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1.
Neuron ; 109(7): 1067-1069, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33831359

RESUMO

Axonal degeneration is controlled by the TIR domain NADase SARM1. In this issue of Neuron, Figley et al. (2021) reveal a key regulatory mechanism that controls SARM1's enzymatic activity, providing insight into how NAD+ biosynthesis by the NMNAT2 enzyme protects axons, and a new therapeutic path to tune SARM1 activity.


Assuntos
Proteínas do Domínio Armadillo , NAD , Proteínas do Domínio Armadillo/genética , Axônios , Proteínas do Citoesqueleto/genética , NAD+ Nucleosidase
2.
Crit Rev Oncol Hematol ; 160: 103283, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33667657

RESUMO

Glioblastoma, the most common primary brain malignancy, is an exceptionally fatal cancer. Lack of suitable biomarkers and efficient treatment largely contribute to the therapy failure. Cytoskeletal proteins are crucial proteins in glioblastoma pathogenesis and can potentially serve as biomarkers and therapeutic targets. Among them, GFAP, has gained most attention as potential diagnostic biomarker, while vimentin and microtubules are considered as prospective therapeutic targets. Microtubules represent one of the best anti-cancer targets due to their critical role in cell proliferation. Despite testing in clinical trials, the efficiency of taxanes, epothilones, vinca-domain binding drugs, colchicine-domain binding drugs and γ-tubulin binding drugs remains to be confirmed. Moreover, tumor treating field that disrupts microtubules draw attention because of its high efficiency and is called "the fourth cancer treatment modality". Thereby, because of the involvement of cytoskeleton in key physiological and pathological processes, its therapeutic potential in glioblastoma is currently extensively investigated.


Assuntos
Glioblastoma , Biomarcadores , Proteínas do Citoesqueleto/genética , Glioblastoma/diagnóstico , Glioblastoma/tratamento farmacológico , Humanos , Estudos Prospectivos , Tubulina (Proteína)
4.
Biomed Environ Sci ; 34(2): 139-151, 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33685573

RESUMO

Objective: The underlying mechanism of Ezrin in ovarian cancer (OVCA) is far from being understood. Therefore, this study aimed to assess the role of Ezrin in OVCA cells (SKOV3 and CaOV3) and investigate the associated molecular mechanisms. Methods: We performed Western blotting, reverse transcription-quantitative polymerase chain reaction, MTT, cell colony, cell wound healing, transwell migration and invasion, RhoA and Rac active pull down assays, and confocal immunofluorescence experiments to evaluate the functions and molecular mechanisms of Ezrin overexpression or knockdown in the proliferation and metastasis of OVCA cells. Results: The ectopic expression of Ezrin significantly increased cell proliferation, invasiveness, and epithelial-mesenchymal transition (EMT) in OVCA cells. By contrast, the knockdown of endogenous Ezrin prevented OVCA cell proliferation, invasiveness, and EMT. Lastly, we observed that Ezrin can positively regulate the active forms of RhoA rather than Rac-1 in OVCA cells, thereby promoting robust stress fiber formation. Conclusion: Our results indicated that Ezrin regulates OVCA cell proliferation and invasiveness by modulating EMT and induces actin stress fiber formation by regulating Rho-GTPase activity, which provides novel insights into the treatment of the OVCA.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas do Citoesqueleto/genética , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Invasividade Neoplásica , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Fibras de Estresse/genética , Fibras de Estresse/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo
5.
Nat Commun ; 12(1): 1903, 2021 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-33771994

RESUMO

Aberrant regulation of microRNAs (miRNAs) has been implicated in the pathogenesis of Alzheimer's disease (AD), but most abnormally expressed miRNAs found in AD are not regulated by synaptic activity. Here we report that dysfunction of miR-135a-5p/Rock2/Add1 results in memory/synaptic disorder in a mouse model of AD. miR-135a-5p levels are significantly reduced in excitatory hippocampal neurons of AD model mice. This decrease is tau dependent and mediated by Foxd3. Inhibition of miR-135a-5p leads to synaptic disorder and memory impairments. Furthermore, excess Rock2 levels caused by loss of miR-135a-5p plays an important role in the synaptic disorder of AD via phosphorylation of Ser726 on adducin 1 (Add1). Blocking the phosphorylation of Ser726 on Add1 with a membrane-permeable peptide effectively rescues the memory impairments in AD mice. Taken together, these findings demonstrate that synaptic-related miR-135a-5p mediates synaptic/memory deficits in AD via the Rock2/Add1 signaling pathway, illuminating a potential therapeutic strategy for AD.


Assuntos
Doença de Alzheimer/genética , Proteínas do Citoesqueleto/genética , Transtornos da Memória/genética , MicroRNAs/genética , Sinapses/metabolismo , Quinases Associadas a rho/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Animais , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Sinapses/fisiologia , Quinases Associadas a rho/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
6.
Neuron ; 109(7): 1118-1136.e11, 2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33657413

RESUMO

Axon degeneration is a central pathological feature of many neurodegenerative diseases. Sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) is a nicotinamide adenine dinucleotide (NAD+)-cleaving enzyme whose activation triggers axon destruction. Loss of the biosynthetic enzyme NMNAT2, which converts nicotinamide mononucleotide (NMN) to NAD+, activates SARM1 via an unknown mechanism. Using structural, biochemical, biophysical, and cellular assays, we demonstrate that SARM1 is activated by an increase in the ratio of NMN to NAD+ and show that both metabolites compete for binding to the auto-inhibitory N-terminal armadillo repeat (ARM) domain of SARM1. We report structures of the SARM1 ARM domain bound to NMN and of the homo-octameric SARM1 complex in the absence of ligands. We show that NMN influences the structure of SARM1 and demonstrate via mutagenesis that NMN binding is required for injury-induced SARM1 activation and axon destruction. Hence, SARM1 is a metabolic sensor responding to an increased NMN/NAD+ ratio by cleaving residual NAD+, thereby inducing feedforward metabolic catastrophe and axonal demise.


Assuntos
Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/metabolismo , Axônios/patologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , NAD/metabolismo , Degeneração Neural/genética , Degeneração Neural/patologia , Mononucleotídeo de Nicotinamida/metabolismo , Animais , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Conformação Proteica
7.
Nat Commun ; 12(1): 1351, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33649372

RESUMO

Exon junction complexes (EJCs) mark untranslated spliced mRNAs and are crucial for the mRNA lifecycle. An imbalance in EJC dosage alters mouse neural stem cell (mNSC) division and is linked to human neurodevelopmental disorders. In quiescent mNSC and immortalized human retinal pigment epithelial (RPE1) cells, centrioles form a basal body for ciliogenesis. Here, we report that EJCs accumulate at basal bodies of mNSC or RPE1 cells and decline when these cells differentiate or resume growth. A high-throughput smFISH screen identifies two transcripts accumulating at centrosomes in quiescent cells, NIN and BICD2. In contrast to BICD2, the localization of NIN transcripts is EJC-dependent. NIN mRNA encodes a core component of centrosomes required for microtubule nucleation and anchoring. We find that EJC down-regulation impairs both pericentriolar material organization and ciliogenesis. An EJC-dependent mRNA trafficking towards centrosome and basal bodies might contribute to proper mNSC division and brain development.


Assuntos
Centrossomo/metabolismo , Cílios/metabolismo , Éxons/genética , Transporte de RNA , RNA Mensageiro/metabolismo , Animais , Autoantígenos/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , RNA Helicases DEAD-box/metabolismo , Fator de Iniciação 4A em Eucariotos/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo
8.
Nat Genet ; 53(3): 392-402, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33589840

RESUMO

Genome-wide association studies have discovered numerous genomic loci associated with Alzheimer's disease (AD); yet the causal genes and variants are incompletely identified. We performed an updated genome-wide AD meta-analysis, which identified 37 risk loci, including new associations near CCDC6, TSPAN14, NCK2 and SPRED2. Using three SNP-level fine-mapping methods, we identified 21 SNPs with >50% probability each of being causally involved in AD risk and others strongly suggested by functional annotation. We followed this with colocalization analyses across 109 gene expression quantitative trait loci datasets and prioritization of genes by using protein interaction networks and tissue-specific expression. Combining this information into a quantitative score, we found that evidence converged on likely causal genes, including the above four genes, and those at previously discovered AD loci, including BIN1, APH1B, PTK2B, PILRA and CASS4.


Assuntos
Doença de Alzheimer/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Mapeamento Cromossômico , Proteínas do Citoesqueleto/genética , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Desequilíbrio de Ligação , Microglia/fisiologia , Proteínas Oncogênicas/genética , Polimorfismo de Nucleotídeo Único , Mapas de Interação de Proteínas/genética , Locos de Características Quantitativas , Fatores de Risco , Tetraspaninas/genética
9.
Int J Mol Sci ; 22(4)2021 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567659

RESUMO

FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.


Assuntos
Proteínas de Bactérias/química , Proteínas do Citoesqueleto/química , Citoesqueleto/química , Microscopia de Força Atômica/métodos , Multimerização Proteica , Staphylococcus aureus/metabolismo , Conformação Proteica , Staphylococcus aureus/química
10.
Nat Commun ; 12(1): 919, 2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568673

RESUMO

Single-molecule localization microscopy (SMLM) enabling the investigation of individual proteins on molecular scales has revolutionized how biological processes are analysed in cells. However, a major limitation of imaging techniques reaching single-protein resolution is the incomplete and often unknown labeling and detection efficiency of the utilized molecular probes. As a result, fundamental processes such as complex formation of distinct molecular species cannot be reliably quantified. Here, we establish a super-resolution microscopy framework, called quantitative single-molecule colocalization analysis (qSMCL), which permits the identification of absolute molecular quantities and thus the investigation of molecular-scale processes inside cells. The method combines multiplexed single-protein resolution imaging, automated cluster detection, in silico data simulation procedures, and widely applicable experimental controls to determine absolute fractions and spatial coordinates of interacting species on a true molecular level, even in highly crowded subcellular structures. The first application of this framework allowed the identification of a long-sought ternary adhesion complex-consisting of talin, kindlin and active ß1-integrin-that specifically forms in cell-matrix adhesion sites. Together, the experiments demonstrate that qSMCL allows an absolute quantification of multiplexed SMLM data and thus should be useful for investigating molecular mechanisms underlying numerous processes in cells.


Assuntos
Proteínas do Citoesqueleto/química , Integrina beta1/química , Proteínas Musculares/química , Imagem Individual de Molécula/métodos , Talina/química , Animais , Adesão Celular , Linhagem Celular , Humanos , Camundongos , Imagem Individual de Molécula/instrumentação
11.
Medicine (Baltimore) ; 100(4): e23788, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33530176

RESUMO

ABSTRACT: Gastric cancer is the fifth most common cancer and the third leading cause of cancer-related mortality globally. Abnormal DNA methylation is closely related to gastric cancer. The purpose of the study was to investigate the methylation of the SYNE1 and MAGI2 gene promoter and its relationship with the clinical-pathological factors, chemotherapy efficacy, and survival, thus providing a new biomarker for the prognosis and chemotherapy efficacy in gastric cancer.The methylation status of SYNE1 and MAGI2 in gastric cancer and adjacent tissues was detected by MSP method in 70 cases of advanced gastric cancer paraffin specimens.The methylation rate of the SYNE1 and MAGI2 gene promoter region was higher in gastric cancer tissues compared with adjacent tissues. The methylation status of SYNE1 was associated with the age at diagnosis and the size of the primary tumors, but no clinical or pathological factors have been found to be related with the methylation status of MAGI2 promoter. A high level of SYNE1 promoter methylation was associated with poorer chemotherapy efficacy in recurrent patients with gastric cancer. Thirty-three percent of the 70 patients exhibited highly methylated MAGI2; in this group, the median progression-free survival time was 4.1 months, shorter than those with negative methylated MAGI2 whose PFS was 5.1 months.MAGI2 is more methylated in gastric cancer than in adjacent tissues suggesting that hypermethylation changes in MAGI2 may be one of the mechanisms of tumorigenesis in gastric cancer. The methylation status of the SYNE1 and MAGI2 promoter regions may affect the chemotherapy efficacy of advanced gastric cancer. The prognosis of MAGI2-negative patients was better than that of positive ones, suggesting that MAGI2 may be an independent prognostic factor for PFS in patients with advanced gastric cancer.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas do Citoesqueleto/genética , Metilação de DNA , Guanilato Quinases/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , Neoplasias Gástricas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/mortalidade
12.
Int J Mol Sci ; 22(3)2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33572860

RESUMO

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal dystrophy, renal cysts, obesity and polydactyly. BBS genes have been implicated in ciliogenesis, hedgehog signaling and retinal pigment epithelium maturation. BBS1 and BBS5 are members of the BBSome, implicated in cilia transport of proteins, and BBS10 is a member of the chaperonin-complex, mediating BBSome assembly. In this study, involvement of BBS1, BBS5 and BBS10 in ciliogenesis and hedgehog signaling were investigated in BBS-defective patient fibroblasts as well as in RPE-hTERT cells following siRNA-mediated knockdown of the BBS genes. Furthermore, the ability of BBS1-defective induced pluripotent stem-cells (iPSCs) to differentiate into RPE cells was assessed. We report that cells lacking functional BBS5 or BBS10 have a reduced number of primary cilia, whereas cells lacking functional BBS1 display shorter primary cilia compared to wild-type cells. Hedgehog signaling was substantially impaired and Smoothened, a component of hedgehog signaling, was trapped inside the cilia of the BBS-defective cells, even in the absence of Smoothened agonist. Preliminary results demonstrated the ability of BBS1-defective iPSC to differentiate into RPE-65 expressing RPE-like cells. The BBS1-/--defective RPE-like cells were less pigmented, compared to RPE-like cells differentiated from control iPSCs, indicating an impact of BBS1 on RPE maturation.


Assuntos
Síndrome de Bardet-Biedl/metabolismo , Chaperoninas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Síndrome de Bardet-Biedl/patologia , Linhagem Celular , Cílios/metabolismo , Cílios/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transdução de Sinais
13.
Methods Mol Biol ; 2218: 219-244, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606235

RESUMO

Zebrafish embryos, with their large size (>0.5 mm) and accessibility, are valuable tools for investigating core cellular processes. Many of those processes, such as cell division, asymmetric inheritance of cellular components, and structural dynamics involved in cell motility and morphology rely on cytoskeletal rearrangements and associated macromolecules. In addition to the protein-rich cytoskeleton, the early embryo is packed with maternally deposited RNA, which serves essential roles in establishing cell polarity, cell fate, and cell organization. Here, we present methods for visualizing endogenous RNA along with cytoskeletal structures, including microtubules and filamentous actin (F-actin) in the context of an intact vertebrate embryo. Each of the four protocols described herein (embryo fixation, RNA probe design/synthesis, double fluorescent in situ hybridization with tubulin immunofluorescence, and fluorescent in situ hybridization with phalloidin labeling of F-actin) are intended for optimal preservation and visualization of both the cytoskeleton and RNAs of interest. These methods can also be modified and applied to a broad range of other uses.


Assuntos
Citoesqueleto/metabolismo , Embrião não Mamífero/metabolismo , Hibridização in Situ Fluorescente/métodos , RNA/metabolismo , Peixe-Zebra/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Divisão Celular/fisiologia , Proteínas do Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Proteínas de Peixe-Zebra/metabolismo
14.
Environ Health Prev Med ; 26(1): 8, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33451279

RESUMO

BACKGROUND: Prenatal stress can cause neurobiological and behavioral defects in offspring; environmental factors play a crucial role in regulating the development of brain and behavioral; this study was designed to test and verify whether an enriched environment can repair learning and memory impairment in offspring rats induced by prenatal stress and to explore its mechanism involving the expression of insulin-like growth factor-2 (IGF-2) and activity-regulated cytoskeletal-associated protein (Arc) in the hippocampus of the offspring. METHODS: Rats were selected to establish a chronic unpredictable mild stress (CUMS) model during pregnancy. Offspring were weaned on 21st day and housed under either standard or an enriched environment. The learning and memory ability were tested using Morris water maze and Y-maze. The expression of IGF-2 and Arc mRNA and protein were respectively measured by using RT-PCR and Western blotting. RESULTS: There was an elevation in the plasma corticosterone level of rat model of maternal chronic stress during pregnancy. Maternal stress's offspring exposed to an enriched environment could decrease their plasma corticosterone level and improve their weight. The offspring of maternal stress during pregnancy exhibited abnormalities in Morris water maze and Y-maze, which were improved in an enriched environment. The expression of IGF-2, Arc mRNA, and protein in offspring of maternal stress during pregnancy was boosted and some relationships existed between these parameters after being exposed enriched environment. CONCLUSIONS: The learning and memory impairment in offspring of prenatal stress can be rectified by the enriched environment, the mechanism of which is related to the decreasing plasma corticosterone and increasing hippocampal IGF-2 and Arc of offspring rats following maternal chronic stress during pregnancy.


Assuntos
Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica , Fator de Crescimento Insulin-Like II/genética , Deficiências da Aprendizagem/genética , Transtornos da Memória/genética , Proteínas do Tecido Nervoso/genética , Efeitos Tardios da Exposição Pré-Natal/genética , Meio Social , Estresse Psicológico/genética , Animais , Proteínas do Citoesqueleto/metabolismo , Feminino , Hipocampo/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Aprendizagem , Deficiências da Aprendizagem/psicologia , Masculino , Transtornos da Memória/psicologia , Proteínas do Tecido Nervoso/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/psicologia , Distribuição Aleatória , Ratos , Ratos Wistar
15.
DNA Cell Biol ; 40(2): 332-347, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393844

RESUMO

Neuroblastoma (NB) has the highest incidence of all extracranial solid tumors in children and is highly lethal. This study aims to establish a prognostic model of NB with MYCN-related genes. We determined the gene expression profiles of 900 NB samples from the UCSC database and four Gene Expression Omnibus (GEO) data sets, and performed a comprehensive bioinformatics analysis and clinical sample verification. After univariate Cox regression, least absolute shrinkage and selection operator (Lasso), and multivariate Cox regression analyses, four (AKR1C1, CHD5, PDE4DIP, and PRKACB) genes were finally selected and used to construct a risk score prognostic model. In the UCSC data set, the high-risk group exhibited a significantly worse prognosis than the low-risk group. In addition, the nomogram, which includes prognostic markers and clinical factors, demonstrates high prognostic value. Finally, the differential expression of the four genes in the model was verified by quantitative real-time PCR in clinical tissues. These findings of MYCN-related genes provide a new and reliable prognostic model for NB related to MYCN.


Assuntos
Biomarcadores Tumorais/genética , Biologia Computacional , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/diagnóstico , Neuroblastoma/genética , Reação em Cadeia da Polimerase em Tempo Real , 20-Hidroxiesteroide Desidrogenases/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas do Citoesqueleto/genética , DNA Helicases/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas do Tecido Nervoso/genética , Prognóstico
16.
Life Sci ; 269: 119045, 2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33453238

RESUMO

AIM: To determine the role of MICAL2 in myofibroblasts differentiation and epidural fibrosis. BACKGROUND: Epidural fibrosis (EF) may develop following laminectomy and aberrant myofibroblasts differentiation and excessive extracellular matrix (ECM) accumulation play key roles in the formation of EF. Dense epidural fibrosis results to the poor surgical outcomes and failed back surgery syndrome (FBSS), and there is no effective treatment available. Molecule interacting with Casl2 (MICAL2) has been demonstrated to participate in multiple cellular processes by regulating actin cytoskeleton dynamics. However, its role in epidural fibrosis remains totally unverified. MATERIALS AND METHODS: The potential functions and mechanisms of MICAL2 were explored using western blotting, immunofluorescence and lentivirus infection. KEY FINDINGS: In our study, we determined that the MICAL2 expression was elevated in epidural fibrotic tissues and TGF-ß1-stimulated fibroblasts. Moreover, knockdown of MICAL2 using MICAL2-specific short hairpin RNA attenuated TGF-ß1-induced myofibroblasts differentiation and epidural fibrosis both in vitro and vivo, as indicated by decreased scar formation, reduced collagen production and down-regulated expression of α-SMA, collagen-1 and fibronectin. We also demonstrated that MICAL2 knockdown affected the migratory capability of fibroblasts in vitro. By further mechanistic research, we revealed that the MRTF-A nuclear translocation was inhibited in response to the knockdown of MICAL2 in fibroblasts and MICAL2 served as a pro-fibrotic factor in an SRF/MRTF-A-dependent manner. SIGNIFICANCE: In conclusion, our results indicated that MICAL2 mediated myofibroblasts differentiation and promoted epidural fibrogenesis via SRF/MRTF-A signaling pathway, suggesting manipulation of MICAL2 activity as a novel alternative strategy for the prevention of epidural fibrosis.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Espaço Epidural/patologia , Fibrose/patologia , Regulação da Expressão Gênica , Miofibroblastos/patologia , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Proteínas do Citoesqueleto/genética , Espaço Epidural/metabolismo , Feminino , Fibrose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Fator de Resposta Sérica/genética , Fatores de Transcrição/genética
17.
Nat Commun ; 12(1): 480, 2021 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-33473127

RESUMO

The cytoplasm in mammalian cells is considered homogeneous. In this study, we report that the cytoplasmic fluidity is regulated in the blebbing cells; the cytoplasm of rapidly expanding membrane blebs is more disordered than the cytoplasm of retracting blebs. The increase of cytoplasmic fluidity in the expanding bleb is caused by a sharp rise in the calcium concentration. The STIM-Orai1 pathway regulates this rapid and restricted increase of calcium in the expanding blebs. Conversely, activated ERM protein binds to Orai1 to inhibit the store-operated calcium entry in retracting blebs, which results in decreased in cytoplasmic calcium, rapid reassembly of the actin cortex.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Animais , Sinalização do Cálcio/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/antagonistas & inibidores , Células HEK293 , Humanos , Proteínas de Membrana/fisiologia
18.
Exp Eye Res ; 203: 108400, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33347868

RESUMO

Current research on healthy corneal stromal cells will typically use primary cells as they are the most representative of in vivo behaviour. Primary cells are normally isolated from the limbus of discarded donor peripheral corneal tissue left over from transplantation (due to its relative abundance). Therefore, the central part of the cornea is less used in research as this tissue is usually used for transplantation. In some cases, although rare, the whole cornea, can become available for research. It is important to keep in mind that these corneas often have longer storage time, but the use of the central tissue for research is even more interesting, as knowing what cells are being transplanted into recipients would be highly relevant. To this end, stromal cells were extracted from both the limbus and central button of healthy corneas donated for research. This allowed for important comparison between central and limbal cells in culture. Of interest here was the extraction method of stromal cells from the donor tissue. The two most common methods of extraction are enzyme digestion and explant migration. However, no work has been done to understand how each method relatively affects the extracted cells. The extraction method and location from which stromal cells are harvested seems to have a significant effect on the cell adherence, survival, and gene expression of the stromal cells in culture. Enzyme digested cells showed that limbal and central cells had different gene expressions prior to culture, with gene such as ALDH3A1 being much more expressed in limbal cells. Enzyme digesting the limbal ring seems to yield the hardiest populations of stromal cells, a desirable trait in the culture of primary cells.


Assuntos
Separação Celular/métodos , Ceratócitos da Córnea/fisiologia , Substância Própria/citologia , Limbo da Córnea/citologia , Técnicas de Cultura de Células , Sobrevivência Celular/fisiologia , Meios de Cultura Livres de Soro , Proteínas do Citoesqueleto/genética , Regulação da Expressão Gênica/fisiologia , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Doadores de Tecidos
19.
Methods Mol Biol ; 2220: 201-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975777

RESUMO

The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.


Assuntos
Células Epiteliais/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/patologia , Microscopia de Fluorescência/métodos , Linhagem Celular , Proteínas do Citoesqueleto/análise , Células Epiteliais/patologia , Imunofluorescência/métodos , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia
20.
Nucleic Acids Res ; 49(2): 621-635, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33337475

RESUMO

The integration of retroviral reverse transcripts into the chromatin of the cells that they infect is required for virus replication. Retroviral integration has far-reaching consequences, from perpetuating deadly human diseases to molding metazoan evolution. The lentivirus human immunodeficiency virus 1 (HIV-1), which is the causative agent of the AIDS pandemic, efficiently infects interphase cells due to the active nuclear import of its preintegration complex (PIC). To enable integration, the PIC must navigate the densely-packed nuclear environment where the genome is organized into different chromatin states of varying accessibility in accordance with cellular needs. The HIV-1 capsid protein interacts with specific host factors to facilitate PIC nuclear import, while additional interactions of viral integrase, the enzyme responsible for viral DNA integration, with cellular nuclear proteins and nucleobases guide integration to specific chromosomal sites. HIV-1 integration favors transcriptionally active chromatin such as speckle-associated domains and disfavors heterochromatin including lamina-associated domains. In this review, we describe virus-host interactions that facilitate HIV-1 PIC nuclear import and integration site targeting, highlighting commonalities among factors that participate in both of these steps. We moreover discuss how the nuclear landscape influences HIV-1 integration site selection as well as the establishment of active versus latent virus infection.


Assuntos
HIV-1/fisiologia , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Integração Viral , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas do Capsídeo/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Cromatina/genética , Cromatina/metabolismo , Citoplasma/metabolismo , Citoplasma/virologia , Proteínas do Citoesqueleto/metabolismo , Transcriptase Reversa do HIV/fisiologia , HIV-1/enzimologia , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Interfase , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Poro Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Conformação Proteica , Domínios Proteicos , Fatores de Transcrição/deficiência , Fatores de Transcrição/fisiologia , Integração Viral/genética , Integração Viral/fisiologia , Latência Viral , Replicação Viral , Fatores de Poliadenilação e Clivagem de mRNA/deficiência , Fatores de Poliadenilação e Clivagem de mRNA/fisiologia
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