Nuclear lamins are usually classified as A-type (lamins A and C) or B-type (lamins B1 and B2). A-type lamins have been implicated in multiple genetic diseases but are not required for cell growth or development. In contrast, B-type lamins have been considered essential in eukaryotic cells, with crucial roles in DNA replication and in the formation of the mitotic spindle. Knocking down the genes for B-type lamins (LMNB1, LMNB2) in HeLa cells has been reported to cause apoptosis. In the current study, we created conditional knockout alleles for mouse Lmnb1 and Lmnb2, with the goal of testing the hypothesis that B-type lamins are crucial for the growth and viability of mammalian cells in vivo. Using the keratin 14-Cre transgene, we bred mice lacking the expression of both Lmnb1 and Lmnb2 in skin keratinocytes (Lmnb1Δ/ΔLmnb2Δ/Δ). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1Δ/ΔLmnb2Δ/Δ mice, and lamin B1 and lamin B2 proteins were undetectable. But despite an absence of B-type lamins in keratinocytes, the skin and hair of Lmnb1Δ/ΔLmnb2Δ/Δ mice developed normally and were free of histological abnormalities, even in 2-year-old mice. After an intraperitoneal injection of bromodeoxyuridine (BrdU), similar numbers of BrdU-positive keratinocytes were observed in the skin of wild-type and Lmnb1Δ/ΔLmnb2Δ/Δ mice. Lmnb1Δ/ΔLmnb2Δ/Δ keratinocytes did not exhibit aneuploidy, and their growth rate was normal in culture. These studies challenge the concept that B-type lamins are essential for proliferation and vitality of eukaryotic cells.
Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2–deficiency in mice results in severe brain abnormalities. While the early stages of forebrain development in lamin B2–deficient mice appear to be normal, cortical neurons fail to migrate and organize into proper layers within the cerebral cortex. The morphogenesis of the hippocampus and cerebellum is also severely impaired. These phenotypes are reminiscent of lissencephaly, a human brain developmental disorder characterized by an abnormal neuronal migration. Most mutations in lissencephaly patients affect cytoplasmic regulators of nuclear translocation, which is a crucial step in neuronal migration. The phenotypes of lamin B2–deficient mice suggest that lamin B2 may also play a key role in nuclear translocation. Potential mechanisms for lamin B2 involvement, which include mechanical and non-mechanical roles, and participation in LINC complexes in the nuclear envelope, are discussed along with evidence that lamins B1 and B2 play distinct, cell-specific functions.
nuclear envelope; nuclear lamina; lamin B2; LINC complex; nesprin; SUN; lissencephaly; neuronal migration; cortical neurons; nuclear translocation
Nuclear lamins are major components of the nuclear lamina, and play essential roles in supporting the nucleus and organizing nuclear structures. While a large number of clinically important mutations have been mapped to the LMNA gene in humans, very few mutations have been associated with the B-type lamins. We have shown that lamin B2-deficiency in mice results in severe brain abnormalities. While the early stages of forebrain development in lamin B2-deficient mice appear to be normal, cortical neurons fail to migrate and organize into proper layers within the cerebral cortex. The morphogenesis of the hippocampus and cerebellum is also severely impaired. These phenotypes are reminiscent of lissencephaly, a human brain developmental disorder characterized by an abnormal neuronal migration. Most mutations in lissencephaly patients affect cytoplasmic regulators of nuclear translocation, which is a crucial step in neuronal migration. The phenotypes of lamin B2-deficient mice suggest that lamin B2 may also play a key role in nuclear translocation. Potential mechanisms for lamin B2 involvement, which include mechanical and non-mechanical roles and participation in LINC complexes in the nuclear envelope, are discussed along with evidence that lamins B1 and B2 play distinct, cell-specific functions.
nuclear envelope; nuclear lamina; lamin B2; LINC complex; nesprin; SUN; lissencephaly; neuronal migration; cortical neurons; nuclear translocation
B-type lamins, the major components of the nuclear lamina, are believed to be essential for cell proliferation and survival. We found that mouse embryonic stem cells (ESCs) do not need any lamins for self-renewal and pluripotency. Although genome-wide lamin-B binding profiles correlate with reduced gene expression, such binding is not directly required for gene silencing in ESCs or trophectoderm cells. However, B-type lamins are required for proper organogenesis. Defects in spindle orientation in neural progenitor cells and migration of neurons probably cause brain disorganizations found in lamin-B null mice. Thus, our studies not only disprove several prevailing views of lamin-Bs but also establish a foundation for redefining the function of the nuclear lamina in the context of tissue building and homeostasis.
Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C–only mice (Lmna+/+), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna+/+ mice were entirely healthy, and Lmna+/+ cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl–prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A–related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single LmnaLCO allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24–/– mice. Moreover, treating Zmpste24–/– cells with a prelamin A–specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.
The B-type lamins are widely assumed to be essential for mammalian cells. In part, this assumption is based on a highly cited study that found that RNAi-mediated knockdown of lamin B1 or lamin B2 in HeLa cells arrested cell growth and led to apoptosis. Studies indicating that B-type lamins play roles in DNA replication, the formation of the mitotic spindle, chromatin organization and regulation of gene expression have fueled the notion that B-type lamins must be essential. But surprisingly, this idea had never been tested with genetic approaches. Earlier this year, a research group from UCLA reported the development of genetically modified mice that lack expression of both Lmnb1 and Lmnb2 in skin keratinocytes (a cell type that proliferates rapidly and participates in complex developmental programs). They reasoned that if lamins B1 and B2 were truly essential, then keratinocyte-specific lamin B1/lamin B2 knockout mice would exhibit severe pathology. Contrary to expectations, the skin and hair of lamin B1/lamin B2-deficient mice were quite normal, indicating that the B-type lamins are dispensable in some cell types. The same UCLA research group has gone on to show that lamin B1 and lamin B2 are critical for neuronal migration in the developing brain and for neuronal survival. The absence of either lamin B1 or lamin B2, or the absence of both B-type lamins, results in severe neurodevelopmental abnormalities.
lamin B1; lamin B2; nuclear envelope; nuclear lamina
Neuronal migration is a fundamental component of brain development whose failure is associated with various neurological and psychiatric disorders. Reelin is essential for the stereotypical inside-out sequential lamination of the neocortex, but the molecular mechanisms of its action still remain unclear. Here we show that regulation of Notch activity plays an important part in Reelin signal-dependent neuronal migration. We found that Reelin-deficient mice have reduced levels of the cleaved form of Notch intracellular domain (Notch ICD) and that loss of Notch signaling in migrating neurons results in migration and morphology defects. Further, overexpression of Notch ICD mitigates the laminar and morphological abnormalities of migrating neurons in Reeler. Finally, our in vitro biochemical studies show that Reelin signaling inhibits Notch ICD degradation via Dab1. Together, our results indicate that neuronal migration in the developing cerebral cortex requires a Reelin-Notch interaction.
migration; cerebral cortex; Reelin; Notch
Adult-onset autosomal-dominant leukodystrophy (ADLD) is a progressive and fatal
neurological disorder characterized by early autonomic dysfunction, cognitive
impairment, pyramidal tract and cerebellar dysfunction, and white matter loss in the
central nervous system. ADLD is caused by duplication of the LMNB1
gene, which results in increased lamin B1 transcripts and protein expression. How
duplication of LMNB1 leads to myelin defects is unknown. To address
this question, we developed a mouse model of ADLD that overexpresses lamin B1. These
mice exhibited cognitive impairment and epilepsy, followed by age-dependent motor
deficits. Selective overexpression of lamin B1 in oligodendrocytes also resulted in
marked motor deficits and myelin defects, suggesting these deficits are cell
autonomous. Proteomic and genome-wide transcriptome studies indicated that lamin B1
overexpression is associated with downregulation of proteolipid protein, a highly
abundant myelin sheath component that was previously linked to another myelin-related
disorder, Pelizaeus-Merzbacher disease. Furthermore, we found that lamin B1
overexpression leads to reduced occupancy of Yin Yang 1 transcription factor at the
promoter region of proteolipid protein. These studies identify a mechanism by which
lamin B1 overexpression mediates oligodendrocyte cell–autonomous
neuropathology in ADLD and implicate lamin B1 as an important regulator of myelin
formation and maintenance during aging.
Laminopathies are a group of disorders caused by mutations in the LMNA gene that encodes the nuclear lamina proteins, lamin A and lamin C; their pathophysiological basis is unknown. We report that lamin A/C–deficient (Lmna–/–) mice develop rapidly progressive dilated cardiomyopathy (DCM) characterized by left ventricular (LV) dilation and reduced systolic contraction. Isolated Lmna–/– myocytes show reduced shortening with normal baseline and peak amplitude of Ca2+ transients. Lmna–/– LV myocyte nuclei have marked alterations of shape and size with central displacement and fragmentation of heterochromatin; these changes are present but less severe in left atrial nuclei. Electron microscopy of Lmna–/– cardiomyocytes shows disorganization and detachment of desmin filaments from the nuclear surface with progressive disruption of the cytoskeletal desmin network. Alterations in nuclear architecture are associated with defective nuclear function evidenced by decreased SREBP1 import, reduced PPARγ expression, and a lack of hypertrophic gene activation. These findings suggest a model in which the primary pathophysiological mechanism in Lmna–/– mice is defective force transmission resulting from disruption of lamin interactions with the muscle-specific desmin network and loss of cytoskeletal tension. Despite severe DCM, defects in nuclear function prevent Lmna–/– cardiomyocytes from developing compensatory hypertrophy and accelerate disease progression.
The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.
The nuclear lamina is the structural scaffold of the nuclear envelope and is well known for its central role in nuclear organization and maintaining nuclear stability and shape. In the past, a number of severe human disorders have been identified to be associated with mutations in lamins. Extensive research on this topic has provided novel important clues about nuclear lamina function. These studies have contributed to the knowledge that the lamina constitutes a complex multifunctional platform combining both structural and regulatory functions. Here, we report that, in addition to the previously demonstrated significance for somatic cell differentiation and maintenance, the nuclear lamina is also an essential determinant for germ cell development. Both male and female mice lacking the short meiosis-specific A-type lamin C2 have a severely defective meiosis, which at least in the male results in infertility. Detailed analysis revealed that lamin C2 is required for telomere-driven dynamic repositioning of meiotic chromosomes. Loss of lamin C2 affects precise synapsis of the homologs and interferes with meiotic double-strand break repair. Taken together, our data explain how the nuclear lamina contributes to meiotic chromosome behaviour and accurate genome haploidization on a mechanistic level.
Diploid germ line cells have to undergo meiosis to produce haploid gametes. Haploidization involves pairing and recombination of homologous chromosomes as a prerequisite for their proper segregation. Pairing of homologous chromosomes requires their active repositioning within meiotic nuclei, which depends on the interaction of telomeres with the nuclear envelope. This dynamic association is vital for a faithful meiosis and thus crucial for fertility. However, very little is known about the relationship between telomeres and nuclear envelope components. Here, we have investigated the role of the nuclear lamina, a structural scaffold that is intimately associated with the inner nuclear membrane. In somatic cells, the lamina is a key player in chromatin organization and fulfils various functions such as nuclear structure maintenance and regulation of transcription. In order to understand its role in meiosis, we investigated lamin C2, the only A-type lamin isoform expressed in mammalian meiotic cells. We demonstrate that lamin C2 is essential for timely repositioning of meiotic telomeres. In its absence, synapsis of homologous chromosomes and double-strand break repair are severely affected. These multiple meiotic defects lead to infertility in males. We conclude that the nuclear lamina contributes directly to fertility through facilitating meiotic chromosome movements.
Lamins are intermediate filament proteins that form a major component of the nuclear lamina, a protein complex at the surface of the inner nuclear membrane. Numerous clinically diverse conditions, termed laminopathies, have been found to result from mutation of LMNA. In contrast, coding or loss of function mutations of LMNB1, encoding lamin B1, have not been identified in human disease. In mice, polymorphism in Lmnb1 has been shown to modify risk of neural tube defects (NTDs), malformations of the central nervous system that result from incomplete closure of the neural folds.
Mutation analysis by DNA sequencing was performed on all exons of LMNB1 in 239 samples from patients with NTDs from the United Kingdom, Sweden, and United States. Possible functional effects of missense variants were analyzed by bioinformatics prediction and fluorescence in photobleaching.
In NTD patients, we identified two unique missense variants that were predicted to disrupt protein structure/function and represent putative contributory mutations. Fluorescence loss in photobleaching analysis showed that the A436T variant compromised stability of lamin B1 interaction within the lamina.
The genetic basis of human NTDs appears highly heterogenous with possible involvement of multiple predisposing genes. We hypothesize that rare variants of LMNB1 may contribute to susceptibility to NTDs. Birth Defects Research (Part A) 97:398–402, 2013. © 2013 Wiley Periodicals, Inc.
anencephaly; lamin B1; neural tube defects; nuclear lamina; spina bifida
Reelin, an extracellular glycoprotein has an important role in the proper migration and positioning of neurons during brain development. Lack of reelin causes not only disorganized lamination of the cerebral and cerebellar cortex but also malpositioning of mesencephalic dopaminergic (mDA) neurons. However, the accurate role of reelin in the migration and positioning of mDA neurons is not fully elucidated. In this study, reelin-deficient reeler mice exhibited a significant loss of mDA neurons in the substantia nigra pars compacta (SNc) and a severe alteration of cell distribution in the retrorubal field (RRF). This abnormality was also found in Dab1-deficinet, yotari mice. Stereological analysis revealed that total number of mDA neurons was not changed compared to wild type, suggesting that the loss of mDA neurons in reeler may not be due to the neurogenesis of mDA neurons. We also found that formation of PSA-NCAM-positive tangential nerve fibers rather than radial glial fibers was greatly reduced in the early developmental stage (E14.5) of reeler. These findings provide direct evidence that the alteration in distribution pattern of mDA neurons in the reeler mesencephalon mainly results from the defect of the lateral migration using tangential fibers as a scaffold.
Reelin; dopaminergic neurons; radial glia; tangential fibers; neuronal migration
Tuberous sclerosis complex (TSC) is an autosomal dominant, tumor predisposition disorder characterized by significant neurodevelopmental brain lesions, such as tubers and subependymal nodules. The neuropathology of TSC is often associated with seizures and intellectual disability. To learn about the developmental perturbations that lead to these brain lesions, we created a mouse model that selectively deletes the Tsc2 gene from radial glial progenitor cells in the developing cerebral cortex and hippocampus. These Tsc2 mutant mice were severely runted, developed post-natal megalencephaly and died between 3 and 4 weeks of age. Analysis of brain pathology demonstrated cortical and hippocampal lamination defects, hippocampal heterotopias, enlarged dysplastic neurons and glia, abnormal myelination and an astrocytosis. These histologic abnormalities were accompanied by activation of the mTORC1 pathway as assessed by increased phosphorylated S6 in brain lysates and tissue sections. Developmental analysis demonstrated that loss of Tsc2 increased the subventricular Tbr2-positive basal cell progenitor pool at the expense of early born Tbr1-positive post-mitotic neurons. These results establish the novel concept that loss of function of Tsc2 in radial glial progenitors is one initiating event in the development of TSC brain lesions as well as underscore the importance of Tsc2 in the regulation of neural progenitor pools. Given the similarities between the mouse and the human TSC lesions, this model will be useful in further understanding TSC brain pathophysiology, testing potential therapies and identifying other genetic pathways that are altered in TSC.
The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.
emerin; muscular dystrophy; nuclear envelope; lamins
Heterotopic or aberrantly positioned cortical neurons are associated with epilepsy and intellectual disability. Various mouse models exist with forms of heterotopia, but the composition and state of cells developing in heterotopic bands has been little studied. Dcx knockout (KO) mice show hippocampal CA3 pyramidal cell lamination abnormalities, appearing from the age of E17.5, and mice suffer from spontaneous epilepsy. The Dcx KO CA3 region is organized in two distinct pyramidal cell layers, resembling a heterotopic situation, and exhibits hyperexcitability. Here, we characterized the abnormally organized cells in postnatal mouse brains. Electron microscopy confirmed that the Dcx KO CA3 layers at postnatal day (P) 0 are distinct and separated by an intermediate layer devoid of neuronal somata. We found that organization and cytoplasm content of pyramidal neurons in each layer were altered compared to wild type (WT) cells. Less regular nuclei and differences in mitochondria and Golgi apparatuses were identified. Each Dcx KO CA3 layer at P0 contained pyramidal neurons but also other closely apposed cells, displaying different morphologies. Quantitative PCR and immunodetections revealed increased numbers of oligodendrocyte precursor cells (OPCs) and interneurons in close proximity to Dcx KO pyramidal cells. Immunohistochemistry experiments also showed that caspase-3 dependent cell death was increased in the CA1 and CA3 regions of Dcx KO hippocampi at P2. Thus, unsuspected ultrastructural abnormalities and cellular heterogeneity may lead to abnormal neuronal function and survival in this model, which together may contribute to the development of hyperexcitability.
Progeroid phenotypes are mainly encountered in 2 types of syndromes: in laminopathies, which are characterized by nuclear shape abnormalities due to lamin A alteration, and in DNA damage response defect syndromes. Because lamin A dysregulation leads to DNA damages, it has been proposed that senescence occurs in both types of syndromes through the accumulation of damages. We recently showed that elevated oxidative stress is responsible for lamin B1 accumulation, nuclear shape alteration and senescence in the DDR syndrome, ataxia telangiectasia (A-T). Interestingly, overexpression of lamin B1 in wild type cells is sufficient to induce senescence without the induction of DNA damages. Here, we will discuss the importance of controlling the lamins level in order for maintenance nuclear architecture and we will comment the relationships of lamins with other senescence mechanisms. Finally, we will describe emerging data reporting redox control by lamins, leading us to propose a general mechanism by which reactive oxygen species can induce senescence through lamin dysregulation and NSA.
senescence; lamin B1; lamin A; nuclear shape alteration; ataxia telangiectasia; oxidative stress; DNA damage; telomeres; laminopathies
Polymicrogyria is a relatively common but poorly understood defect of cortical development characterized by numerous small gyri and a thick disorganized cortical plate lacking normal lamination. We show an association between bilateral asymmetrical polymicrogyria and de novo mutations in a β-tubulin gene, TUBB2B, in four patients and a 27 GW (gestational week) fetus. Neuropathological examination of the fetus revealed an absence of cortical lamination associated with the presence of ectopic neuronal cells in the white matter, and in the leptomeningeal spaces due to breaches in the pial basement membrane. In utero RNAi-based inactivation demonstrates that TUBB2B is required for neuronal migration. We also show that two disease-associated mutations lead to an impaired formation of tubulin heterodimers. These observations, together with previous data, demonstrate that disruption of microtubule-based processes underlies a large spectrum of neuronal migration disorders that includes not only lissencephaly/pachygyria, but also polymicrogyria malformations.
Interaction of lamins with chromatin and transcription factors regulate transcription. Oct-1 has previously been shown to colocalize partly with B-type lamins and is essential for transcriptional regulation of oxidative stress response genes. Using sequential extraction, co-immunoprecipitation (IP), fluorescence loss in photobleaching, and fluorescence resonance energy transfer, we confirm Oct-1–lamin B1 association at the nuclear periphery and show that this association is lost in Lmnb1Δ/Δ cells. We show that several Oct-1–dependent genes, including a subset involved in oxidative stress response, are dysregulated in Lmnb1Δ/Δ cells. Electrophoretic mobility shift assay and chromatin IP reveal that Oct-1 binds to the putative octamer-binding sequences of the dysregulated genes and that this activity is increased in cells lacking functional lamin B1. Like Oct1−/− cells, Lmnb1Δ/Δ cells have elevated levels of reactive oxygen species and are more susceptible to oxidative stress. Sequestration of Oct-1 at the nuclear periphery by lamin B1 may be a mechanism by which the nuclear envelope can regulate gene expression and contribute to the cellular response to stress, development, and aging.
Mutations in the lamin A/C (LMNA) gene, which encodes nuclear membrane proteins, cause a variety of human conditions including dilated cardiomyopathy (DCM) with associated cardiac conduction system disease. To investigate mechanisms responsible for electrophysiologic and myocardial phenotypes caused by dominant human LMNA mutations, we performed longitudinal evaluations in heterozygous Lmna+/- mice. Despite one normal allele, Lmna+/- mice had 50% of normal cardiac lamin A/C levels and developed cardiac abnormalities. Conduction system function was normal in neonatal Lmna+/- mice but by 4 weeks of age AV nodal myocytes had abnormally shaped nuclei and active apoptosis. Telemetric and in vivo electrophysiologic studies in 10 week-old Lmna+/- mice showed atrioventricular (AV) conduction defects and both atrial and ventricular arrhythmias, analogous to those observed in humans with heterozygous LMNA mutations. Isolated myocytes from 12-month old Lmna+/- mice exhibited impaired contractility. In vivo cardiac studies of aged Lmna+/- mice revealed DCM; in some mice this occurred without overt conduction system disease. However, neither histopathology nor serum CK levels indicated skeletal muscle pathology. These data demonstrate cardiac pathology due to heterozygous Lmna mutations reflecting a 50% reduction in lamin protein levels. Lamin haploinsufficiency caused early-onset programmed cell death of AV nodal myocytes and progressive electrophysiologic disease. While lamin haploinsufficiency was better tolerated by non-conducting myocytes, ultimately these too succumbed to diminished lamin levels leading to dilated cardiomyopathy, which presumably arose independently from conduction system disease.
Cardiac Conduction System; Arrhythmia; Heart Block; Laminopathies; Dilated cardiomyopathy
Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1–Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal–Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1–Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell–cell interactions of radial glial neuroepithelial progenitors by the Lis1–Nde1 complex is essential for their self renewal during the early phase of corticogenesis.
Hutchinson-Gilford progeria syndrome (HGPS), reportedly a model for normal aging, is a genetic disorder in children marked by dramatic signs suggestive for premature aging. It is usually caused by de novo mutations in the nuclear envelope protein lamin A. Lamins are essential to maintain nuclear integrity and loss of lamin A/C results in increased cellular sensitivity to mechanical strain and defective mechanotransduction signaling. Since increased mechanical sensitivity in vascular cells could contribute to loss of smooth muscle cells and the development of arteriosclerosis—the leading cause of death in HGPS patients—we investigated the effect of mechanical stress on cells from HGPS patients. We found that skin fibroblasts from HGPS patients developed progressively stiffer nuclei with increasing passage number. Importantly, fibroblasts from HGPS patients had decreased viability and increased apoptosis under repetitive mechanical strain, as well as attenuated wound healing, and these defects preceded changes in nuclear stiffness. Treating fibroblasts with farnesyltransferase inhibitors (FTI) restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing directional persistence of migrating cells but did not improve cellular sensitivity to mechanical strain. These data suggest that increased mechanical sensitivity in HGPS cells is unrelated to changes in nuclear stiffness and that increased biomechanical sensitivity could provide a potential mechanism for the progressive loss of vascular smooth muscle cells under physiological strain in HGPS patients.
Hutchinson-Gilford progeria syndrome; farnesyltransferase inhibitors; lamin; cellular mechanics; cell migration; wound healing
The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1). However, the pathways of proteasomal degradation have not been well characterized.
To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells.
Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system.
Changes in the shape of the nuclear lamina are exhibited in senescent cells, as well as in cells expressing mutations in lamina genes. To identify cells with defects in the nuclear lamina we developed an imaging method that quantifies the intensity and curvature of the nuclear lamina. We show that this method accurately describes changes in the nuclear lamina. Spatial changes in nuclear lamina coincide with redistribution of lamin A proteins and local reduction in protein mobility in senescent cell. We suggest that local accumulation of lamin A in the nuclear envelope leads to bending of the structure. A quantitative distinction of the nuclear lamina shape in cell populations was found between fresh and senescent cells, and between primary myoblasts from young and old donors. Moreover, with this method mutations in lamina genes were significantly distinct from cells with wild-type genes. We suggest that this method can be applied to identify abnormal cells during aging, in in vitro propagation, and in lamina disorders.
cell senescence; aging cells; apoptosis; nuclear lamina; image processing
Proper development of axonal connections is essential for brain function. A forward genetic screen for mice with defects in thalamocortical development previously isolated a mutant called baffled. Here we describe the axonal defects of baffled in further detail and identify a point mutation in the Hspa5 gene, encoding the endoplasmic reticulum chaperone BiP/GRP78. This hypomorphic mutation of BiP disrupts proper development of the thalamocortical axon projection and other forebrain axon tracts, as well as cortical lamination. In baffled mutant brains, a reduced number of thalamic axons innervate the cortex by the time of birth. Thalamocortical and corticothalamic axons are delayed, overfasciculated, and disorganized along their pathway through the ventral telencephalon. Furthermore, dissociated mutant neurons show reduced axon extension in vitro. Together, these findings demonstrate a sensitive requirement for the ER chaperone BiP/GRP78 during axon outgrowth and pathfinding in the developing mammalian brain.
brain development; axon guidance; cortical lamination; corticothalamic axons; chaperone; Hspa5; mouse; ENU mutant; protein misfolding; UPR; endoplasmic reticulum; heat shock protein