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1.
Stem Cell Res ; 77: 103396, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38522388

RESUMO

Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches.


Assuntos
Desmina , Células-Tronco Pluripotentes Induzidas , Mutação , Células-Tronco Pluripotentes Induzidas/metabolismo , Humanos , Desmina/metabolismo , Desmina/genética , Linhagem Celular , Sistemas CRISPR-Cas , Técnicas de Introdução de Genes , Diferenciação Celular
2.
PLoS Biol ; 21(8): e3002261, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37590318

RESUMO

Epithelial-mesenchymal transition (EMT) is an early event in cell dissemination from epithelial tissues. EMT endows cells with migratory, and sometimes invasive, capabilities and is thus a key process in embryo morphogenesis and cancer progression. So far, matrix metalloproteinases (MMPs) have not been considered as key players in EMT but rather studied for their role in matrix remodelling in later events such as cell migration per se. Here, we used Xenopus neural crest cells to assess the role of MMP28 in EMT and migration in vivo. We show that a catalytically active MMP28, expressed by neighbouring placodal cells, is required for neural crest EMT and cell migration. We provide strong evidence indicating that MMP28 is imported in the nucleus of neural crest cells where it is required for normal Twist expression. Our data demonstrate that MMP28 can act as an upstream regulator of EMT in vivo raising the possibility that other MMPs might have similar early roles in various EMT-related contexts such as cancer, fibrosis, and wound healing.


Assuntos
Transição Epitelial-Mesenquimal , Crista Neural , Movimento Celular , Núcleo Celular , Epitélio
3.
Development ; 147(7)2020 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-32280063

RESUMO

Matrix metalloproteinases have a broad spectrum of substrates ranging from extracellular matrix components and adhesion molecules to chemokines and growth factors. Despite being mostly secreted, MMPs have been detected in the cytosol, the mitochondria or the nucleus. Although most of the attention is focused on their role in matrix remodeling, the diversity of their substrates and their complex trafficking open the possibility for non-canonical functions. Yet in vivo examples and experimental demonstration of the physiological relevance of such activities are rare. Here, we have used chick neural crest (NC) cells, a highly migratory stem cell population likened to invasive cancer cells, as a model for physiological epithelial-mesenchymal transition (EMT). We demonstrate that MMP14 is required for NC delamination. Interestingly, this role is independent of its cytoplasmic tail and of its catalytic activity. Our in vivo data indicate that, in addition to being a late pro-invasive factor, MMP14 is also likely to be an early player, owing to its role in EMT.


Assuntos
Matriz Extracelular/metabolismo , Lamina Tipo A/metabolismo , Metaloproteinase 14 da Matriz/fisiologia , Crista Neural/metabolismo , Animais , Animais Geneticamente Modificados , Caderinas/metabolismo , Catálise , Células Cultivadas , Embrião de Galinha , Transição Epitelial-Mesenquimal/fisiologia
4.
Dev Dyn ; 244(12): 1538-49, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26335666

RESUMO

BACKGROUND: Development of the pronephros in Xenopus laevis is largely dependent on retinoic acid signaling at the time of kidney field specification with the simultaneous occurrence of a necessary calcium signaling. At the crossroads of these two signaling pathways, we studied the role of Hspa9 (heat shock 70 kDa protein 9) encoding a mitochondrial chaperone in pronephros development. RESULTS: We first showed that Hspa9 is highly expressed in the pronephros territory and elongating nephric duct. We then observed that upon reduced retinoic acid signaling hspa9 expression was reduced as pax8 and pax2. Overexpression of hspa9 enlarged the pax8 positive pronephros territory, leading to a larger pronephric tubule. Loss of function of hspa9 in the kidney field using morpholino approach severely reduced pax8 expression and pronephros formation. Phenotypic rescue was achieved by co-injection of the full-length murine Hspa9 mRNA. However, no rescue was observed when Hspa9 mRNA lacking the mitochondrial-targeting sequence was injected, as this truncated form is able to interfere with pronephros formation when injected solely. CONCLUSIONS: Hspa9 is an important mediator for pronephros development through modulation of pax8. Mitochondrial functions of hspa9 are likely to be involved in specification of pronephric cell fate.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Mitocondriais/metabolismo , Pronefro/embriologia , Proteínas de Xenopus/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Choque Térmico HSP70/genética , Proteínas Mitocondriais/genética , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Pronefro/metabolismo , Transdução de Sinais/fisiologia , Tretinoína/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
5.
Dev Cell ; 34(4): 421-34, 2015 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-26235046

RESUMO

Contact inhibition of locomotion (CIL) is the process through which cells move away from each other after cell-cell contact, and it contributes to malignant invasion and developmental migration. Various cell types exhibit CIL, whereas others remain in contact after collision and may form stable junctions. To investigate what determines this differential behavior, we study neural crest cells, a migratory stem cell population whose invasiveness has been likened to cancer metastasis. By comparing pre-migratory and migratory neural crest cells, we show that the switch from E- to N-cadherin during EMT is essential for acquisition of CIL behavior. Loss of E-cadherin leads to repolarization of protrusions, via p120 and Rac1, resulting in a redistribution of forces from intercellular tension to cell-matrix adhesions, which break down the cadherin junction. These data provide insight into the balance of physical forces that contributes to CIL in cells in vivo.


Assuntos
Caderinas/metabolismo , Movimento Celular , Polaridade Celular , Inibição de Contato , Transição Epitelial-Mesenquimal , Crista Neural/citologia , Animais , Fenômenos Biomecânicos , Cateninas/metabolismo , Junções Intercelulares/metabolismo , Ligação Proteica , Xenopus laevis , Proteínas rac1 de Ligação ao GTP/metabolismo , delta Catenina
6.
Biochim Biophys Acta ; 1793(6): 1033-40, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19135096

RESUMO

In vertebrates, the formation of the nervous system starts at gastrulation with a process called neural induction. This process requires, at least in part, the inhibition of BMP signalling in the ectoderm by noggin, as well as FGF receptor activation and Ca2+ signalling. Our studies with Xenopus embryos suggest that an increase in intracellular Ca2+ concentration ([Ca2+]i), via dihydropyridine-sensitive Ca2+ channels (DHP-sensitive Ca2+ channels) is necessary and sufficient to direct the ectodermal cells toward a neural fate, and that Ca2+ directly controls the expression of neural genes. The mechanism by which the DHP-sensitive Ca2+ channels are activated during neural induction remains unknown. One possible mechanism is via the activation of FGF signalling. Using isolated ectoderm tissue, here we demonstrated that FGF-4 depolarises the membrane of ectodermal cells and induces an increase in [Ca2+]i. This Ca2+ increase can be blocked by SU5402, an FGF receptor inhibitor, and by DHP-sensitive Ca2+ channel antagonists. These inhibitors also block the induction of neural genes. We discuss a possible gating mechanism for the activation of DHP-sensitive Ca2+ channels via the FGF signalling pathway, which involves arachidonic acid and TRPC1 channel activation.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Transdução de Sinais/fisiologia , Xenopus laevis , Animais , Ácido Araquidônico/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Indução Embrionária , Pirróis/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/genética
7.
Cancer Biol Ther ; 6(8): 1318-23, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17660712

RESUMO

Overexpression and activation of TPM3-ALK tyrosine kinase fusion protein is a causal oncogenic event in the development of Anaplastic Large Cell Lymphoma and Inflammatory Myofibroblastic ALK-positive tumors. Thus, the development of ALK specific tyrosine kinase inhibitors is a current therapeutic challenge. Animal models are essential to assess, in vivo, the efficiency of ALK-oncogene inhibitors and to identify new and/or additional therapeutic targets in the ALK tumorigenesis pathway. Using the tetracycline system to allow conditional and concomitant TPM3-ALK and luciferase expression, we have developed a unique transplant model for bioluminescent TPM3-ALK-induced fibroblastic tumors in athymic nude mice. The reversible TPM3-ALK expression allowed us to demonstrate that this oncogene is essential for the tumor growth and its maintenance. In addition, we showed that this model could be used to precisely assess tumor growth inhibition upon ALK chemical inactivation. As proof of principle, we used the general tyrosine kinase inhibitor herbimycin A to inhibit ALK oncoprotein activity. As expected, herbimycin A treatment reduced tumor growth as assessed both by tumor volume measurement and bioluminescent imaging. We conclude that this transplant model for TPM3-ALK-induced tumors represents a valuable tool not only to accurately and rapidly evaluate in vivo ALK-targeted therapies but also to gain insight into the mechanism of ALK-positive tumor development.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Modelos Animais de Doenças , Linfoma Anaplásico de Células Grandes/tratamento farmacológico , Camundongos , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tropomiosina/antagonistas & inibidores , Quinase do Linfoma Anaplásico , Animais , Antibióticos Antineoplásicos/uso terapêutico , Benzoquinonas/farmacologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Genes Reporter , Lactamas Macrocíclicas/farmacologia , Luciferases/análise , Luciferases/genética , Substâncias Luminescentes/análise , Linfoma Anaplásico de Células Grandes/enzimologia , Camundongos Nus , Transplante de Neoplasias , Proteínas de Fusão Oncogênica/análise , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/análise , Receptores Proteína Tirosina Quinases , Rifabutina/análogos & derivados , Tropomiosina/análise
9.
Biochemistry ; 43(39): 12585-91, 2004 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-15449948

RESUMO

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.


Assuntos
Permeabilidade da Membrana Celular , Detergentes , Proteínas de Escherichia coli/síntese química , Canais Iônicos/síntese química , Mecanotransdução Celular , Permeabilidade da Membrana Celular/genética , Sistema Livre de Células/química , Cetomacrogol , Ácidos Cólicos , Ácido Desoxicólico , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos , Glucosídeos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Mecanotransdução Celular/genética , Octoxinol , Técnicas de Patch-Clamp , Polímeros/síntese química , Polímeros/metabolismo , Polissorbatos , Solubilidade
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