RESUMEN
Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson-Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced "donut formation" and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.
Asunto(s)
Antineoplásicos/farmacología , Núcleo Celular , Centrosoma , Mitosis/efectos de los fármacos , Prenilación de Proteína/efectos de los fármacos , Huso Acromático , Animales , Antígenos/biosíntesis , Antígenos/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/patología , Centrosoma/metabolismo , Centrosoma/patología , Células Hep G2 , Humanos , Lamina Tipo B/biosíntesis , Lamina Tipo B/genética , Ratones , Ratones Transgénicos , Mitosis/genética , Progeria/genética , Progeria/metabolismo , Progeria/patología , Prenilación de Proteína/genética , Huso Acromático/genética , Huso Acromático/metabolismo , Huso Acromático/patologíaRESUMEN
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.
Asunto(s)
Compartimento Celular , Laminas/metabolismo , Membrana Nuclear/patología , Animales , Proteínas Bacterianas/metabolismo , División Celular , Dextranos/metabolismo , Regulación de la Expresión Génica , Humanos , Lamina Tipo A/metabolismo , Proteínas Luminiscentes/metabolismo , Sustancias Macromoleculares/metabolismo , Ratones , Peso Molecular , Membrana Nuclear/ultraestructura , Señales de Localización Nuclear , Transportador 1 de Catión Orgánico/metabolismo , Transporte de ProteínasRESUMEN
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.
Asunto(s)
Adipocitos/citología , Adipogénesis , Citoesqueleto/metabolismo , Lámina Nuclear/metabolismo , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/ultraestructura , Animales , Células Cultivadas , Citoesqueleto/ultraestructura , Humanos , Inmunohistoquímica , Laminas/análisis , Laminas/biosíntesis , Ratones , Lámina Nuclear/ultraestructuraRESUMEN
Thioredoxin-interacting protein (Txnip), originally characterized as an inhibitor of thioredoxin, is now known to be a critical regulator of glucose metabolism in vivo. Txnip is a member of the alpha-arrestin protein family; the alpha-arrestins are related to the classical beta-arrestins and visual arrestins. Txnip is the only alpha-arrestin known to bind thioredoxin, and it is not known whether the metabolic effects of Txnip are related to its ability to bind thioredoxin or related to conserved alpha-arrestin function. Here we show that wild type Txnip and Txnip C247S, a Txnip mutant that does not bind thioredoxin in vitro, both inhibit glucose uptake in mature adipocytes and in primary skin fibroblasts. Furthermore, we show that Txnip C247S does not bind thioredoxin in cells, using thiol alkylation to trap the Txnip-thioredoxin complex. Because Txnip function was independent of thioredoxin binding, we tested whether inhibition of glucose uptake was conserved in the related alpha-arrestins Arrdc4 and Arrdc3. Both Txnip and Arrdc4 inhibited glucose uptake and lactate output, while Arrdc3 had no effect. Structure-function analysis indicated that Txnip and Arrdc4 inhibit glucose uptake independent of the C-terminal WW-domain binding motifs, recently identified as important in yeast alpha-arrestins. Instead, regulation of glucose uptake was intrinsic to the arrestin domains themselves. These data demonstrate that Txnip regulates cellular metabolism independent of its binding to thioredoxin and reveal the arrestin domains as crucial structural elements in metabolic functions of alpha-arrestin proteins.
Asunto(s)
Arrestina/metabolismo , Arrestinas/fisiología , Proteínas Portadoras/fisiología , Regulación de la Expresión Génica , Tiorredoxinas/fisiología , Secuencia de Aminoácidos , Animales , Fibroblastos/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Lamina Tipo A/fisiología , Lipodistrofia Parcial Familiar/metabolismo , Mutación Missense , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Precursores de Proteínas/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Predisposición Genética a la Enfermedad , Humanos , Peróxido de Hidrógeno/farmacología , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Complejos Multiproteicos , Especies Reactivas de Oxígeno/metabolismoAsunto(s)
Proteínas de Unión al ADN/fisiología , Corazón/fisiopatología , Proteínas de la Membrana/fisiología , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Distrofina/genética , Distrofina/metabolismo , Distrofina/fisiología , Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Corazón/crecimiento & desarrollo , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos mdx , Ratones Noqueados , Modelos Biológicos , Miocardio/metabolismo , Miocardio/patología , Disfunción Ventricular Izquierda/genética , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
Laminopathies, mainly caused by mutations in the LMNA gene, are a group of inherited diseases with a highly variable penetrance; i.e., the disease spectrum in persons with identical LMNA mutations range from symptom-free conditions to severe cardiomyopathy and progeria, leading to early death. LMNA mutations cause nuclear abnormalities and cellular fragility in response to cellular mechanical stress, but the genotype/phenotype correlations in these diseases remain unclear. Consequently, tools such as mutation analysis are not adequate for predicting the course of the disease. Here, we employ growth substrate stiffness to probe nuclear fragility in cultured dermal fibroblasts from a laminopathy patient with compound progeroid syndrome. We show that culturing of these cells on substrates with stiffness higher than 10 kPa results in malformations and even rupture of the nuclei, while culture on a soft substrate (3 kPa) protects the nuclei from morphological alterations and ruptures. No malformations were seen in healthy control cells at any substrate stiffness. In addition, analysis of the actin cytoskeleton organization in this laminopathy cells demonstrates that the onset of nuclear abnormalities correlates to an increase in cytoskeletal tension. Together, these data indicate that culturing of these LMNA mutated cells on substrates with a range of different stiffnesses can be used to probe the degree of nuclear fragility. This assay may be useful in predicting patient-specific phenotypic development and in investigations on the underlying mechanisms of nuclear and cellular fragility in laminopathies.
Asunto(s)
Núcleo Celular/metabolismo , Lamina Tipo A/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Cardiomiopatías/genética , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Línea Celular , Forma del Núcleo Celular/fisiología , Fibroblastos/citología , Fibroblastos/metabolismo , Heterocigoto , Humanos , Lamina Tipo A/genética , Mutación , Progeria/genética , Progeria/metabolismo , Progeria/patologíaAsunto(s)
Dermatosis Facial/diagnóstico , Enfermedades de la Uña/diagnóstico , Administración Cutánea , Antibacterianos/administración & dosificación , Diagnóstico Diferencial , Dermatosis Facial/complicaciones , Dermatosis Facial/patología , Femenino , Pie , Mano , Humanos , Persona de Mediana Edad , Enfermedades de la Uña/complicaciones , Enfermedades de la Uña/patologíaRESUMEN
While the structure of chromatin and its physical properties have been well studied on isolated chromatin fibres and DNA strands in vitro, its organization and function in the intact interphase nucleus is less clear. Chromatin organization is critical for transcriptional regulation and DNA replication, and mounting evidence suggests that cells respond to changes in the mechanical environment with alterations in nuclear architecture that are accompanied by modifications in gene expression. Since the nucleus forms part of a continuous physical network spanning the extracellular matrix, the cytoskeleton and the nuclear envelope, environmentally mediated forces can be transmitted to the nucleus and induce deformations of the chromatin.Here, we describe a subset of techniques that can be applied to probe nuclear mechanics, ranging from micropipette aspiration to strain experiments on living cells. These experiments probe the physical properties of the nuclear envelope, the nucleoplasm, and the chromatin. We discuss the advantages and disadvantages of each technique and elaborate on their use to examine specific aspects of chromatin. In the end, a combination of these technologies can provide important insights into the delicate relationship between form and function of chromatin organization in the living cell.
Asunto(s)
Cromatina/fisiología , Animales , Fenómenos Biomecánicos , Núcleo Celular/fisiología , Citoesqueleto/fisiología , Matriz Extracelular/fisiología , Humanos , Microscopía de Fuerza Atómica/métodos , Microscopía Fluorescente/métodos , Modelos Biológicos , Membrana Nuclear/fisiología , Reología/métodos , Estrés Mecánico , SucciónRESUMEN
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 maintaining 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 restored nuclear stiffness in HGPS cells and accelerated the wound healing response in HGPS and healthy control cells by increasing the directional persistence of migrating cells. However, farnesyltransferase inhibitors 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.
Asunto(s)
Farnesiltransferasa/antagonistas & inhibidores , Metionina/análogos & derivados , Progeria/patología , Adolescente , Anciano de 80 o más Años , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Masculino , Metionina/farmacología , Proteínas Nucleares/metabolismo , Progeria/metabolismo , Sensibilidad y Especificidad , Piel/patología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Birt-Hogg-Dubé (BHD) syndrome is an autosomal-dominantly inherited cancer syndrome characterized by fibrofolliculomas, lung cysts leading to pneumothorax, and chromophobic/oncocytic renal cell carcinoma. The disease is caused by heterozygous mutations in the BHD gene encoding folliculin and all mutations reported putatively lead to protein truncation. Although the function of folliculin is unknown, it is thought to be a tumor suppressor, with loss of heterozygosity (LOH) initiating tumor formation. Here, we report on four novel BHD gene mutations, including two splice-site mutations, in patients presenting with skin lesions only. We further show that LOH cannot be detected in fibrofolliculomas from three patients, suggesting that for the manifestation of cutaneous tumors in BHD syndrome haplo-insufficiency of folliculin is sufficient to initiate uncontrolled growth. Renal microscopic oncocytosis in BHD is considered as a precursor to malignant kidney tumors and may likewise be the result of haplo-insufficiency, with somatic second-hit mutations or LOH giving rise to malignancy later in life.
Asunto(s)
Carcinoma de Células Renales/genética , Enfermedades del Cabello/genética , Neoplasias Renales/genética , Pérdida de Heterocigocidad , Enfermedades Pulmonares/genética , Mutación , Síndromes Neoplásicos Hereditarios/diagnóstico , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Neoplasias Cutáneas/genética , Proteínas Supresoras de Tumor/genética , Adulto , Anciano , Carcinoma de Células Renales/diagnóstico , Femenino , Enfermedades del Cabello/diagnóstico , Humanos , Neoplasias Renales/diagnóstico , Enfermedades Pulmonares/diagnóstico , Masculino , Persona de Mediana Edad , Síndromes Neoplásicos Hereditarios/genética , Neoplasias Cutáneas/diagnósticoRESUMEN
LMNA-associated progeroid syndromes have been reported with both recessive and dominant inheritance. We report a 2-year-old boy with an apparently typical Hutchinson-Gilford progeria syndrome (HGPS) due to compound heterozygous missense mutations (p.T528M and p.M540T) in LMNA. Both mutations affect a conserved region within the C-terminal globular domain of A-type lamins, defining a progeria hot spot. The nuclei of the patient showed no prelamin A accumulation. In general, the nuclear phenotype did not correspond to that previously described for HGPS. Instead, honeycomb figures predominated and nuclear blebs with reduced/absent expression of B-type lamins could be detected. The healthy heterozygous parents showed similar nuclear changes, although in a smaller percentage of nuclei. Treatment with a farnesylation inhibitor resulted in accumulation of prelamin A at the nuclear periphery, in annular nuclear membrane plaques and in intra/trans-nuclear membrane invaginations. In conclusion, these findings suggest a critical role for the C-terminal globular lamin A/C region in nuclear structure and support a major contribution of abnormal assembly to the progeroid phenotype. In contrast to earlier suggestions, we show that prelamin A accumulation is not the major determinant of the progeroid phenotype.