RESUMO
This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris-ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.
Assuntos
Carcinoma de Células Escamosas , Pepsina A , Humanos , Ácido Edético , 5-Metilcitosina , Epigênese Genética , DNA/genética , Metilação de DNA , Antígenos , Citratos , CitosinaRESUMO
SOX2 expression in high-grade cervical intraepithelial neoplasia (CIN3) and cervical squamous cell carcinoma is increased compared to that in the normal cervical epithelium. However, data on the expression and histological distribution of SOX2 in squamous epithelium during progression of CIN are largely lacking. We studied SOX2 expression throughout the epithelium in 53 cases of CIN1, 2, and 3. In general, SOX2 expression increased and expanded from basal/parabasal to the intermediate/superficial compartment during early stages of progression of CIN. An unexpected, specific expression pattern was found in areas classified as CIN2 and CIN3. This pattern was characterized by the absence or low expression of SOX2 in the basal/parabasal compartment and variable levels in the intermediate and superficial compartments. It was significantly associated with CIN3 (p = 0.009), not found in CIN1 and only seen in part of the CIN2 lesions. When the different patterns were correlated with the genetic make-up and presence of HPV, the CIN3-related pattern contained HPV-positive cells in the basal/parabasal cell compartment that were disomic. This is in contrast to the areas exhibiting the CIN1 and CIN2 related patterns, which frequently exhibited aneusomic cells. Based on their SOX2 localisation pattern, CIN1 and CIN2 could be delineated from CIN3. These data shed new light on the pathogenesis and dynamics of progression in premalignant cervical lesions, as well as on the target cells in the epithelium for HPV infection.
Assuntos
Infecções por Papillomavirus , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Feminino , Humanos , Fatores de Transcrição SOXB1/genéticaRESUMO
A- and B-type lamins are type V intermediate filament proteins. Mutations in the genes encoding these lamins cause rare diseases, collectively called laminopathies. A fraction of the cells obtained from laminopathy patients show aberrations in the localization of each lamin subtype, which may represent only the minority of the lamina disorganization. To get a better insight into more delicate and more abundant lamina abnormalities, the lamin network can be studied using super-resolution microscopy. We compared confocal scanning laser microscopy and stimulated emission depletion (STED) microscopy in combination with different fluorescence labeling approaches for the study of the lamin network. We demonstrate the suitability of an immunofluorescence staining approach when using STED microscopy, by determining the lamin layer thickness and the degree of lamin A and B1 colocalization as detected in fixed fibroblasts (co-)stained with lamin antibodies or (co-)transfected with EGFP/YFP lamin constructs. This revealed that immunofluorescence staining of cells does not lead to consequent changes in the detected lamin layer thickness, nor does it influence the degree of colocalization of lamin A and B1, when compared to the transfection approach. Studying laminopathy patient dermal fibroblasts (LMNA c.1130G>T (p.(Arg377Leu)) variant) confirmed the suitability of immunofluorescence protocols in STED microscopy, which circumvents the need for less convenient transfection steps. Furthermore, we found a significant decrease in lamin A/C and B1 colocalization in these patient fibroblasts, compared to normal human dermal fibroblasts. We conclude that super-resolution light microscopy combined with immunofluorescence protocols provides a potential tool to detect structural lamina differences between normal and laminopathy patient fibroblasts.
Assuntos
Proteínas de Filamentos Intermediários/metabolismo , Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Laminopatias/patologia , Membrana Nuclear/metabolismo , Células 3T3 , Animais , Linhagem Celular , Fibroblastos/metabolismo , Imunofluorescência , Proteínas de Filamentos Intermediários/genética , Lamina Tipo A/genética , Lamina Tipo B/genética , Laminopatias/genética , Camundongos , Microscopia ConfocalRESUMO
In adherent cells, the relevance of a physical mechanotransduction pathway provided by the perinuclear actin cap stress fibers has recently emerged. Here, we investigate the impact of a functional actin cap on the cellular adaptive response to topographical cues and uniaxial cyclic strain. Lmna-deficient fibroblasts are used as a model system because they do not develop an intact actin cap, but predominantly form a basal layer of actin stress fibers underneath the nucleus. We observe that topographical cues induce alignment in both normal and Lmna-deficient fibroblasts, suggesting that the topographical signal transmission occurs independently of the integrity of the actin cap. By contrast, in response to cyclic uniaxial strain, Lmna-deficient cells show a compromised strain avoidance response, which is completely abolished when topographical cues and uniaxial strain are applied along the same direction. These findings point to the importance of an intact and functional actin cap in mediating cellular strain avoidance.
Assuntos
Actinas/metabolismo , Lamina Tipo A/deficiência , Modelos Biológicos , Estresse Mecânico , Estresse Fisiológico , Actinina , Animais , Anisotropia , Forma Celular , Embrião de Mamíferos/citologia , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Lamina Tipo A/metabolismo , Camundongos , Miosinas/metabolismo , Fosforilação , Fibras de Estresse/metabolismo , Fatores de TempoRESUMO
Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.
Assuntos
Conexinas/genética , Eritroceratodermia Variável/genética , Mutação de Sentido Incorreto , Morte Celular , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/biossíntese , Conexina 43/genética , Retículo Endoplasmático/ultraestrutura , Epiderme/patologia , Eritroceratodermia Variável/patologia , Genes Dominantes , Estudos de Associação Genética , Ácido Glicirretínico/análogos & derivados , Ácido Glicirretínico/farmacologia , Células HeLa , Humanos , Queratinócitos , Necrose , Transporte ProteicoRESUMO
Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder where patients are predisposed to kidney cancer, lung and kidney cysts and benign skin tumors. BHD is caused by heterozygous mutations affecting folliculin (FLCN), a conserved protein that is considered a tumor suppressor. Previous research has uncovered multiple roles for FLCN in cellular physiology, yet it remains unclear how these translate to BHD lesions. Since BHD manifests hallmark characteristics of ciliopathies, we speculated that FLCN might also have a ciliary role. Our data indicate that FLCN localizes to motile and non-motile cilia, centrosomes and the mitotic spindle. Alteration of FLCN levels can cause changes to the onset of ciliogenesis, without abrogating it. In three-dimensional culture, abnormal expression of FLCN disrupts polarized growth of kidney cells and deregulates canonical Wnt signalling. Our findings further suggest that BHD-causing FLCN mutants may retain partial functionality. Thus, several BHD symptoms may be due to abnormal levels of FLCN rather than its complete loss and accordingly, we show expression of mutant FLCN in a BHD-associated renal carcinoma. We propose that BHD is a novel ciliopathy, its symptoms at least partly due to abnormal ciliogenesis and canonical Wnt signalling.
Assuntos
Síndrome de Birt-Hogg-Dubé/fisiopatologia , Cílios/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Sequência de Bases , Síndrome de Birt-Hogg-Dubé/genética , Linhagem Celular , Polaridade Celular , Proliferação de Células , Centrossomo/fisiologia , Cílios/patologia , Humanos , Rim/fisiologia , Microtúbulos/fisiologia , Dados de Sequência Molecular , Análise de Sequência de DNA , Via de Sinalização WntRESUMO
Not long after the discovery of lamin proteins, it became clear that not all lamin subtypes are ubiquitously expressed in cells and tissues. Especially, A-type lamins showed an inverse correlation with proliferation and were thus initially called statins. Here we compare the findings of both A- and B-type lamin expression in various normal tissues and their neoplastic counterparts. Based on immunocytochemistry it becomes clear that lamin expression patterns are much more complicated than initially assumed: while normally proliferative cells are devoid of A-type lamin expression, many neoplastic tissues do show prominent A-type lamin expression. Conversely, cells that do not proliferate can be devoid of lamin expression. Yet, within the different types of tissues and tumors, lamins can be used to distinguish between tumor subtypes. The link between the appearance of A-type lamins in differentiation and the appearance of A-type lamins in a tumor likely relates the proliferative capacity of the tumor to its differentiation state.While lamins are targets for degradation in the apoptotic process, and accordingly are often used as markers for apoptosis, intriguing studies on an active role of lamins in the initiation or the prevention of apoptosis have been published recently and give rise to a renewed interest in the role of lamins in cancer.
Assuntos
Apoptose/fisiologia , Neoplasias/fisiopatologia , Lâmina Nuclear/fisiologia , Diferenciação Celular , Feminino , Humanos , Lamina Tipo A/genética , Lamina Tipo A/fisiologia , Masculino , Mutação , Neoplasias/classificação , Neoplasias/patologiaRESUMO
Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Miócitos Cardíacos/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Animais , Antígenos CD36/metabolismo , Linhagem Celular , Gorduras na Dieta/farmacologia , Expressão Gênica , Cardiopatias/metabolismo , Cardiopatias/patologia , Insulina/farmacologia , Insulina/fisiologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Contração Miocárdica , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Palmitatos/farmacologia , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
The nuclear lamina provides structural support to the nucleus and has a central role in nuclear organization and gene regulation. Defects in its constituents, the lamins, lead to a class of genetic diseases collectively referred to as laminopathies. Using live cell imaging, we observed the occurrence of intermittent, non-lethal ruptures of the nuclear envelope in dermal fibroblast cultures of patients with different mutations of lamin A/C. These ruptures, which were absent in normal fibroblasts, could be mimicked by selective knockdown as well as knockout of LMNA and were accompanied by the loss of cellular compartmentalization. This was demonstrated by the influx of cytoplasmic transcription factor RelA and regulatory protein Cyclin B1 into the nucleus, and efflux of nuclear transcription factor OCT1 and nuclear structures containing the promyelocytic leukemia (PML) tumour suppressor protein to the cytoplasm. While recovery of enhanced yellow fluorescent protein-tagged nuclear localization signal in the nucleus demonstrated restoration of nuclear membrane integrity, part of the mobile PML structures became permanently translocated to the cytoplasm. These satellite PML structures were devoid of the typical PML body components, such as DAXX, SP100 or SUMO1. Our data suggest that nuclear rupture and loss of compartmentalization may add to cellular dysfunction and disease development in various laminopathies.
Assuntos
Compartimento Celular , Laminas/metabolismo , Membrana Nuclear/patologia , Animais , Proteínas de Bactérias/metabolismo , Divisão Celular , Dextranos/metabolismo , Regulação da Expressão Gênica , Humanos , Lamina Tipo A/metabolismo , Proteínas Luminescentes/metabolismo , Substâncias Macromoleculares/metabolismo , Camundongos , Peso Molecular , Membrana Nuclear/ultraestrutura , Sinais de Localização Nuclear , Transportador 1 de Cátions Orgânicos/metabolismo , Transporte ProteicoRESUMO
BACKGROUND: Dilated cardiomyopathy (DCM) was considered a monogenetic disease that can be caused by over 60 genes. Evidence suggests that the combination of multiple pathogenic variants leads to greater disease severity and earlier onset. So far, not much is known about the prevalence and disease course of multiple pathogenic variants in patients with DCM. To gain insight into these knowledge gaps, we (1) systematically collected clinical information from a well-characterized DCM cohort and (2) created a mouse model. METHODS: Complete cardiac phenotyping and genotyping was performed in 685 patients with consecutive DCM. Compound heterozygous digenic (LMNA [lamin]/titin deletion A-band) with monogenic (LMNA/wild-type) and wild-type/wild-type mice were created and phenotypically followed over time. RESULTS: One hundred thirty-one likely pathogenic/pathogenic (LP/P) variants in robust DCM-associated genes were found in 685 patients with DCM (19.1%) genotyped for the robust genes. Three of the 131 patients had a second LP/P variant (2.3%). These 3 patients had a comparable disease onset, disease severity, and clinical course to patients with DCM with one LP/P. The LMNA/Titin deletion A-band mice had no functional differences compared with the LMNA/wild-type mice after 40 weeks of follow-up, although RNA-sequencing suggests increased cardiac stress and sarcomere insufficiency in the LMNA/Titin deletion A-band mice. CONCLUSIONS: In this study population, 2.3% of patients with DCM with one LP/P also have a second LP/P in a different gene. Although the second LP/P does not seem to influence the disease course of DCM in patients and mice, the finding of a second LP/P can be of importance to their relatives.
Assuntos
Cardiomiopatia Dilatada , Humanos , Animais , Camundongos , Cardiomiopatia Dilatada/epidemiologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Conectina/genética , Prevalência , Mutação , GenótipoRESUMO
In human diploid fibroblasts (HDFs), expression of lamina-associated polypeptide 2 alpha (LAP2alpha) upon entry and exit from G(0) is tightly correlated with phosphorylation and subnuclear localization of retinoblastoma protein (Rb). Phosphoisoforms of Rb and LAP2alpha are down-regulated in G(0). Although RbS780 phosphoform and LAP2alpha are up-regulated upon reentry into G(1) and colocalize in the nucleoplasm, RbS795 migrates between nucleoplasmic and speckle compartments. In HDFs, which are null for lamins A/C, LAP2alpha is mislocalized within nuclear aggregates, and this is correlated with cell cycle arrest and accumulation of Rb within speckles. Nuclear retention of nucleoplasmic Rb during G(1) phase but not of speckle-associated Rb depends on lamin A/C. siRNA knock down of LAP2alpha or lamin A/C in HDFs leads to accumulation of Rb in speckles and G(1) arrest, probably because of activation of a cell cycle checkpoint. Our results suggest that LAP2alpha and lamin A/C are involved in controlling Rb localization and phosphorylation, and a lack or mislocalization of either protein leads to cell cycle arrest in HDFs.
Assuntos
Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Ciclo Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/análise , Proteínas de Ligação a DNA/genética , Fibroblastos/química , Fibroblastos/citologia , Humanos , Espaço Intranuclear/química , Espaço Intranuclear/metabolismo , Antígeno Ki-67/metabolismo , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Lamina Tipo B/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Mutação , Proteínas Nucleares/análise , Proteínas Nucleares/metabolismo , Octoxinol/química , Fosforilação , RNA Interferente Pequeno/genética , Proteína do Retinoblastoma/análise , Proteína do Retinoblastoma/metabolismo , Ribonucleoproteínas/análise , Ribonucleoproteínas/metabolismo , Fatores de Processamento de Serina-Arginina , Solubilidade , Spliceossomos/química , Spliceossomos/metabolismoRESUMO
Invaginations of the nuclear membrane occur in different shapes, sizes, and compositions. Part of these pleiomorphic invaginations make up the nucleoplasmic reticulum (NR), while others are merely nuclear folds. We define the NR as tubular invaginations consisting of either both the inner and outer nuclear membrane, or only the inner nuclear membrane. Specifically, invaginations of both the inner and outer nuclear membrane are also called type II NR, while those of only the inner nuclear membrane are defined as type I NR. The formation and structure of the NR is determined by proteins associated to the nuclear membrane, which induce a high membrane curvature leading to tubular invaginations. Here we review and discuss the current knowledge of nuclear invaginations and the NR in particular. An increase in tubular invaginations of the nuclear envelope is associated with several pathologies, such as laminopathies, cancer, (reversible) heart failure, and Alzheimer's disease. Furthermore, viruses can induce both type I and II NR. In laminopathies, the amount of A-type lamins throughout the nucleus is generally decreased or the organization of lamins or lamin-associated proteins is disturbed. Also, lamin overexpression or modulation of lamin farnesylation status impacts NR formation, confirming the importance of lamin processing in NR formation. Virus infections reorganize the nuclear lamina via (de)phosphorylation of lamins, leading to an uneven thickness of the nuclear lamina and in turn lobulation of the nuclear membrane and the formation of invaginations of the inner nuclear membrane. Since most studies on the NR have been performed with cell cultures, we present additional proof for the existence of these structures in vivo, focusing on a variety of differentiated cardiovascular and hematopoietic cells. Furthermore, we substantiate the knowledge of the lamin composition of the NR by super-resolution images of the lamin A/C and B1 organization. Finally, we further highlight the essential role of lamins in NR formation by demonstrating that (over)expression of lamins can induce aberrant NR structures.
RESUMO
BACKGROUND: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.
Assuntos
Códon sem Sentido , Fibroblastos/fisiologia , Lamina Tipo A/genética , Membrana Nuclear/genética , Progéria/genética , Linhagem Celular , Forma Celular , Fibroblastos/patologia , Humanos , Imuno-Histoquímica , Lamina Tipo A/deficiência , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Progéria/metabolismo , Progéria/patologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Valores de Referência , Pele/citologia , Fenômenos Fisiológicos da PeleRESUMO
A thorough understanding of fat cell biology is necessary to counter the epidemic of obesity. Although molecular pathways governing adipogenesis are well delineated, the structure of the nuclear lamina and nuclear-cytoskeleton junction in this process are not. The identification of the 'linker of nucleus and cytoskeleton' (LINC) complex made us consider a role for the nuclear lamina in adipose conversion. We herein focused on the structure of the nuclear lamina and its coupling to the vimentin network, which forms a cage-like structure surrounding individual lipid droplets in mature adipocytes. Analysis of a mouse and human model system for fat cell differentiation showed fragmentation of the nuclear lamina and subsequent loss of lamins A, C, B1 and emerin at the nuclear rim, which coincides with reorganization of the nesprin-3/plectin/vimentin complex into a network lining lipid droplets. Upon 18 days of fat cell differentiation, the fraction of adipocytes expressing lamins A, C and B1 at the nuclear rim increased, though overall lamin A/C protein levels were low. Lamin B2 remained at the nuclear rim throughout fat cell differentiation. Light and electron microscopy of a subcutaneous adipose tissue specimen showed striking indentations of the nucleus by lipid droplets, suggestive for an increased plasticity of the nucleus due to profound reorganization of the cellular infrastructure. This dynamic reorganization of the nuclear lamina in adipogenesis is an important finding that may open up new venues for research in and treatment of obesity and nuclear lamina-associated lipodystrophy.
Assuntos
Adipócitos/citologia , Adipogenia , Citoesqueleto/metabolismo , Lâmina Nuclear/metabolismo , Células 3T3-L1 , Adipócitos/metabolismo , Adipócitos/ultraestrutura , Animais , Células Cultivadas , Citoesqueleto/ultraestrutura , Humanos , Imuno-Histoquímica , Laminas/análise , Laminas/biossíntese , Camundongos , Lâmina Nuclear/ultraestruturaRESUMO
BACKGROUND: Standing desks have been brought into the education environment to reduce sedentary behavior among students. The current study explored the effects of standing in tutorial group meetings on learning among undergraduate students. METHODS: Ninety-six participants were randomly allocated to a Sit or Stand group, with 2 h tutorial group meetings scheduled, once or twice per week, for nine weeks. Learning was analyzed using exam grades, concept maps, and tutorial interactions. RESULTS: Overall, the Sit and Stand groups did not differ from each other in terms of learning, measured through their exam, concept map, and the use of learning-oriented interactions. CONCLUSION: Standing in tutorial group meetings neither enhanced nor compromised learning. Considering the health risks associated with prolonged sedentary behavior, offering standing tutorial group meetings to undergraduate students is a recommended solution to break up prolonged sedentary behavior and encourage more physical activity, while maintaining the learning performance of students.
Assuntos
Posição Ortostática , Local de Trabalho , Exercício Físico , Processos Grupais , Humanos , Comportamento SedentárioRESUMO
The nuclear lamins are the major components of the nuclear lamina in the nuclear envelope. Lamins are involved in numerous functions, including a role in providing structural support to the cell and the mechanosensing of the cell. Mutations in the genes encoding for lamins lead to the rare diseases termed laminopathies. However, not only laminopathies show alterations in the nuclear lamina. Deregulation of lamin expression is reported in multiple cancers and several viral infections lead to a disrupted nuclear lamina. The structural and mechanical effects of alterations in the nuclear lamina can partly explain the phenotypes seen in disease, such as muscular weakness in certain laminopathies and transmigration of cancer cells. However, a lot of answers to questions about the relation between changes in the nuclear lamina and disease development remain elusive. Here, we review the current understandings of the contribution of the nuclear lamina in the structural support and mechanosensing of healthy and diseased cells.
Assuntos
Laminas/metabolismo , Lâmina Nuclear/metabolismo , Humanos , Mecanorreceptores/metabolismo , Mutação/genéticaRESUMO
Dunnigan-type familial partial lipodystrophy (FPLD) is a laminopathy characterized by an aberrant fat distribution and a metabolic syndrome for which oxidative stress has recently been suggested as one of the disease-causing mechanisms. In a family affected with FPLD, we identified a heterozygous missense mutation c.1315C>T in the LMNA gene leading to the p.R439C substitution. Cultured patient fibroblasts do not show any prelamin A accumulation and reveal honeycomb-like lamin A/C formations in a significant percentage of nuclei. The mutation affects a region in the C-terminal globular domain of lamins A and C, different from the FPLD-related hot spot. Here, the introduction of an extra cysteine allows for the formation of disulphide-mediated lamin A/C oligomers. This oligomerization affects the interaction properties of the C-terminal domain with DNA as shown by gel retardation assays and causes a DNA-interaction pattern that is distinct from the classical R482W FPLD mutant. Particularly, whereas the R482W mutation decreases the binding efficiency of the C-terminal domain to DNA, the R439C mutation increases it. Electron spin resonance spectroscopy studies show significantly higher levels of reactive oxygen species (ROS) upon induction of oxidative stress in R439C patient fibroblasts compared to healthy controls. This increased sensitivity to oxidative stress seems independent of the oligomerization and enhanced DNA binding typical for R439C, as both the R439C and R482W mutants show a similar and significant increase in ROS upon induction of oxidative stress by H2O2.
Assuntos
Lamina Tipo A/fisiologia , Lipodistrofia Parcial Familiar/metabolismo , Mutação de Sentido Incorreto , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Precursores de Proteínas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Predisposição Genética para Doença , Humanos , Peróxido de Hidrogênio/farmacologia , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Complexos Multiproteicos , Espécies Reativas de Oxigênio/metabolismoRESUMO
CONTEXT: Upon palmitate oversupply, membrane fatty acid-transporter CD36 (SR-B2) permanently translocates from endosomal storage to the sarcolemma, inducing lipotoxicity. CD36 translocation results from endosomal alkalinisation elicited by palmitate-induced disattachment of the cytoplasmic V1-subcomplex from the membrane-integrated V0-subcomplex of vacuolar-type H+-ATPase. OBJECTIVE: Develop a CD36 fluorescent labeling technique as initial step towards live cell imaging. METHODS: Three human CD36 (hCD36) mutants were constructed via insertion of a tetracysteine motif at different positions within the extracellular domain. Constructs were lentivirally transduced for subsequent CD36 labeling with fluorescein-arsenical hairpin-binder (FlAsH). Cell imaging was combined with V0/V1 immunostaining and Western blotting. RESULTS: Transduction of hCD36-wildtype and mutants yielded corresponding proteins in HL-1 cardiomyocytes. Tetracysteine mutant-2 (hCD36-TC2) showed similar fatty acid uptake to wildtype. FlAsH staining revealed a speckled pattern reminiscent of endosomes. We found decreased V1 co-localization with CD36 upon high-palmitate culturing. Conversely, V0 consistently co-localized with CD36. CONCLUSION: hCD36-TC2 is a possible candidate for application of biarsenical dyes in live imaging studies pending further investigation. Our data is compatible with V0/V1 disassembly in high-palmitate-treated cells.
Assuntos
Antígenos CD36/metabolismo , Western Blotting , Endossomos/metabolismo , Células HEK293 , Humanos , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , ATPases Vacuolares Próton-Translocadoras/metabolismoRESUMO
The phenotypic heterogeneity of Lamin A/C (LMNA) variants renders it difficult to classify them. As a consequence, many LMNA variants are classified as variant of unknown significance (VUS). A number of studies reported different types of visible nuclear abnormalities in LMNA-variant carriers, such as herniations, honeycomb-like structures and irregular Lamin staining. In this study, we used lamin A/C immunostaining and nuclear DAPI staining to assess the number and type of nuclear abnormalities in primary dermal fibroblast cultures of laminopathy patients and healthy controls. The total number of abnormal nuclei, which includes herniations, honeycomb-structures, and donut-like nuclei, was found to be the most discriminating parameter between laminopathy and control cell cultures. The percentage abnormal nuclei was subsequently scored in fibroblasts of 28 LMNA variant carriers, ranging from (likely) benign to (likely) pathogenic variant. Using this method, 27 out of 28 fibroblast cell cultures could be classified as either normal (n = 14) or laminopathy (n = 13) and no false positive results were obtained. The obtained specificity was 100% (CI 40-100%) and sensitivity 77% (46-95%). We conclude that assessing the percentage of abnormal nuclei is a quick and reliable method, which aids classification or confirms pathogenicity of identified LMNA variants causing formation of aberrant lamin A/C protein.
Assuntos
Núcleo Celular/patologia , Fibroblastos/patologia , Testes Genéticos/métodos , Lamina Tipo A/genética , Células Cultivadas , Citogenética/métodos , Fibroblastos/metabolismo , HumanosRESUMO
BACKGROUND: The Neural Cell Adhesion Molecule (NCAM) is a glycoprotein expressed as 120, 140 and/or 180 kDa isoforms, all derived through alternative splicing of a single gene. NCAM 120 contains no intracellular domain, whereas NCAM 140 and 180 have different intracellular domains determined by alternative splicing of exon 18. NCAM has been described as a biomarker to discriminate small cell lung cancer (SCLC) from non-SCLC (NSCLC). However, peripheral blood mononuclear cells (PBMC) also express NCAM. We studied the expression of NCAM splice variants in cell lines, tumor tissues and control cells. METHODS: Using reverse transcriptase-PCR we evaluated the expression of NCAM exon 18 splice variants in lung cancers cell lines, control cell lines, PBMC of healthy controls and SCLC tissue. In addition we studied the expression of the NCAM exon 18 encoded protein (E18) in SCLC by immunocytochemistry and flow cytometry using an E18-specific monoclonal antibody obtained by hybridoma fusion of E18-immunized mouse spleen cells. Finally we looked at immune responses to E18 in mice. RESULTS: We found expression of RNA encoding the NCAM 180 variant in all SCLC cell lines. NCAM exon 18 was not expressed in 23/28 (82%) of the other tumor and leukemia cell lines tested and PBMC. Next, we also evaluated the expression of NCAM exon 18 in human SCLC tissue. Expression of NCAM exon 18 in 8 of the 10 (80%) SCLC biopsy samples was found. The newly raised E18-specific antibodies stained NCAM at the adherent junctions between adjacent cells in SCLC cell lines. The data demonstrate the intracellular location of E18 in SCLC. Furthermore, a specific cytotoxic T cell (CTL) response and significant antibody titers were found in mice upon immunization with recombinant E18 and its encoding DNA. CONCLUSIONS: The results of this study can be applied in the diagnosis and immunotherapy of SCLC. A larger study investigating E18 as a marker for SCLC is indicated.