins renders traditional immunoprecipitation-dependent methods such as pulse-chase analysis ineffective for comparing the relative stabilities of mutant and wild type lamins. Here, we employ a novel platform for inferring differences in lamin stability, which is based on normalization to a co-translated reporter protein following porcine teschovirus-1 2A peptide-mediated co-translational cleavage. The results obtained using this method support the notion that progerin is more stable than lamin A. Moreover, treatment of FTI reduces progerin relative stability to the level of wild type lamin A.

LMNA missense mutations causing familial partial lipodystrophy do not lead to an accumulation of prelamin A

ABSTRACTA variety of missense mutations in LMNA (the gene for lamin C and prelamin A) cause familial partial lipodystrophy (FPLD), a disease associated with reduced adipose tissue, particularly in the limbs. Several studies have reported that fibroblasts from FPLD subjects have an accumulation of prelamin A. Those findings were intriguing but also perplexing because many of the LMNA missense mutations associated with lipodystrophy are located in sequences distant from the sequences required for the farnesylation of prelamin A and ZMPSTE24-mediated conversion of prelamin A to mature lamin A. Here, we revisited the issue of prelamin A accumulation in the setting of FPLD mutations. We used western blots with lamin A/C antibodies and prelamin A–specific monoclonal antibodies to assess prelamin A levels in wild-type fibroblasts and fibroblasts carrying LMNA mutations associated with lipodystrophy (R482W, I299V, C591F, T528M). None of the mutant fibroblasts exhibited an accumulation of prelamin A. Also, the amount of prelamin A accumulation in response to lopinavir (an inhibitor of ZMPSTE24) was similar in wild-type and mutant fibroblasts. Thus, the LMNA lipodystrophy mutations that we examined did not lead to prelamin A accumulation, nor did they render those cells more susceptible to prelamin A accumulation when ZMPSTE24 was inhibited by lopinavir.

Cloning and Characterization of Sf9 Cell Lamin and the Lamin Conformational Changes during Autographa californica multiple nucleopolyhedrovirus Infection.

At present, the details of lamina alterations after baculovirus infection remain elusive. In this study, a lamin gene in the Sf9 cell line of Spodoptera frugiperda was cloned. The open reading frame (orf) of the Sf9 lamin was 1860 bp and encoded a protein with a molecular weight of 70 kDa. A transfection assay with a red fluorescence protein (rfp)-lamin fusion protein indicated that Sf9 lamin was localized in the nuclear rim. Transmission electron microscopy observations indicated that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsids may pass through the nuclear envelope. Immunofluorescence assay indicated that the lamina showed a ruffled staining pattern with the formation of invaginations in the Sf9 cells infected with AcMNPV, while it was evenly distributed at the nuclear periphery of mock-infected cells. Western blotting results indicated that the total amount of lamin in the baculovirus-infected Sf9 cells was significantly decreased compared with the mock-infected cells. These results imply that AcMNPV infection induces structural and biochemical rearrangements of lamina of Sf9 cells.

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope.

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein.

A peculiar lamin in a peculiar mammal: Expression of lamin LIII in platypus (Ornithorhynchus anatinus)

Platypus (Ornithorhynchus anatinus) holds a unique phylogenetic position at the base of the mammalian lineage due to an amalgamation of mammalian and sauropsid-like features. Here we describe the set of four lamin genes for platypus. Lamins are major components of the nuclear lamina, which constitutes a main component of the nucleoskeleton and is involved in a wide range of nuclear functions. Vertebrate evolution was accompanied by an increase in the number of lamin genes from a single gene in their closest relatives, the tunicates and cephalochordates, to four genes in the vertebrate lineage. Of the four genes the LIII gene is characterized by the presence of two alternatively spliced CaaX-encoding exons. In amphibians and fish LIII is the major lamin protein in oocytes and early embryos. The LIII gene is conserved throughout the vertebrate lineage, with the notable exception of marsupials and placental mammals, which have lost the LIII gene. Here we show that platypus has retained an LIII gene, albeit with a significantly altered structure and with a radically different expression pattern. The platypus LIII gene contains only a single CaaX-encoding exon and the head domain together with coil 1a and part of coil1b of the platypus LIII protein is replaced by a novel short non-helical N-terminus. It is expressed exclusively in the testis. These features resemble those of male germ cell-specific lamins in placental mammals, in particular those of lamin C2. Our data suggest (i) that the specific functions of LIII, which it fulfills in all other vertebrates, is no longer required in mammals and (ii) once it had been freed from these functions has undergone structural alterations and has adopted a new functionality in monotremes.

Polyphyly of nuclear lamin genes indicates an early eukaryotic origin of the metazoan-type intermediate filament proteins.

The nuclear lamina is a protein meshwork associated with the inner side of the nuclear envelope contributing structural, signalling and regulatory functions. Here, I report on the evolution of an important component of the lamina, the lamin intermediate filament proteins, across the eukaryotic tree of life. The lamins show a variety of protein domain and sequence motif architectures beyond the classical α-helical rod, nuclear localisation signal, immunoglobulin domain and CaaX motif organisation, suggesting extension and adaptation of functions in many species. I identified lamin genes not only in metazoa and Amoebozoa as previously described, but also in other opisthokonts including Ichthyosporea and choanoflagellates, in oomycetes, a sub-family of Stramenopiles, and in Rhizaria, implying that they must have been present very early in eukaryotic evolution if not even the last common ancestor of all extant eukaryotes. These data considerably extend the current perception of lamin evolution and have important implications with regard to the evolution of the nuclear envelope.