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1.
Int J Mol Sci ; 22(19)2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34638534

RESUMO

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 Confocal
2.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502098

RESUMO

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.


Assuntos
Adesão Celular , Lamina Tipo A/metabolismo , Miofibroblastos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Lamina Tipo A/genética , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Nanotubos/química , Pinças Ópticas , Ratos , Ratos Sprague-Dawley
3.
Stem Cell Res ; 56: 102530, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34507144

RESUMO

LAMIN A/C, encoded by the LMNA gene, supports the normal structure of the cell nucleus and regulates the connection between the nucleus and the cytoskeleton as a component of the nucleus envelope. The loss of expression and function of the LMNA gene would lead to the occurrence of congenital muscular dystrophy and Emery-Dreifuss muscular dystrophy which are collectively named as laminopathies. Here, we report a human induced pluripotent stem cell (iPSC) line (EHTJUi005-A-3) generated from a wild iPSC (EHTJUi005-A) with homozygous knockout of the gene LMNA through CRISPR/Cas9. This iPSC line provides a useful research model for studying laminopathies disease.


Assuntos
Células-Tronco Pluripotentes Induzidas , Laminopatias , Distrofia Muscular de Emery-Dreifuss , Sistemas CRISPR-Cas/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Mutação , Tecnologia
4.
Nat Commun ; 12(1): 4722, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354059

RESUMO

Mutations in the LaminA gene are a common cause of monogenic dilated cardiomyopathy. Here we show that mice with a cardiomyocyte-specific Lmna deletion develop cardiac failure and die within 3-4 weeks after inducing the mutation. When the same Lmna mutations are induced in mice genetically deficient in the LINC complex protein SUN1, life is extended to more than one year. Disruption of SUN1's function is also accomplished by transducing and expressing a dominant-negative SUN1 miniprotein in Lmna deficient cardiomyocytes, using the cardiotrophic Adeno Associated Viral Vector 9. The SUN1 miniprotein disrupts binding between the endogenous LINC complex SUN and KASH domains, displacing the cardiomyocyte KASH complexes from the nuclear periphery, resulting in at least a fivefold extension in lifespan. Cardiomyocyte-specific expression of the SUN1 miniprotein prevents cardiomyopathy progression, potentially avoiding the necessity of developing a specific therapeutic tailored to treating each different LMNA cardiomyopathy-inducing mutation of which there are more than 450.


Assuntos
Cardiomiopatia Dilatada/genética , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Dependovirus/genética , Feminino , Humanos , Lamina Tipo A/deficiência , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução Genética
5.
Biochim Biophys Acta Gene Regul Mech ; 1864(10): 194746, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34419630

RESUMO

Lamin A/C proteins, major components of the nuclear lamina, are encoded by the LMNA gene. These proteins have multiple cellular functions, including DNA transcription and replication, chromatin organization, regulation of the cell cycle, and apoptosis. Mutations in LMNA are associated with a variety of diseases called laminopathies. LMNA has implications in cancer; however, its mechanisms of dysregulation in cancer cells are not yet fully understood. In this study, among the LMNA transcript variants, we focused on a transcriptional variant 6 (termed LMNA-V6), which contains unique 3 exons upstream of exon 1 of LMNA. The promoter region of LMNA-V6 formed multiple G-quadruplexes and increased its transcriptional activity. Moreover, LMNA-V6 negatively regulated other LMNA mRNA variants, lamin A and lamin C, via direct interaction with their promoter. Knockdown of LMNA-V6 decreased the proliferation of colon cancer cells, whereas overexpression of the unique 3 exons of LMNA-V6 increased cell growth. Furthermore, microarray gene expression profiling showed that alteration of LMNA-V6 levels influenced the expression of p53 in colon cancer cells. Taken together, the results suggest that LMNA-V6 may be a novel functional RNA whose expression is regulated through multiple G-quadruplexes in colon cancer cells.


Assuntos
Neoplasias do Colo/genética , Quadruplex G , Regulação Neoplásica da Expressão Gênica , Lamina Tipo A/genética , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Humanos , Lamina Tipo A/metabolismo , Isoformas de RNA/genética , Isoformas de RNA/metabolismo , Splicing de RNA , Transcrição Genética
6.
J Cell Sci ; 134(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34387316

RESUMO

Dephosphorylation of lamin A, which triggers nuclear lamina reconstitution, is crucial for the completion of mitosis. However, the specific phosphatase and regulatory mechanism that allow timely lamin A dephosphorylation remain unclear. Here, we report that RepoMan (also known as CDCA2), a regulatory subunit of protein phosphatase 1γ (PP1γ) is transiently modified with SUMO-2 at K762 during late telophase. SUMOylation of RepoMan markedly enhanced its binding affinity with lamin A. Moreover, SUMOylated RepoMan contributes to lamin A recruitment to telophase chromosomes and dephosphorylation of the mitotic lamin A phosphorylation. Expression of a SUMO-2 mutant that has a defective interaction with the SUMO-interacting motif (SIM) resulted in failure of the lamin A and RepoMan association, along with abrogation of lamin A dephosphorylation and subsequent nuclear lamina formation. These findings strongly suggest that RepoMan recruits lamin A through SUMO-SIM interaction. Thus, transient SUMOylation of RepoMan plays an important role in the spatiotemporal regulation of lamin A dephosphorylation and the subsequent nuclear lamina formation at the end of mitosis.


Assuntos
Lamina Tipo A , Sumoilação , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Mitose , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Telófase
7.
J Orthop Surg Res ; 16(1): 413, 2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193225

RESUMO

BACKGROUND: The network of intermediate filament proteins underlying the inner nuclear membrane forms the nuclear lamin. A- and B-type lamins are the major components of the nuclear lamina. Lamins function in many nuclear activities. The role of lamin A and transcription factors (NF-kB) as anti-apoptotic is well documented. Recently, lamin A has also been considered as a mechanosensor protein that is able to maintain nuclear integrity from mechanical insults. We aimed to verify how lamin A expression varies in healthy cuff cells and in those with different-sized tears where various mechanical stresses are present. METHODS: Forty-three patients with rotator cuff tear (RCT) [23M-20F, mean age (SD): 63.5 (6.1)] were enrolled. Tissue samples excised from the most medial point of tear margins were analyzed for lamin A expression by immunohistochemistry. Controls were represented by samples obtained by normal supraspinatus tendons excised from patients submitted to reverse shoulder prosthesis implant [8M-7F, mean age (SD): 67.9 (7.1)]. The intensity of staining was graded, and an H-score was assigned. Statistical analysis was performed. RESULTS: Our study revealed a moderate intensity of lamin A in the healthy cuff tendons, a higher expression of this protein in the small tears, and a significant decrease of lamin A with increasing tear size (p < 0.0001). CONCLUSIONS: Our study emphasizes the importance of early repair of small RCTs since nuclear stability is maintained, and the cellular function is protected by lamin A overexpression. High re-tear of massive cuff repair could be due to cellular apoptosis and nuclear modifications induced by lamin A lack. LEVEL OF EVIDENCE: III.


Assuntos
Lamina Tipo A/metabolismo , Lesões do Manguito Rotador/metabolismo , Manguito Rotador/citologia , Tenócitos/metabolismo , Idoso , Apoptose , Estudos de Casos e Controles , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Lesões do Manguito Rotador/patologia
8.
Cells ; 10(7)2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202258

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disease caused by a mutation in LMNA. The mutation generates a truncated and farnesylated form of prelamin A, called progerin. Affected individuals develop several features of normal aging, including lipodystrophy caused by the loss of general subcutaneous fat. To determine whether premature cellular senescence is responsible for the altered adipogenesis in patients with HGPS, we evaluated the differentiation of HGPS skin-derived precursor stem cells (SKPs) into adipocytes. The SKPs were isolated from primary human HGPS and normal fibroblast cultures, with senescence of 5 and 30%. We observed that the presence of high numbers of senescent cells reduced SKPs' adipogenic differentiation potential. Treatment with baricitinib, a JAK-STAT inhibitor, ameliorated the ability of HGPS SKPs to differentiate into adipocytes. Our findings suggest that the development of lipodystrophy in patients with HGPS may be associated with an increased rate of cellular senescence and chronic inflammation.


Assuntos
Lamina Tipo A/metabolismo , Progéria/patologia , Pele/patologia , Células-Tronco/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipócitos/patologia , Adipogenia/efeitos dos fármacos , Adolescente , Animais , Azetidinas/farmacologia , Compostos de Boro , Diferenciação Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Criança , Pré-Escolar , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Interleucina-8/metabolismo , Janus Quinases/antagonistas & inibidores , Janus Quinases/metabolismo , Masculino , Camundongos , PPAR gama/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Pirazóis/farmacologia , Células-Tronco/efeitos dos fármacos , Sulfonamidas/farmacologia
9.
Sci Rep ; 11(1): 12695, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135352

RESUMO

Caspase-6 (Casp6) is implicated in Alzheimer disease (AD) cognitive impairment and pathology. Hippocampal atrophy is associated with cognitive impairment in AD. Here, a rare functional exonic missense CASP6 single nucleotide polymorphism (SNP), causing the substitution of asparagine with threonine at amino acid 73 in Casp6 (Casp6N73T), was associated with hippocampal subfield CA1 volume preservation. Compared to wild type Casp6 (Casp6WT), recombinant Casp6N73T altered Casp6 proteolysis of natural substrates Lamin A/C and α-Tubulin, but did not alter cleavage of the Ac-VEID-AFC Casp6 peptide substrate. Casp6N73T-transfected HEK293T cells showed elevated Casp6 mRNA levels similar to Casp6WT-transfected cells, but, in contrast to Casp6WT, did not accumulate active Casp6 subunits nor show increased Casp6 enzymatic activity. Electrophysiological and morphological assessments showed that Casp6N73T recombinant protein caused less neurofunctional damage and neurodegeneration in hippocampal CA1 pyramidal neurons than Casp6WT. Lastly, CASP6 mRNA levels were increased in several AD brain regions confirming the implication of Casp6 in AD. These studies suggest that the rare Casp6N73T variant may protect against hippocampal atrophy due to its altered catalysis of natural protein substrates and intracellular instability thus leading to less Casp6-mediated damage to neuronal structure and function.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Região CA1 Hipocampal/patologia , Caspase 6/genética , Caspase 6/metabolismo , Polimorfismo de Nucleotídeo Único , Transmissão Sináptica , Doença de Alzheimer/enzimologia , Substituição de Aminoácidos , Encéfalo/enzimologia , Encéfalo/patologia , Caspase 1/genética , Caspase 1/metabolismo , Caspase 6/química , Precursores Enzimáticos/metabolismo , Células HEK293 , Hipocampo , Humanos , Lamina Tipo A/metabolismo , Mutação de Sentido Incorreto , Degeneração Neural , Células Piramidais/citologia , Células Piramidais/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismo
10.
Cells ; 10(5)2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064612

RESUMO

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that recapitulates many symptoms of physiological aging and precipitates death. Patients develop severe vascular alterations, mainly massive vascular smooth muscle cell loss, vessel stiffening, calcification, fibrosis, and generalized atherosclerosis, as well as electrical, structural, and functional anomalies in the heart. As a result, most HGPS patients die of myocardial infarction, heart failure, or stroke typically during the first or second decade of life. No cure exists for HGPS, and therefore it is of the utmost importance to define the mechanisms that control disease progression in order to develop new treatments to improve the life quality of patients and extend their lifespan. Since the discovery of the HGPS-causing mutation, several animal models have been generated to study multiple aspects of the syndrome and to analyze the contribution of different cell types to the acquisition of the HGPS-associated cardiovascular phenotype. This review discusses current knowledge about cardiovascular features in HGPS patients and animal models and the molecular and cellular mechanisms through which progerin causes cardiovascular disease.


Assuntos
Doenças Cardiovasculares/genética , Doenças Cardiovasculares/metabolismo , Modelos Animais de Doenças , Progéria/genética , Envelhecimento/metabolismo , Animais , Aterosclerose , Sistema Cardiovascular , Ensaios Clínicos como Assunto , Citoesqueleto/metabolismo , Endotélio Vascular/metabolismo , Fibrose , Cardiopatias/metabolismo , Humanos , Lamina Tipo A/metabolismo , Camundongos , Músculo Liso/metabolismo , Músculo Liso Vascular/metabolismo , Infarto do Miocárdio/metabolismo , Fenótipo , Acidente Vascular Cerebral/complicações , Calcificação Vascular
11.
Diabetes ; 70(9): 1970-1984, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34088712

RESUMO

Mechanisms by which autosomal recessive mutations in Lmna cause familial partial lipodystrophy type 2 (FPLD2) are poorly understood. To investigate the function of lamin A/C in adipose tissue, we created mice with an adipocyte-specific loss of Lmna (Lmna ADKO). Although Lmna ADKO mice develop and maintain adipose tissues in early postnatal life, they show a striking and progressive loss of white and brown adipose tissues as they approach sexual maturity. Lmna ADKO mice exhibit surprisingly mild metabolic dysfunction on a chow diet, but on a high-fat diet they share many characteristics of FPLD2 including hyperglycemia, hepatic steatosis, hyperinsulinemia, and almost undetectable circulating adiponectin and leptin. Whereas Lmna ADKO mice have reduced regulated and constitutive bone marrow adipose tissue with a concomitant increase in cortical bone, FPLD2 patients have reduced bone mass and bone mineral density compared with controls. In cell culture models of Lmna deficiency, mesenchymal precursors undergo adipogenesis without impairment, whereas fully differentiated adipocytes have increased lipolytic responses to adrenergic stimuli. Lmna ADKO mice faithfully reproduce many characteristics of FPLD2 and thus provide a unique animal model to investigate mechanisms underlying Lmna-dependent loss of adipose tissues.


Assuntos
Adipócitos/metabolismo , Adipogenia/fisiologia , Lamina Tipo A/genética , Lipodistrofia Parcial Familiar/genética , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Densidade Óssea/fisiologia , Modelos Animais de Doenças , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/metabolismo , Camundongos , Camundongos Knockout
12.
J Virol ; 95(17): e0087321, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34133898

RESUMO

Nuclear envelope budding in herpesvirus nuclear egress may be negatively regulated, since the pUL31/pUL34 nuclear egress complex heterodimer can induce membrane budding without capsids when expressed ectopically or on artificial membranes in vitro, but not in the infected cell. We have previously described a pUL34 mutant that contained alanine substitutions at R158 and R161 and that showed impaired growth, impaired pUL31/pUL34 interaction, and unregulated budding. Here, we determine the phenotypic contributions of the individual substitutions to these phenotypes. Neither substitution alone was able to reproduce the impaired growth or nuclear egress complex (NEC) interaction phenotypes. Either substitution, however, could fully reproduce the unregulated budding phenotype, suggesting that misregulated budding may not substantially impair virus replication. In addition, the R158A substitution caused relocalization of the NEC to intranuclear punctate structures and recruited lamin A/C to these structures, suggesting that this residue might be important for recruitment of kinases for dispersal of nuclear lamins. IMPORTANCE Herpesvirus nuclear egress is a complex, regulated process coordinated by two virus proteins that are conserved among the herpesviruses that form a heterodimeric nuclear egress complex (NEC). The NEC drives budding of capsids at the inner nuclear membrane and recruits other viral and host cell proteins for disruption of the nuclear lamina, membrane scission, and fusion. The structural basis of individual activities of the NEC, apart from membrane budding, are not clear, nor is the basis of the regulation of membrane budding. Here, we explore the properties of NEC mutants that have an unregulated budding phenotype, determine the significance of that regulation for virus replication, and also characterize a structural requirement for nuclear lamina disruption.


Assuntos
Herpes Simples/virologia , Herpesvirus Humano 1/fisiologia , Mutação , Lâmina Nuclear/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Chlorocebus aethiops , Herpes Simples/genética , Herpes Simples/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/patologia , Membrana Nuclear/virologia , Lâmina Nuclear/patologia , Lâmina Nuclear/virologia , Células Vero , Proteínas Virais/genética , Liberação de Vírus
13.
Mol Cell Biol ; 41(7): e0064820, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-33972393

RESUMO

Paclitaxel is a key member of the Taxane (paclitaxel [originally named taxol], docetaxel/Taxotere) family of successful drugs used in the current treatment of several solid tumors, including ovarian cancer. The molecular target of paclitaxel has been identified as tubulin, and paclitaxel binding alters the dynamics and thus stabilizes microtubule bundles. Traditionally, the anticancer mechanism of paclitaxel has been thought to originate from its interfering with the role of microtubules in mitosis, resulting in mitotic arrest and subsequent apoptosis. However, recent evidence suggests that paclitaxel operates in cancer therapies via an as-yet-undefined mechanism rather than as a mitotic inhibitor. We found that paclitaxel caused a striking break up of nuclei (referred to as multimicronucleation) in malignant ovarian cancer cells but not in normal cells, and susceptibility to undergo nuclear fragmentation and cell death correlated with a reduction in nuclear lamina proteins, lamin A/C. Lamin A/C proteins are commonly lost, reduced, or heterogeneously expressed in ovarian cancer, accounting for the aberration of nuclear shape in malignant cells. Mouse ovarian epithelial cells isolated from lamin A/C-null mice were highly sensitive to paclitaxel and underwent nuclear breakage, compared to control wild-type cells. Forced overexpression of lamin A/C led to resistance to paclitaxel-induced nuclear breakage in cancer cells. Additionally, paclitaxel-induced multimicronucleation occurred independently of cell division that was achieved by either the withdrawal of serum or the addition of mitotic inhibitors. These results provide a new understanding for the mitotis-independent mechanism for paclitaxel killing of cancer cells, where paclitaxel induces nuclear breakage in malignant cancer cells that have a malleable nucleus but not in normal cells that have a stiffer nuclear envelope. As such, we identify that reduced nuclear lamin A/C protein levels correlate with nuclear shape deformation and are a key determinant of paclitaxel sensitivity of cancer cells.


Assuntos
Lamina Tipo A/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Animais , Antimitóticos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Lamina Tipo A/metabolismo , Camundongos Transgênicos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Neoplasias Ovarianas/patologia , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
14.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34028539

RESUMO

While diverse cellular components have been identified as mechanotransduction elements, the deformation of the nucleus itself is a critical mechanosensory mechanism, implying that nuclear stiffness is essential in determining responses to intracellular and extracellular stresses. Although the nuclear membrane protein lamin A/C is known to contribute to nuclear stiffness, bulk moduli of nuclei have not been reported for various levels of lamin A/C. Here, we measure the nuclear bulk moduli as a function of lamin A/C expression and applied osmotic stress, revealing a linear dependence within the range of 2-4 MPa. We also find that the nuclear compression is anisotropic, with the vertical axis of the nucleus being more compliant than the minor and major axes in the substrate plane. We then related the spatial distribution of lamin A/C with submicron 3D nuclear envelope deformation, revealing that local areas of the nuclear envelope with higher density of lamin A/C have correspondingly lower local deformations. These findings describe the complex dispersion of nuclear deformations as a function of lamin A/C expression and distribution, implicating a lamin A/C role in mechanotransduction. This article has an associated First Person interview with the first author of the paper.


Assuntos
Lamina Tipo A , Mecanotransdução Celular , Núcleo Celular/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Membrana Nuclear/metabolismo
17.
Sci Rep ; 11(1): 10099, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980953

RESUMO

Ataxia telangiectasia (AT) is a rare genetic neurodegenerative disease. To date, there is no available cure for the illness, but the use of glucocorticoids has been shown to alleviate the neurological symptoms associated with AT. While studying the effects of dexamethasone (dex) in AT fibroblasts, by chance we observed that the nucleoplasmic Lamin A/C was affected by the drug. In addition to the structural roles of A-type lamins, Lamin A/C has been shown to play a role in the regulation of gene expression and cell cycle progression, and alterations in the LMNA gene is cause of human diseases called laminopathies. Dex was found to improve the nucleoplasmic accumulation of soluble Lamin A/C and was capable of managing the large chromatin Lamin A/C scaffolds contained complex, thus regulating epigenetics in treated cells. In addition, dex modified the interactions of Lamin A/C with its direct partners lamin associated polypeptide (LAP) 2a, Retinoblastoma 1 (pRB) and E2F Transcription Factor 1 (E2F1), regulating local gene expression dependent on E2F1. These effects were differentially observed in both AT and wild type (WT) cells. To our knowledge, this is the first reported evidence of the role of dex in Lamin A/C dynamics in AT cells, and may represent a new area of research regarding the effects of glucocorticoids on AT. Moreover, future investigations could also be extended to healthy subjects or to other pathologies such as laminopathies since glucocorticoids may have other important effects in these contexts as well.


Assuntos
Ataxia Telangiectasia/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dexametasona/farmacologia , Fator de Transcrição E2F1/metabolismo , Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Salivares Ricas em Prolina/metabolismo , Ataxia Telangiectasia/tratamento farmacológico , Ataxia Telangiectasia/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição E2F1/genética , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Lamina Tipo A/genética , Proteínas de Membrana/genética , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Salivares Ricas em Prolina/genética
18.
Int J Mol Sci ; 22(8)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923914

RESUMO

Intermediate filaments are major components of the cytoskeleton. Desmin and synemin, cytoplasmic intermediate filament proteins and A-type lamins, nuclear intermediate filament proteins, play key roles in skeletal and cardiac muscle. Desmin, encoded by the DES gene (OMIM *125660) and A-type lamins by the LMNA gene (OMIM *150330), have been involved in striated muscle disorders. Diseases include desmin-related myopathy and cardiomyopathy (desminopathy), which can be manifested with dilated, restrictive, hypertrophic, arrhythmogenic, or even left ventricular non-compaction cardiomyopathy, Emery-Dreifuss Muscular Dystrophy (EDMD2 and EDMD3, due to LMNA mutations), LMNA-related congenital Muscular Dystrophy (L-CMD) and LMNA-linked dilated cardiomyopathy with conduction system defects (CMD1A). Recently, mutations in synemin (SYNM gene, OMIM *606087) have been linked to cardiomyopathy. This review will summarize clinical and molecular aspects of desmin-, lamin- and synemin-related striated muscle disorders with focus on LMNA and DES-associated clinical entities and will suggest pathogenetic hypotheses based on the interplay of desmin and lamin A/C. In healthy muscle, such interplay is responsible for the involvement of this network in mechanosignaling, nuclear positioning and mitochondrial homeostasis, while in disease it is disturbed, leading to myocyte death and activation of inflammation and the associated secretome alterations.


Assuntos
Cardiomiopatias/genética , Cardiomiopatias/patologia , Proteínas de Filamentos Intermediários/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Mutação/genética , Miocárdio/metabolismo , Miocárdio/patologia , Animais , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo
19.
Med Sci (Paris) ; 37(4): 413-416, 2021 Apr.
Artigo em Francês | MEDLINE | ID: mdl-33908864

RESUMO

Hutchinson-Gilford Progeria (acute premature aging) is caused by a de novo point mutation in the lamin A gene. Recently, this mutation has been accurately corrected by base editing in patient cell lines and in a mouse model, resulting in nearly complete reversal to a normal phenotype. This success opens the perspective for clinical applications in Progeria and other diseases.


Assuntos
Lamina Tipo A/genética , Mutação Puntual , Progéria/terapia , Reparo Gênico Alvo-Dirigido , Animais , Modelos Animais de Doenças , Imunofluorescência , Edição de Genes/métodos , Humanos , Lamina Tipo A/metabolismo , Camundongos , Progéria/genética
20.
Sci Rep ; 11(1): 9122, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907225

RESUMO

Werner syndrome (WRN) is a rare progressive genetic disorder, caused by functional defects in WRN protein and RecQ4L DNA helicase. Acceleration of the aging process is initiated at puberty and the expected life span is approximately the late 50 s. However, a Wrn-deficient mouse model does not show premature aging phenotypes or a short life span, implying that aging processes differ greatly between humans and mice. Gene expression analysis of WRN cells reveals very similar results to gene expression analysis of Hutchinson Gilford progeria syndrome (HGPS) cells, suggesting that these human progeroid syndromes share a common pathological mechanism. Here we show that WRN cells also express progerin, an abnormal variant of the lamin A protein. In addition, we reveal that duplicated sequences of human WRN (hWRN) from exon 9 to exon 10, which differ from the sequence of mouse WRN (mWRN), are a natural inhibitor of progerin. Overexpression of hWRN reduced progerin expression and aging features in HGPS cells. Furthermore, the elimination of progerin by siRNA or a progerin-inhibitor (SLC-D011 also called progerinin) can ameliorate senescence phenotypes in WRN fibroblasts and cardiomyocytes, derived from WRN-iPSCs. These results suggest that progerin, which easily accumulates under WRN-deficient conditions, can lead to premature aging in WRN and that this effect can be prevented by SLC-D011.


Assuntos
Lamina Tipo A/metabolismo , Progéria/patologia , Helicase da Síndrome de Werner/metabolismo , Síndrome de Werner/genética , Adulto , Senilidade Prematura/genética , Animais , Linhagem Celular , Senescência Celular/efeitos dos fármacos , Criança , Modelos Animais de Doenças , Feminino , Fibroblastos/patologia , Expressão Gênica , Humanos , Masculino , Camundongos Mutantes , Progéria/genética , Isoformas de Proteínas , Síndrome de Werner/patologia , Helicase da Síndrome de Werner/genética
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