RESUMO
Nuclear segmentation is a hallmark feature of mammalian neutrophil differentiation, but the mechanisms that control this process are poorly understood. Gene expression in maturing neutrophils requires combinatorial actions of lineage-restricted and more widely expressed transcriptional regulators. Examples include interactions of the widely expressed ETS transcription factor, GA-binding protein (GABP), with the relatively lineage-restricted E-twenty-six (ETS) factor, PU.1, and with CCAAT enhancer binding proteins, C/EBPα and C/EBPε. Whether such cooperative interactions between these transcription factors also regulate the expression of genes encoding proteins that control nuclear segmentation is unclear. We investigated the roles of ETS and C/EBP family transcription factors in regulating the gene encoding the lamin B receptor (LBR), an inner nuclear membrane protein whose expression is required for neutrophil nuclear segmentation. Although C/EBPε was previously shown to bind the Lbr promoter, surprisingly, we found that neutrophils derived from Cebpe null mice exhibited normal Lbr gene and protein expression. Instead, GABP provided transcriptional activation through the Lbr promoter in the absence of C/EBPε, and activities supported by GABP were greatly enhanced by either C/EBPε or PU.1. Both GABP and PU.1 bound Ets sites in the Lbr promoter in vitro, and in vivo within both early myeloid progenitors and differentiating neutrophils. These findings demonstrate that GABP, PU.1, and C/EBPε cooperate to control transcription of the gene encoding LBR, a nuclear envelope protein that is required for the characteristic lobulated morphology of mature neutrophils.
Assuntos
Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Granulócitos/citologia , Receptores Citoplasmáticos e Nucleares/biossíntese , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Núcleo Celular , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Receptor de Lamina BRESUMO
There are roughly 14 distinct heritable autosomal dominant diseases associated with mutations in lamins A/C, including Emery-Dreifuss muscular dystrophy (EDMD). The mechanical model proposes that the lamin mutations change the mechanical properties of muscle nuclei, leading to cell death and tissue deterioration. Here, we developed an experimental protocol that analyzes the effect of disease-linked lamin mutations on the response of nuclei to mechanical strain in living Caenorhabditis elegans We found that the EDMD mutation L535P disrupts the nuclear mechanical response specifically in muscle nuclei. Inhibiting lamin prenylation rescued the mechanical response of the EDMD nuclei, reversed the muscle phenotypes and led to normal motility. The LINC complex and emerin were also required to regulate the mechanical response of C. elegans nuclei. This study provides evidence to support the mechanical model and offers a potential future therapeutic approach towards curing EDMD.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Laminas , Modelos Biológicos , Movimento , Distrofia Muscular de Emery-Dreifuss , Mutação de Sentido Incorreto , Proteínas Nucleares , Fenótipo , Substituição de Aminoácidos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Laminas/genética , Laminas/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Prenilação de Proteína/genéticaRESUMO
Nuclear pore complexes (NPCs) serve as the gateway of the cell nucleus. These macromolecular assemblies form selective aqueous translocation channels permitting the free diffusion of small molecules, as well as receptor-mediated transport of large cargoes. Over the past decade, major progress has been made in both the structural determination of individual nucleoporins and subcomplexes by X-ray crystallography and in the structural analysis of the entire NPC by cryo-electron tomography (cryo-ET). The metazoan NPC structure from Xenopus laevis oocytes was recently resolved up to 20 Å by combining cryo-ET with advanced image processing techniques, revealing for the first time the architecture of the central channel. Here, we discuss the structure of the Xenopus laevis NPC and consider future perspectives that will eventually allow reconstructing the scaffold and gate of the NPC with higher resolution and identifying its transport-relevant regions. This will eventually allow us to describe the structure of the NPC 'in action'.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear , Oócitos , Proteínas de Xenopus/metabolismo , Animais , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Oócitos/metabolismo , Oócitos/ultraestrutura , Xenopus laevisRESUMO
Lamins are intermediate filament proteins that form a fibrous meshwork, called the nuclear lamina, between the inner nuclear membrane and peripheral heterochromatin of metazoan cells. The assembly and incorporation of lamin A/C into the lamina, as well as their various functions, are still not well understood. Here, we employed designed ankyrin repeat proteins (DARPins) as new experimental tools for lamin research. We screened for DARPins that specifically bound to lamin A/C, and interfered with lamin assembly in vitro and with incorporation of lamin A/C into the native lamina in living cells. The selected DARPins inhibited lamin assembly and delocalized A-type lamins to the nucleoplasm without modifying lamin expression levels or the amino acid sequence. Using these lamin binders, we demonstrate the importance of proper integration of lamin A/C into the lamina for nuclear mechanical properties and nuclear envelope integrity. Finally, our study provides evidence for cell-type-specific differences in lamin functions.
Assuntos
Núcleo Celular/metabolismo , Laminas/metabolismo , Membrana Nuclear/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Humanos , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismoRESUMO
Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made between mutant lamins and the Nrf2 signaling pathway, suggesting new avenues of therapeutic intervention that include regulation of protein folding and metabolism, as well as maintenance of redox homoeostasis.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lamina Tipo A/genética , Distrofias Musculares/genética , Fator 2 Relacionado a NF-E2/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Núcleo Celular , Drosophila/genética , Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína 1 Associada a ECH Semelhante a Kelch , Lamina Tipo A/metabolismo , Músculo Esquelético/metabolismo , Mutação , Fator 2 Relacionado a NF-E2/genética , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Estresse Oxidativo , Conformação Proteica , Dobramento de Proteína , Proteína Sequestossoma-1RESUMO
Lamin proteins are the major constituents of the nuclear lamina, a proteinaceous network that lines the inner nuclear membrane. Primarily, the nuclear lamina provides structural support for the nucleus and the nuclear envelope; however, lamins and their associated proteins are also involved in most of the nuclear processes, including DNA replication and repair, regulation of gene expression, and signaling. Mutations in human lamin A and associated proteins were found to cause a large number of diseases, termed 'laminopathies.' These diseases include muscular dystrophies, lipodystrophies, neuropathies, and premature aging syndromes. Despite the growing number of studies on lamins and their associated proteins, the molecular organization of lamins in health and disease is still elusive. Likewise, there is no comprehensive view how mutations in lamins result in a plethora of diseases, selectively affecting different tissues. Here, we discuss some of the structural aspects of lamins and the nuclear lamina organization, in light of recent results.
Assuntos
Laminas/química , Lâmina Nuclear/química , Humanos , Laminas/genética , Microscopia Eletrônica de Transmissão , Modelos Biológicos , MutaçãoRESUMO
Eukaryotic cells have a layer of heterochromatin at the nuclear periphery. To investigate mechanisms regulating chromatin distribution, we analyzed heterochromatin organization in different tissues and species, including mice with mutations in the lamin B receptor (Lbr) and lamin A (Lmna) genes that encode nuclear envelope (NE) proteins. We identified LBR- and lamin-A/C-dependent mechanisms tethering heterochromatin to the NE. The two tethers are sequentially used during cellular differentiation and development: first the LBR- and then the lamin-A/C-dependent tether. The absence of both LBR and lamin A/C leads to loss of peripheral heterochromatin and an inverted architecture with heterochromatin localizing to the nuclear interior. Myoblast transcriptome analyses indicated that selective disruption of the LBR- or lamin-A-dependent heterochromatin tethers have opposite effects on muscle gene expression, either increasing or decreasing, respectively. These results show how changes in NE composition contribute to regulating heterochromatin positioning, gene expression, and cellular differentiation during development.
Assuntos
Heterocromatina/metabolismo , Lamina Tipo A/metabolismo , Desenvolvimento Muscular , Mioblastos/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Perfilação da Expressão Gênica , Camundongos , Mioblastos/citologia , Membrana Nuclear/metabolismo , Receptor de Lamina BRESUMO
Lamins are intermediate filament proteins that assemble into a meshwork underneath the inner nuclear membrane, the nuclear lamina. Mutations in the LMNA gene, encoding lamins A and C, cause a variety of diseases collectively called laminopathies. The disease mechanism for these diverse conditions is not well understood. Since lamins A and C are fundamental determinants of nuclear structure and stability, we tested whether defects in nuclear mechanics could contribute to the disease development, especially in laminopathies affecting mechanically stressed tissue such as muscle. Using skin fibroblasts from laminopathy patients and lamin A/C-deficient mouse embryonic fibroblasts stably expressing a broad panel of laminopathic lamin A mutations, we found that several mutations associated with muscular dystrophy and dilated cardiomyopathy resulted in more deformable nuclei; in contrast, lamin mutants responsible for diseases without muscular phenotypes did not alter nuclear deformability. We confirmed our results in intact muscle tissue, demonstrating that nuclei of transgenic Drosophila melanogaster muscle expressing myopathic lamin mutations deformed more under applied strain than controls. In vivo and in vitro studies indicated that the loss of nuclear stiffness resulted from impaired assembly of mutant lamins into the nuclear lamina. Although only a subset of lamin mutations associated with muscular diseases caused increased nuclear deformability, almost all mutations tested had defects in force transmission between the nucleus and cytoskeleton. In conclusion, our results indicate that although defective nuclear stability may play a role in the development of muscle diseases, other factors, such as impaired nucleo-cytoskeletal coupling, likely contribute to the muscle phenotype.
Assuntos
Citoesqueleto/metabolismo , Lamina Tipo A/genética , Músculos/metabolismo , Doenças Musculares/genética , Mutação , Lâmina Nuclear/metabolismo , Animais , Células Cultivadas , Citoesqueleto/química , Citoesqueleto/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/química , Lamina Tipo A/metabolismo , Camundongos , Camundongos Knockout , Músculos/química , Doenças Musculares/metabolismo , Lâmina Nuclear/química , Lâmina Nuclear/genética , Estabilidade ProteicaRESUMO
Neutrophils are characterized by their distinct nuclear shape, which is thought to facilitate the transit of these cells through pore spaces less than one-fifth of their diameter. We used human promyelocytic leukemia (HL-60) cells as a model system to investigate the effect of nuclear shape in whole cell deformability. We probed neutrophil-differentiated HL-60 cells lacking expression of lamin B receptor, which fail to develop lobulated nuclei during granulopoiesis and present an in vitro model for Pelger-Huët anomaly; despite the circular morphology of their nuclei, the cells passed through micron-scale constrictions on similar timescales as scrambled controls. We then investigated the unique nuclear envelope composition of neutrophil-differentiated HL-60 cells, which may also impact their deformability; although lamin A is typically down-regulated during granulopoiesis, we genetically modified HL-60 cells to generate a subpopulation of cells with well defined levels of ectopic lamin A. The lamin A-overexpressing neutrophil-type cells showed similar functional characteristics as the mock controls, but they had an impaired ability to pass through micron-scale constrictions. Our results suggest that levels of lamin A have a marked effect on the ability of neutrophils to passage through micron-scale constrictions, whereas the unusual multilobed shape of the neutrophil nucleus is less essential.
Assuntos
Membrana Nuclear/metabolismo , Movimento Celular , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Forma do Núcleo Celular , Expressão Gênica , Células HL-60 , Humanos , Lamina Tipo A/biossíntese , Lamina Tipo A/genética , Técnicas Analíticas Microfluídicas , Infiltração de Neutrófilos , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Membrana Nuclear/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Tretinoína/farmacologia , Tretinoína/fisiologia , Receptor de Lamina BRESUMO
Visualizing the dynamic molecular architecture of cells is instrumental for answering fundamental questions in cellular and structural biology. Although modern microscopy techniques, including fluorescence and conventional electron microscopy, have allowed us to gain insights into the molecular organization of cells, they are limited in their ability to visualize multicomponent complexes in their native environment. Cryo-electron tomography (cryo-ET) allows cells, and the macromolecular assemblies contained within, to be reconstructed in situ, at a resolution of 2-6 nm. By combining cryo-ET with super-resolution fluorescence microscopy approaches, it should be possible to localize proteins with high precision inside cells and so elucidate a more realistic view of cellular processes. Thus, cryo-ET may bridge the resolution gap between cellular and structural biology.
Assuntos
Células/citologia , Células/ultraestrutura , Tomografia com Microscopia Eletrônica/métodos , Animais , Microscopia Crioeletrônica , Citoesqueleto/ultraestrutura , Tomografia com Microscopia Eletrônica/instrumentação , Adesões Focais/ultraestrutura , Humanos , Substâncias Macromoleculares , Microscopia de Fluorescência , Poro Nuclear/ultraestruturaRESUMO
The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm. NPCs fuse the inner and outer nuclear membranes to form aqueous translocation channels that allow the free diffusion of small molecules and ions, as well as receptor-mediated transport of large macromolecules. The NPC regulates nucleocytoplasmic transport of macromolecules, utilizing soluble receptors that identify and present cargo to the NPC, in a highly selective manner to maintain cellular functions. The NPC is composed of multiple copies of approximately 30 different proteins, termed nucleoporins, which assemble to form one of the largest multiprotein assemblies in the cell. In this review, we address structural and functional aspects of this fundamental cellular machinery.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/química , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Humanos , Membrana Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Transporte ProteicoRESUMO
Lamin B receptor (LBR) is a bifunctional nuclear membrane protein with N-terminal lamin B and chromatin-binding domains plus a C-terminal sterol Δ(14) reductase domain. LBR expression increases during neutrophil differentiation, and deficient expression disrupts neutrophil nuclear lobulation characteristic of Pelger-Huët anomaly. Thus, LBR plays a critical role in regulating myeloid differentiation, but how the two functional domains of LBR support this role is currently unclear. We previously identified abnormal proliferation and deficient functional maturation of promyelocytes (erythroid, myeloid, and lymphoid [EML]-derived promyelocytes) derived from EML-ic/ic cells, a myeloid model of ichthyosis (ic) bone marrow that lacks Lbr expression. In this study, we provide new evidence that cholesterol biosynthesis is important to myeloid cell growth and is supported by the sterol reductase domain of Lbr. Cholesterol biosynthesis inhibitors caused growth inhibition of EML cells that increased in EML-derived promyelocytes, whereas cells lacking Lbr exhibited complete growth arrest at both stages. Lipid production increased during wild-type neutrophil maturation, but ic/ic cells exhibited deficient levels of lipid and cholesterol production. Ectopic expression of a full-length Lbr in EML-ic/ic cells rescued both nuclear lobulation and growth arrest in cholesterol starvation conditions. Lipid production also was rescued, and a deficient respiratory burst was corrected. Expression of just the C-terminal sterol reductase domain of Lbr in ic/ic cells also improved each of these phenotypes. Our data support the conclusion that the sterol Δ(14) reductase domain of LBR plays a critical role in cholesterol biosynthesis and that this process is essential to both myeloid cell growth and functional maturation.
Assuntos
Colesterol/imunologia , Metabolismo dos Lipídeos/imunologia , Células Progenitoras Mieloides/imunologia , Mielopoese/imunologia , Receptores Citoplasmáticos e Nucleares/imunologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Colesterol/biossíntese , Colesterol/genética , Metabolismo dos Lipídeos/genética , Camundongos , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Mielopoese/genética , Estrutura Terciária de Proteína , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptor de Lamina BRESUMO
In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.(1) On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.(2) Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.(3) To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling.
Assuntos
Núcleo Celular/fisiologia , Interfase/fisiologia , Análise de Célula Única/métodos , Animais , Fenômenos Biomecânicos , Fenômenos Biofísicos , Humanos , Camundongos , Agulhas , Análise de Célula Única/instrumentaçãoRESUMO
Over the past two decades, the biomechanical properties of cells have emerged as key players in a broad range of cellular functions, including migration, proliferation, and differentiation. Although much of the attention has focused on the cytoskeletal networks and the cell's microenvironment, relatively little is known about the contribution of the cell nucleus. Here, we present an overview of the structural elements that determine the physical properties of the nucleus and discuss how changes in the expression of nuclear components or mutations in nuclear proteins can not only affect nuclear mechanics but also modulate cytoskeletal organization and diverse cellular functions. These findings illustrate that the nucleus is tightly integrated into the surrounding cellular structure. Consequently, changes in nuclear structure and composition are highly relevant to normal development and physiology and can contribute to many human diseases, such as muscular dystrophy, dilated cardiomyopathy, (premature) aging, and cancer.
Assuntos
Núcleo Celular/fisiologia , Fenômenos Fisiológicos Celulares/fisiologia , Doença/etiologia , Lâmina Nuclear/fisiologia , Proteínas Nucleares/metabolismo , Adaptação Fisiológica/fisiologia , Animais , Fenômenos Biomecânicos/fisiologia , Núcleo Celular/ultraestrutura , Humanos , Lâmina Nuclear/ultraestrutura , Proteínas Nucleares/ultraestruturaRESUMO
Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.
Assuntos
Cromatina/química , Evolução Molecular , Animais , Anticorpos Monoclonais/imunologia , Arabidopsis , Autoanticorpos/imunologia , Caenorhabditis elegans , Linhagem Celular Tumoral , Cromatina/metabolismo , Drosophila , Histonas/química , Histonas/metabolismo , Humanos , Interfase , Camundongos , Microscopia de Fluorescência , Nucleossomos/imunologiaRESUMO
Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.
Assuntos
Núcleo Celular/enzimologia , Retículo Endoplasmático/enzimologia , Proteínas de Membrana/metabolismo , Proteínas Mutantes/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Actinas/metabolismo , Aminoácidos/metabolismo , Apoptose , Autofagia , Linhagem Celular , Núcleo Celular/ultraestrutura , Sobrevivência Celular , Colesterol/metabolismo , Retículo Endoplasmático/ultraestrutura , Humanos , Laminas/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico , Transfecção , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Receptor de Lamina BRESUMO
The nuclear envelope (NE) is a barrier that separates nuclear from cytoplasmic processes. It is composed of an inner and outer nuclear membrane (INM, ONM), separated by the perinuclear space (PNS). The ONM is contiguous with the endoplasmic reticulum (ER), and thus, the lumen of the NE and that of the ER constitute one compartment. The lamin B receptor (LBR) is a NE protein that has a central structural role as a linker of the INM, the lamina and chromatin, and a less well characterized functional role as a sterol reductase. In a recent study, we reported that the forced expression of mutant variants of LBR in some cell types induces a separation of the INM from the outer nuclear envelope concomitantly with a separation of ER membranes, whereas in other cells no separation is observed. In this extra view, we speculate about the mechanism that leads to this fundamental disruption of NE and ER structure. Our observations furthermore raise the question to what extent LBR contributes to the establishment or maintenance of the ER and PNS luminal compartment, and how a single mutant protein can so drastically interfere with its regular organization.
Assuntos
Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/patologia , Núcleo Celular/ultraestrutura , Cromatina/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Perfilação da Expressão Gênica , Células HeLa , Humanos , Mutação , Membrana Nuclear/ultraestrutura , Osteocondrodisplasias/genética , Anomalia de Pelger-Huët/genética , Fenótipo , Receptor de Lamina BRESUMO
The lamin B receptor (LBR) is an inner nuclear membrane protein with a structural function interacting with chromatin and lamins, and an enzymatic function as a sterol reductase. Heterozygous LBR mutations cause nuclear hyposegmentation in neutrophils (Pelger anomaly), while homozygous mutations cause prenatal death with skeletal defects and abnormal sterol metabolism (Greenberg dysplasia). It has remained unclear whether the lethality in Greenberg dysplasia is due to cholesterol defects or altered nuclear morphology.To answer this question we characterized two LBR missense mutations and showed that they cause Greenberg dysplasia. Both mutations affect residues that are evolutionary conserved among sterol reductases. In contrast to wildtype LBR, both mutations failed to rescue C14 sterol reductase deficient yeast, indicating an enzymatic defect. We found no Pelger anomaly in the carrier parent excluding marked effects on nuclear structure. We studied Lbr in mouse embryos and demonstrate expression in skin and the developing skeletal system consistent with sites of histological changes in Greenberg dysplasia. Unexpectedly we found in disease-relevant cell types not only nuclear but also cytoplasmatic LBR localization. The cytoplasmatic LBR staining co-localized with ER-markers and is thus consistent with the sites of endogeneous sterol synthesis. We conclude that LBR missense mutations can abolish sterol reductase activity, causing lethal Greenberg dysplasia but not Pelger anomaly. The findings separate the metabolic from the structural function and indicate that the sterol reductase activity is essential for human intrauterine development.
Assuntos
Osteocondrodisplasias/genética , Anomalia de Pelger-Huët/genética , Receptores Citoplasmáticos e Nucleares/genética , Animais , Linhagem Celular Tumoral , Fibroblastos/metabolismo , Genótipo , Células HeLa , Heterozigoto , Homozigoto , Humanos , Camundongos , Mutação de Sentido Incorreto , Membrana Nuclear/metabolismo , Osteocondrodisplasias/patologia , Oxirredutases/genética , Oxirredutases/metabolismo , Anomalia de Pelger-Huët/patologia , Fenótipo , Receptores Citoplasmáticos e Nucleares/análise , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Receptor de Lamina BRESUMO
The principal human blood granulocyte (neutrophil) possesses a lobulated and deformable nucleus, important to facilitate rapid egress from blood vessels as these cells migrate to sites of bacterial or fungal infection. This unusual nuclear shape is a product of elevated levels of an integral membrane protein of the nuclear envelope lamin B receptor (LBR) and of decreased amounts of lamin A/C. In humans, a genetic deficiency of LBR produces Pelger-Huët anomaly, resulting in blood neutrophils that exhibit hypolobulated nuclei with redistributed heterochromatin. Structural changes in nuclear architecture occur during granulopoiesis within bone marrow. The exact mechanisms of this nuclear shape change and of heterochromatin redistribution remain largely unknown. As a tool to facilitate analysis of these mechanisms, a stable LBR knockdown subline of HL-60 cells was established. During in vitro granulopoiesis induced with retinoic acid, the LBR knockdown cells retain an ovoid shaped nucleus with reduced levels of lamin A/C; while, the parent cells develop highly lobulated nuclei. In contrast, macrophage forms induced in LBR knockdown cells by in vitro treatment with phorbol ester were indistinguishable from the parent cells, judged by both nuclear shape and attached cell morphology. The capability of differentiation of LBR knockdown HL-60 cells should facilitate a detailed analysis of the molecular relationship between LBR levels, granulocyte nuclear shape and heterochromatin distribution.