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1.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31602295

RESUMO

Cytoplasmic intermediate filaments (IFs) surround the nucleus and are often anchored at membrane sites to form effectively transcellular networks. Mutations in IF proteins (IFps) have revealed mechanical roles in epidermis, muscle, liver, and neurons. At the same time, there have been phenotypic surprises, illustrated by the ability to generate viable and fertile mice null for a number of IFp-encoding genes, including vimentin. Yet in humans, the vimentin ( VIM) gene displays a high probability of intolerance to loss-of-function mutations, indicating an essential role. A number of subtle and not so subtle IF-associated phenotypes have been identified, often linked to mechanical or metabolic stresses, some of which have been found to be ameliorated by the over-expression of molecular chaperones, suggesting that such phenotypes arise from what might be termed "orphan" effects as opposed to the absence of the IF network per se, an idea originally suggested by Toivola et al. and Pekny and Lane.


Assuntos
Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/genética , Animais , Humanos , Camundongos , Mutação , Fenótipo
2.
PLoS Biol ; 17(7): e3000390, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31323028

RESUMO

Processes of molecular innovation require tinkering and shifting in the function of existing genes. How this occurs in terms of molecular evolution at long evolutionary scales remains poorly understood. Here, we analyse the natural history of a vast group of membrane-associated molecular systems in Bacteria and Archaea-the type IV filament (TFF) superfamily-that diversified in systems involved in flagellar or twitching motility, adhesion, protein secretion, and DNA uptake. The phylogeny of the thousands of detected systems suggests they may have been present in the last universal common ancestor. From there, two lineages-a bacterial and an archaeal-diversified by multiple gene duplications, gene fissions and deletions, and accretion of novel components. Surprisingly, we find that the 'tight adherence' (Tad) systems originated from the interkingdom transfer from Archaea to Bacteria of a system resembling the 'EppA-dependent' (Epd) pilus and were associated with the acquisition of a secretin. The phylogeny and content of ancestral systems suggest that initial bacterial pili were engaged in cell motility and/or DNA uptake. In contrast, specialised protein secretion systems arose several times independently and much later in natural history. The functional diversification of the TFF superfamily was accompanied by genetic rearrangements with implications for genetic regulation and horizontal gene transfer: systems encoded in fewer loci were more frequently exchanged between taxa. This may have contributed to their rapid evolution and spread across Bacteria and Archaea. Hence, the evolutionary history of the superfamily reveals an impressive catalogue of molecular evolution mechanisms that resulted in remarkable functional innovation and specialisation from a relatively small set of components.


Assuntos
Citoesqueleto/genética , DNA/metabolismo , Transferência Genética Horizontal/genética , Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/genética , Archaea/classificação , Archaea/genética , Archaea/metabolismo , Bactérias/classificação , Bactérias/genética , Bactérias/metabolismo , Adesão Celular/genética , Citoesqueleto/metabolismo , DNA/genética , Evolução Molecular , Proteínas de Filamentos Intermediários/classificação , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/classificação , Filamentos Intermediários/metabolismo , Movimento , Filogenia , Transporte Proteico/genética
3.
Cells ; 8(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31126068

RESUMO

Intermediate filament (IF) proteins make up the largest family of cytoskeletal proteins in metazoans, and are traditionally known for their roles in fostering structural integrity in cells and tissues. Remarkably, individual IF genes are tightly regulated in a fashion that reflects the type of tissue, its developmental and differentiation stages, and biological context. In cancer, IF proteins serve as diagnostic markers, as tumor cells partially retain their original signature expression of IF proteins. However, there are also characteristic alterations in IF gene expression and protein regulation. The use of high throughput analytics suggests that tumor-associated alterations in IF gene expression have prognostic value. Parallel research is also showing that IF proteins directly and significantly impact several key cellular properties, including proliferation, death, migration, and invasiveness, with a demonstrated impact on the development, progression, and characteristics of various tumors. In this review, we draw from recent studies focused on three IF proteins most associated with cancer (keratins, vimentin, and nestin) to highlight how several "hallmarks of cancer" described by Hanahan and Weinberg are impacted by IF proteins. The evidence already in hand establishes that IF proteins function beyond their classical roles as markers and serve as effectors of tumorigenesis.


Assuntos
Carcinogênese/metabolismo , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Queratinas/metabolismo , Metástase Neoplásica/fisiopatologia , Nestina/metabolismo , Vimentina/metabolismo , Animais , Carcinogênese/genética , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/imunologia , Inflamação/metabolismo , Queratinas/genética , Queratinas/imunologia , Camundongos , Metástase Neoplásica/genética , Neovascularização Patológica/metabolismo , Nestina/genética , Vimentina/genética
4.
J Clin Invest ; 128(10): 4604-4621, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30106752

RESUMO

Regulatory T cells (Tregs) are critical for maintaining immune homeostasis. However, current Treg immunotherapies do not optimally treat inflammatory diseases in patients. Understanding the cellular processes that control Treg function may allow for the augmentation of therapeutic efficacy. In contrast to activated conventional T cells, in which protein kinase C-θ (PKC-θ) localizes to the contact point between T cells and antigen-presenting cells, in human and mouse Tregs, PKC-θ localizes to the opposite end of the cell in the distal pole complex (DPC). Here, using a phosphoproteomic screen, we identified the intermediate filament vimentin as a PKC-θ phospho target and show that vimentin forms a DPC superstructure on which PKC-θ accumulates. Treatment of mouse Tregs with either a clinically relevant PKC-θ inhibitor or vimentin siRNA disrupted vimentin and enhanced Treg metabolic and suppressive activity. Moreover, vimentin-disrupted mouse Tregs were significantly better than controls at suppressing alloreactive T cell priming in graft-versus-host disease (GVHD) and GVHD lethality, using a complete MHC-mismatch mouse model of acute GVHD (C57BL/6 donor into BALB/c host). Interestingly, vimentin disruption augmented the suppressor function of PKC-θ-deficient mouse Tregs. This suggests that enhanced Treg activity after PKC-θ inhibition is secondary to effects on vimentin, not just PKC-θ kinase activity inhibition. Our data demonstrate that vimentin is a key metabolic and functional controller of Treg activity and provide proof of principle that disruption of vimentin is a feasible, translationally relevant method to enhance Treg potency.


Assuntos
Células Apresentadoras de Antígenos/imunologia , Doença Enxerto-Hospedeiro/imunologia , Filamentos Intermediários/imunologia , Ativação Linfocitária , Linfócitos T Reguladores/imunologia , Vimentina/imunologia , Animais , Células Apresentadoras de Antígenos/patologia , Modelos Animais de Doenças , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/patologia , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Proteína Quinase C-theta/genética , Proteína Quinase C-theta/imunologia , Linfócitos T Reguladores/patologia , Vimentina/genética
5.
Annu Rev Cell Dev Biol ; 34: 1-28, 2018 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-30059630

RESUMO

Intermediate filaments (IFs) are one of the three major elements of the cytoskeleton. Their stability, intrinsic mechanical properties, and cell type-specific expression patterns distinguish them from actin and microtubules. By providing mechanical support, IFs protect cells from external forces and participate in cell adhesion and tissue integrity. IFs form an extensive and elaborate network that connects the cell cortex to intracellular organelles. They act as a molecular scaffold that controls intracellular organization. However, IFs have been revealed as much more than just rigid structures. Their dynamics is regulated by multiple signaling cascades and appears to contribute to signaling events in response to cell stress and to dynamic cellular functions such as mitosis, apoptosis, and migration.


Assuntos
Biologia Celular/tendências , Citoplasma/genética , Filamentos Intermediários/genética , Microtúbulos/genética , Actinas/química , Actinas/genética , Citoplasma/química , Citoesqueleto/química , Citoesqueleto/genética , Proteína Glial Fibrilar Ácida/genética , Humanos , Filamentos Intermediários/química , Microtúbulos/química , Mitose/genética , Transdução de Sinais/genética
6.
Sci Rep ; 8(1): 4072, 2018 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-29511223

RESUMO

Cytoskeleton plays a vital role in stress tolerance; however, involvement of intermediate filaments (IFs) in such a response remains elusive in crop plants. This study provides clear evidence about the unique involvement of IFs in cellular protection against abiotic stress in rice. Transcript abundance of Oryza sativa intermediate filament (OsIF) encoding gene showed 2-10 fold up-regulation under different abiotic stress. Overexpression of OsIF in transgenic rice enhanced tolerance to salinity and heat stress, while its knock-down (KD) rendered plants more sensitive thereby indicating the role of IFs in promoting survival under stress. Seeds of OsIF overexpression rice germinated normally in the presence of high salt, showed better growth, maintained chloroplast ultrastructure and favourable K+/Na+ ratio than the wild type (WT) and KD plants. Analysis of photosynthesis and chlorophyll a fluorescence data suggested better performance of both photosystem I and II in the OsIF overexpression rice under salinity stress as compared to the WT and KD. Under salinity and high temperature stress, OsIF overexpressing plants could maintain significantly high yield, while the WT and KD plants could not. Further, metabolite profiling revealed a 2-4 fold higher accumulation of proline and trehalose in OsIF overexpressing rice than WT, under salinity stress.


Assuntos
Resposta ao Choque Térmico , Filamentos Intermediários/metabolismo , Oryza/fisiologia , Fotossíntese , Salinidade , Estresse Fisiológico , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Filamentos Intermediários/genética , Metabolômica , Oryza/efeitos dos fármacos , Oryza/genética , Oryza/efeitos da radiação , Prolina/análise , RNA Mensageiro/análise , Trealose/análise
7.
Am J Physiol Lung Cell Mol Physiol ; 315(1): L102-L115, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29543041

RESUMO

Generation of secondary alveolar septa occurs primarily after birth in humans and is complete in mice postnatally, when mechanical stresses vary as air space pressure oscillates. Alveolar mesenchymal cells deposit elastic fibers, which limit cell strain; although when the elastic fiber network is incomplete, this function is also served by the intracellular cytoskeleton. Intermediate filament proteins support deformation during cell division and migration, which occur during septal elongation. Because platelet-derived growth factor receptor-α (PDGFRα) signaling is essential for alveolar septation, we hypothesized that neuropilin-1 (NRP1) may link PDGFRα to cytoskeletal deformation. During cell migration, NRP1 links receptor tyrosine kinase signaling to cytoskeletal and focal adhesion remodeling. Therefore, we examined the consequences of nrp1 gene deletion in alveolar mesenchymal cells (myofibroblasts and pericytes). NRP1 depletion reduced the proportion of mesenchymal cells that contain nestin and desmin within the subpopulation that lacked PDGFRα but contained PDGFRß. Desmin was reduced at alveolar entry rings, air spaces were enlarged, and surface area was reduced after NRP1 depletion. PDGFRα and NRP1 colocalized to membrane lipid rafts, which are known to contain Src kinase. NRP1 depletion reduced alveolar mesenchymal cell migration and PDGF-A-mediated activation of Src kinase, which may limit accumulation of desmin at septal tips (alveolar entry rings). Cooperation between NRP1 and PDGF signaling is required for secondary septation, and manipulation of NRP1 could promote alveolar regeneration without producing fibrosis.


Assuntos
Filamentos Intermediários/metabolismo , Neuropilina-1/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Alvéolos Pulmonares/crescimento & desenvolvimento , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Animais , Adesões Focais/genética , Adesões Focais/metabolismo , Deleção de Genes , Filamentos Intermediários/genética , Camundongos , Camundongos Transgênicos , Neuropilina-1/genética , Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética
8.
Neurodegener Dis ; 18(2-3): 74-83, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29587262

RESUMO

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is the most common neurodegenerative disorder of the peripheral nervous system. More than 50 genes/loci were found associated with the disease. We found a family with autosomal-dominant CMT2. OBJECTIVE: To reveal the pathogenic gene of the family and further investigate the function of the variant. METHODS: DNA underwent whole-genome linkage analysis for all family members and whole-exome sequencing for 2 affected members. Neurofilament light polypeptide and wild-type or mutant neurofilament heavy polypeptide (NEFH) were co-transfected into SW13 (vim-) cells. The nefh-knockdown zebrafish model was produced by using morpholino antisense oligonucleotides. RESULTS: We identified a novel insertion variant (c.3057insG) in NEFH in the family. The variant led to the loss of a stop codon and an extended 41 amino acids in the protein. Immunofluorescence results revealed that mutant NEFH disrupted the neurofilament network and induced aggregation of NEFH protein. Knockdown of nefh in zebrafish caused a slightly or severely curled tail. The motor ability of nefh-knockdown embryos was impaired or even absent, and the embryos showed developmental defects of axons in motor neurons. The abnormal phenotype and axonal developmental defects could be rescued by injection of human wild-type but not human mutant NEFH mRNA. CONCLUSIONS: We identified a novel stop loss variant in NEFH that is likely pathogenic for CMT2, and the results provide further evidence for the role of an aberrant assembly of neurofilament in CMT.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Exoma/genética , Estudo de Associação Genômica Ampla , Filamentos Intermediários/genética , Mutação/genética , Animais , Axônios/metabolismo , Feminino , Humanos , Filamentos Intermediários/metabolismo , Masculino , Neurônios Motores/metabolismo , Proteínas de Neurofilamentos/genética , Linhagem , Fenótipo , Peixe-Zebra
9.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(6): 639-650, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29571767

RESUMO

We have previously shown that phospholipase D (PLD) pathways have a role in neuronal degeneration; in particular, we found that PLD activation is associated with synaptic injury induced by oxidative stress. In the present study, we investigated the effect of α-synuclein (α-syn) overexpression on PLD signaling. Wild Type (WT) α-syn was found to trigger the inhibition of PLD1 expression as well as a decrease in ERK1/2 phosphorylation and expression levels. Moreover, ERK1/2 subcellular localization was shown to be modulated by WT α-syn in a PLD1-dependent manner. Indeed, PLD1 inhibition was found to alter the neurofilament network and F-actin distribution regardless of the presence of WT α-syn. In line with this, neuroblastoma cells expressing WT α-syn exhibited a degenerative-like phenotype characterized by a marked reduction in neurofilament light subunit (NFL) expression and the rearrangement of the F-actin organization, compared with either the untransfected or the empty vector-transfected cells. The gain of function of PLD1 through the overexpression of its active form had the effect of restoring NFL expression in WT α-syn neurons. Taken together, our findings reveal an unforeseen role for α-syn in PLD regulation: PLD1 downregulation may constitute an early mechanism in the initial stages of WT α-syn-triggered neurodegeneration.


Assuntos
Regulação para Baixo , Regulação Enzimológica da Expressão Gênica , Doença de Parkinson/metabolismo , Fosfolipase D/biossíntese , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Mutação com Ganho de Função , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Fosfolipase D/genética , alfa-Sinucleína/genética
10.
Elife ; 72018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29513221

RESUMO

Intermediate filaments (IF) are a major component of the metazoan cytoskeleton and are essential for normal cell morphology, motility, and signal transduction. Dysregulation of IFs causes a wide range of human diseases, including skin disorders, cardiomyopathies, lipodystrophy, and neuropathy. Despite this pathophysiological significance, how cells regulate IF structure, dynamics, and function remains poorly understood. Here, we show that site-specific modification of the prototypical IF protein vimentin with O-linked ß-N-acetylglucosamine (O-GlcNAc) mediates its homotypic protein-protein interactions and is required in human cells for IF morphology and cell migration. In addition, we show that the intracellular pathogen Chlamydia trachomatis, which remodels the host IF cytoskeleton during infection, requires specific vimentin glycosylation sites and O-GlcNAc transferase activity to maintain its replicative niche. Our results provide new insight into the biochemical and cell biological functions of vimentin O-GlcNAcylation, and may have broad implications for our understanding of the regulation of IF proteins in general.


Assuntos
Acetilglucosamina/genética , Movimento Celular/genética , Citoesqueleto/genética , Filamentos Intermediários/genética , Acetilglucosamina/metabolismo , Animais , Glicosilação , Humanos , N-Acetilglucosaminiltransferases/genética , Fosforilação , Processamento de Proteína Pós-Traducional/genética , Transdução de Sinais , Vimentina/genética
11.
Hum Mol Genet ; 27(8): 1434-1446, 2018 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-29462312

RESUMO

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterized by compromised function of the neuromuscular junction, manifesting with fatigable muscle weakness. Mutations in MYO9A were previously identified as causative for CMS but the precise pathomechanism remained to be characterized. On the basis of the role of MYO9A as an actin-based molecular motor and as a negative regulator of RhoA, we hypothesized that loss of MYO9A may affect the neuronal cytoskeleton, leading to impaired intracellular transport. To investigate this, we used MYO9A-depleted NSC-34 cells (mouse motor neuron-derived cells), revealing altered expression of a number of cytoskeletal proteins important for neuron structure and intracellular transport. On the basis of these findings, the effect on protein transport was determined using a vesicular recycling assay which revealed impaired recycling of a neuronal growth factor receptor. In addition, an unbiased approach utilizing proteomic profiling of the secretome revealed a key role for defective intracellular transport affecting proper protein secretion in the pathophysiology of MYO9A-related CMS. This also led to the identification of agrin as being affected by the defective transport. Zebrafish with reduced MYO9A orthologue expression were treated with an artificial agrin compound, ameliorating defects in neurite extension and improving motility. In summary, loss of MYO9A affects the neuronal cytoskeleton and leads to impaired transport of proteins, including agrin, which may provide a new and unexpected treatment option.


Assuntos
Agrina/metabolismo , Neurônios Motores/metabolismo , Debilidade Muscular/genética , Síndromes Miastênicas Congênitas/genética , Miosinas/genética , Fator de Crescimento Neural/genética , Junção Neuromuscular/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Actinas/genética , Actinas/metabolismo , Agrina/genética , Amidas , Animais , Movimento Celular , Modelos Animais de Doenças , Embrião não Mamífero , Inibidores Enzimáticos , Regulação da Expressão Gênica , Humanos , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neurônios Motores/ultraestrutura , Debilidade Muscular/metabolismo , Debilidade Muscular/patologia , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/patologia , Miosinas/deficiência , Fator de Crescimento Neural/metabolismo , Junção Neuromuscular/ultraestrutura , Transporte Proteico , Piridinas , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Peixe-Zebra , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
12.
Mol Genet Genomic Med ; 6(2): 288-293, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29274115

RESUMO

BACKGROUND: DES mutations cause different cardiac and skeletal myopathies. Most of them are missense mutations. METHODS: Using a next-generation sequencing cardiac 174 gene panel, we identified a novel heterozygous in-frame indel mutation (DES-c.493_520del28insGCGT, p.Q165_A174delinsAS) in a Caucasian patient with cardiomyopathy in combination with atrioventricular block and skeletal myopathy. This indel mutation is located in the coding region of the first exon. Family anamnesis revealed a history of sudden cardiac death. We performed cell transfection experiments and in vitro assembly experiments to prove the pathogenicity of this novel DES indel mutation. RESULTS: These experiments revealed a severe filament formation defect of mutant desmin supporting the pathogenicity. In addition, we labeled a skeletal muscle biopsy from the mutation carrier revealing cytoplasmic desmin positive protein aggregates. In summary, we identified and functionally characterized a pathogenic DES indel mutation causing cardiac and skeletal myopathy. CONCLUSION: Our study has relevance for the clinical and genetic interpretation of further DES indel mutations causing cardiac or skeletal myopathies and might be helpful for risk stratification.


Assuntos
Cardiomiopatias/genética , Desmina/genética , Adulto , Bloqueio Atrioventricular/genética , Sequência de Bases/genética , Desmina/metabolismo , Humanos , Mutação INDEL/genética , Filamentos Intermediários/genética , Masculino , Músculo Esquelético/metabolismo , Doenças Musculares/genética , Linhagem
13.
Brain Res Bull ; 136: 130-138, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28159699

RESUMO

In response to central nervous system (CNS) injury, astrocytes upregulate intermediate filament (nanofilament) proteins GFAP and vimentin. Whereas the intermediate filament upregulation in astrocytes is important for neuroprotection in the acute phase of injury, in some contexts it might inhibit some of the regenerative processes later on. Thus, timely modulation of the astrocyte intermediate filaments was proposed as a strategy to promote brain repair. We used clomipramine, epoxomicin and withaferin A, drugs reported to decrease the expression of GFAP, and assessed their effect on neurosupportive properties and resilience of astrocytes to oxygen and glucose deprivation (OGD). Clomipramine decreased protein levels of GFAP, as well as vimentin and nestin, and did not affect astrocyte resilience to oxidative stress. Withaferin A sensitized astrocytes to OGD. Both clomipramine and epoxomicin promoted the attachment and survival of neurons co-cultured with astrocytes under standard culture conditions. Moreover, epoxomicin increased neurosupportive properties of astrocytes after OGD. Our data point to clomipramine and epoxomicin as potential candidates for astrocyte modulation to improve outcome after CNS injury.


Assuntos
Astrócitos/efeitos dos fármacos , Clomipramina/farmacologia , Filamentos Intermediários/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Vitanolídeos/farmacologia , Animais , Astrócitos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Hipóxia Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Clomipramina/toxicidade , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Glucose/deficiência , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fármacos Neuroprotetores/toxicidade , Oligopeptídeos/farmacologia , Oligopeptídeos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Vitanolídeos/toxicidade
14.
Cell Biol Int ; 42(2): 132-138, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28980752

RESUMO

Intermediate filaments (IFs) are one of the three types of cytoskeletal polymers that resist tensile and compressive forces in cells. They crosslink each other as well as with actin filaments and microtubules by proteins, which include desmin, filamin C, plectin, and lamin (A/C). Mutations in these proteins can lead to a wide range of pathologies, some of which exhibit mechanical failure of the skin, skeletal, or heart muscle.


Assuntos
Filamentos Intermediários/metabolismo , Desmina/metabolismo , Filaminas/metabolismo , Filamentos Intermediários/química , Filamentos Intermediários/genética , Lamina Tipo A/metabolismo , Plectina/metabolismo
15.
Pediatr Surg Int ; 34(2): 217-225, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29043445

RESUMO

PURPOSE: Intermediate filaments (IFs) are a part of the cytoskeleton that extend throughout the cytoplasm of all cells and function in the maintenance of cell-shape by bearing tension and serving as structural components of the nuclear lamina. In normal intestine, IFs provide a tissue-specific three-dimensional scaffolding with unique context-dependent organizational features. The purpose of this study was to evaluate the role of IFs during intestinal adaptation in a rat model of short bowel syndrome (SBS). MATERIALS AND METHODS: Male rats were divided into two groups: Sham rats underwent bowel transection and SBS rats underwent a 75% bowel resection. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined 2 weeks after operation. Illumina's Digital Gene Expression (DGE) analysis was used to determine the cytoskeleton-related gene expression profiling. IF-related genes and protein expression were determined using real-time PCR, Western blotting and immunohistochemistry. RESULTS: Massive small bowel resection resulted in a significant increase in enterocyte proliferation and concomitant increase in cell apoptosis. From the total number of 20,000 probes, 16 cytoskeleton-related genes were investigated. Between these genes, only myosin and tubulin levels were upregulated in SBS compared to sham animals. Between IF-related genes, desmin, vimentin and lamin levels were down-regulated and keratin and neurofilament remain unchanged. The levels of TGF-ß, vimentin and desmin gene and protein were down-regulated in resected rats (vs sham animals). CONCLUSIONS: Two weeks following massive bowel resection in rats, the accelerated cell turnover was accompanied by a stimulated microfilaments and microtubules, and by inhibited intermediate filaments. Resistance to cell compression rather that maintenance of cell-shape by bearing tension are responsible for contraction, motility and postmitotic cell separation in a late stage of intestinal adaptation.


Assuntos
Procedimentos Cirúrgicos do Sistema Digestório , Regulação da Expressão Gênica , Filamentos Intermediários/genética , RNA/genética , Síndrome do Intestino Curto/genética , Animais , Apoptose , Western Blotting , Proliferação de Células , Desmina/biossíntese , Desmina/genética , Modelos Animais de Doenças , Enterócitos/metabolismo , Enterócitos/patologia , Imuno-Histoquímica , Intestino Delgado/metabolismo , Intestino Delgado/patologia , Intestino Delgado/cirurgia , Queratinas/biossíntese , Queratinas/genética , Laminas/biossíntese , Laminas/genética , Masculino , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Síndrome do Intestino Curto/metabolismo , Síndrome do Intestino Curto/cirurgia , Vimentina/biossíntese , Vimentina/genética
16.
PLoS Genet ; 13(9): e1006983, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28880872

RESUMO

Animal-vegetal (AV) polarity of most vertebrate eggs is established during early oogenesis through the formation and disassembly of the Balbiani Body (Bb). The Bb is a structure conserved from insects to humans that appears as a large granule, similar to a mRNP granule composed of mRNA and proteins, that in addition contains mitochondria, ER and Golgi. The components of the Bb, which have amyloid-like properties, include germ cell and axis determinants of the embryo that are anchored to the vegetal cortex upon Bb disassembly. Our lab discovered in zebrafish the only gene known to function in Bb disassembly, microtubule-actin crosslinking factor 1a (macf1a). Macf1 is a conserved, giant multi-domain cytoskeletal linker protein that can interact with microtubules (MTs), actin filaments (AF), and intermediate filaments (IF). In macf1a mutant oocytes the Bb fails to dissociate, the nucleus is acentric, and AV polarity of the oocyte and egg fails to form. The cytoskeleton-dependent mechanism by which Macf1a regulates Bb mRNP granule dissociation was unknown. We found that disruption of AFs phenocopies the macf1a mutant phenotype, while MT disruption does not. We determined that cytokeratins (CK), a type of IF, are enriched in the Bb. We found that Macf1a localizes to the Bb, indicating a direct function in regulating its dissociation. We thus tested if Macf1a functions via its actin binding domain (ABD) and plectin repeat domain (PRD) to integrate cortical actin and Bb CK, respectively, to mediate Bb dissociation at the oocyte cortex. We developed a CRISPR/Cas9 approach to delete the exons encoding these domains from the macf1a endogenous locus, while maintaining the open reading frame. Our analysis shows that Macf1a functions via its ABD to mediate Bb granule dissociation and nuclear positioning, while the PRD is dispensable. We propose that Macf1a does not function via its canonical mechanism of linking two cytoskeletal systems together in dissociating the Bb. Instead our results suggest that Macf1a functions by linking one cytoskeletal system, cortical actin, to another structure, the Bb, where Macf1a is localized. Through this novel linking process, it dissociates the Bb at the oocyte cortex, thus specifying the AV axis of the oocyte and future egg. To our knowledge, this is also the first study to use genome editing to unravel the module-dependent function of a cytoskeletal linker.


Assuntos
Polaridade Celular/genética , Oogênese/genética , Plaquinas/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Citoesqueleto de Actina/genética , Animais , Células Germinativas/crescimento & desenvolvimento , Complexo de Golgi/genética , Humanos , Filamentos Intermediários/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Peixe-Zebra/crescimento & desenvolvimento
17.
Sci Rep ; 7(1): 11628, 2017 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-28912461

RESUMO

Intermediate filaments are involved in stress-related cell mechanical properties and in plasticity via the regulation of focal adhesions (FAs) and the actomyosin network. We investigated whether vimentin regulates endothelial cells (ECs) and vascular smooth muscle cells (SMCs) and thereby influences vasomotor tone and arterial stiffness. Vimentin knockout mice (Vim-/-) exhibited increased expression of laminin, fibronectin, perlecan, collagen IV and VE-cadherin as well as von Willebrand factor deposition in the subendothelial basement membrane. Smooth muscle (SM) myosin heavy chain, α-SM actin and smoothelin were decreased in Vim-/- mice. Electron microscopy revealed a denser endothelial basement membrane and increased SM cell-matrix interactions. Integrin αv, talin and vinculin present in FAs were increased in Vim-/- mice. Phosphorylated FA kinase and its targets Src and ERK1/2 were elevated in Vim-/- mice. Knockout of vimentin, but not of synemin, resulted in increased carotid stiffness and contractility and endothelial dysfunction, independently of blood pressure and the collagen/elastin ratio. The increase in arterial stiffness in Vim-/- mice likely involves vasomotor tone and endothelial basement membrane organization changes. At the tissue level, the results show the implication of FAs both in ECs and vascular SMCs in the role of vimentin in arterial stiffening.


Assuntos
Membrana Basal/metabolismo , Doenças das Artérias Carótidas/etiologia , Doenças das Artérias Carótidas/metabolismo , Regulação da Expressão Gênica , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Rigidez Vascular/genética , Vimentina/deficiência , Animais , Biomarcadores , Pressão Sanguínea , Doenças das Artérias Carótidas/fisiopatologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Modelos Animais de Doenças , Endotélio/metabolismo , Imunofluorescência , Fenômenos Mecânicos , Camundongos , Camundongos Knockout , Microscopia Confocal , Vasodilatação/genética
18.
Exp Mol Med ; 49(5): e332, 2017 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-28524176

RESUMO

Our study aims to explore the effects of lentivirus-mediated microRNA-124 (miR-124) gene-modified bone marrow mesenchymal stem cell (BMSC) transplantation on the repair of spinal cord injury (SCI) in rats. BMSCs were isolated from the bone marrow of rats. The target gene miR-124 was identified using a luciferase-reporter gene assay. Seventy-two rats were selected for construction of the SCI model, and the rats were randomly divided into the blank group, sham group, SCI group, negative control (NC) group, overexpressed miR-124 group and si-PDXK group. The mRNA expression of miR-124 and the mRNA and protein expression of pyridoxal kinase (PDXK) were detected by quantitative real-time polymerase chain reaction and western blotting. The locomotor capacity of the rats was evaluated using the Basso, Beattie and Bresnahan (BBB) scale. Brdu, neuron-specific enolase (NSE), neurofilament (NF) and microtubule-associated protein 2 (MAP2) were detected using immunohistochemistry. The expression levels of thyrotropin-releasing hormone (TRH), prostacyclin (PGI2) and gangliosides (GM) were measured using an enzyme-linked immunosorbent assay. PDXK was identified as the target gene of miR-124. The overexpressed miR-124 group exhibited higher miR-124 expression than the SCI, NC and si-PDXK groups. Compared with the SCI and NC groups, the PDXK expression was downregulated in the overexpressed miR-124 and si-PDXK groups, and the BBB scores were significantly increased 7, 21 and 35 days after transplantation. The double-labeled positive cell densities (Brdu+NSE/NF/MAP2) and the expression levels of TRH, PGI2 and GM in the overexpressed miR-124 group were significantly higher than those in the NC and SCI groups. These results indicated that miR-124 targeted PDXK to accelerate the differentiation of BMSCs into neurocytes and promote SCI repair.


Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , Terapêutica com RNAi , Traumatismos da Medula Espinal/terapia , Regeneração da Medula Espinal , Animais , Células Cultivadas , Epoprostenol/metabolismo , Gangliosídeos/metabolismo , Filamentos Intermediários/genética , Filamentos Intermediários/metabolismo , Lentivirus/genética , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Piridoxal Quinase/genética , Piridoxal Quinase/metabolismo , Ratos , Ratos Sprague-Dawley , Hormônio Liberador de Tireotropina/metabolismo
19.
Cell Stress Chaperones ; 22(4): 613-626, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28470624

RESUMO

Mutations in the small heat shock protein chaperone CRYAB (αB-crystallin/HSPB5) and the intermediate filament protein desmin, phenocopy each other causing cardiomyopathies. Whilst the binding sites for desmin on CRYAB have been determined, desmin epitopes responsible for CRYAB binding and also the parameters that determine CRYAB binding to desmin filaments are unknown. Using a combination of co-sedimentation centrifugation, viscometric assays and electron microscopy of negatively stained filaments to analyse the in vitro assembly of desmin filaments, we show that the binding of CRYAB to desmin is subject to its assembly status, to the subunit organization within filaments formed and to the integrity of the C-terminal tail domain of desmin. Our in vitro studies using a rapid assembly protocol, C-terminally truncated desmin and two disease-causing mutants (I451M and R454W) suggest that CRYAB is a sensor for the surface topology of the desmin filament. Our data also suggest that CRYAB performs an assembly chaperone role because the assembling filaments have different CRYAB-binding properties during the maturation process. We suggest that the capability of CRYAB to distinguish between filaments with different surface topologies due either to mutation (R454W) or assembly protocol is important to understanding the pathomechanism(s) of desmin-CRYAB myopathies.


Assuntos
Desmina/metabolismo , Filamentos Intermediários/metabolismo , Cadeia B de alfa-Cristalina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Desmina/química , Desmina/genética , Desmina/ultraestrutura , Humanos , Filamentos Intermediários/química , Filamentos Intermediários/genética , Filamentos Intermediários/ultraestrutura , Mutação Puntual , Ligação Proteica , Domínios Proteicos
20.
Am J Physiol Gastrointest Liver Physiol ; 312(6): G628-G634, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28360031

RESUMO

Intermediate filament proteins (IFs), such as cytoplasmic keratins in epithelial cells and vimentin in mesenchymal cells and the nuclear lamins, make up one of the three major cytoskeletal protein families. Whether in digestive organs or other tissues, IFs share several unique features including stress-inducible overexpression, abundance, cell-selective and differentiation state expression, and association with >80 human diseases when mutated. Whereas most IF mutations cause disease, mutations in simple epithelial keratins 8, 18, or 19 or in lamin A/C predispose to liver disease with or without other tissue manifestations. Keratins serve major functions including protection from apoptosis, providing cellular and subcellular mechanical integrity, protein targeting to subcellular compartments, and scaffolding and regulation of cell-signaling processes. Keratins are essential for Mallory-Denk body aggregate formation that occurs in association with several liver diseases, whereas an alternate type of keratin and lamin aggregation occurs upon liver involvement in porphyria. IF-associated diseases have no known directed therapy, but high-throughput drug screening to identify potential therapies is an appealing ongoing approach. Despite the extensive current knowledge base, much remains to be discovered regarding IF physiology and pathophysiology in digestive and nondigestive organs.


Assuntos
Doenças do Sistema Digestório/metabolismo , Sistema Digestório/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/metabolismo , Animais , Sistema Digestório/patologia , Sistema Digestório/fisiopatologia , Doenças do Sistema Digestório/genética , Doenças do Sistema Digestório/patologia , Doenças do Sistema Digestório/fisiopatologia , Regulação da Expressão Gênica , Predisposição Genética para Doença , Humanos , Proteínas de Filamentos Intermediários/genética , Filamentos Intermediários/genética , Filamentos Intermediários/patologia , Corpos de Mallory/metabolismo , Corpos de Mallory/patologia , Mutação , Fenótipo , Polimorfismo Genético
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