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1.
Development ; 2024 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-38975838

RESUMO

Cohesin, a chromatin-associated protein complex with four core subunits (Smc1a, Smc3, Rad21 and either Stag1 or 2), has a central role in cell proliferation and gene expression in metazoans. Human developmental disorders termed "cohesinopathies" are characterised by germline mutations in cohesin or its regulators that do not entirely eliminate cohesin function. However, it is not clear if mutations in individual cohesin subunits have independent developmental consequences. Here we show that zebrafish rad21 or stag2b mutants independently influence embryonic tailbud development. Both mutants have altered mesoderm induction, but only homozygous or heterozygous rad21 mutation affects cell cycle gene expression. stag2b mutants have narrower notochords and reduced Wnt signaling in neuromesodermal progenitors as revealed by single cell RNA-sequencing. Stimulation of Wnt signaling rescues transcription and morphology in stag2b, but not rad21 mutants. Our results suggest that mutations altering the quantity versus composition of cohesin have independent developmental consequences, with implications for the understanding and management of cohesinopathies.

2.
Development ; 146(12)2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31126979

RESUMO

Developmental programs that arrange cells and tissues into patterned organs are remarkably robust. In the developing vertebrate retina, for example, neurons reproducibly assemble into distinct layers giving the mature organ its overall structured appearance. This stereotypic neuronal arrangement, termed lamination, is important for efficient neuronal connectivity. Although retinal lamination is conserved in many vertebrates, including humans, how it emerges from single cell behaviour is not fully understood. To shed light on this issue, we here investigated the formation of the retinal horizontal cell layer. Using in vivo light sheet imaging of the developing zebrafish retina, we generated a comprehensive quantitative analysis of horizontal single cell behaviour from birth to final positioning. Interestingly, we find that all parameters analysed, including cell cycle dynamics, migration paths and kinetics, as well as sister cell dispersal, are very heterogeneous. Thus, horizontal cells show individual non-stereotypic behaviour before final positioning. Yet these initially variable cell dynamics always generate the correct laminar pattern. Consequently, our data show that the extent of single cell stochasticity in the lamination of the vertebrate retina is underexplored.


Assuntos
Movimento Celular , Neurônios/citologia , Retina/embriologia , Peixe-Zebra/embriologia , Animais , Blastômeros/citologia , Ciclo Celular , Linhagem da Célula , Processamento de Imagem Assistida por Computador , Cinética , Camundongos , Mitose , Análise de Célula Única , Fuso Acromático , Processos Estocásticos
3.
J Cell Biochem ; 120(3): 3124-3136, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30272820

RESUMO

The cholesterol hydroxylase/lyase (CHL) system, consisting of cytochrome P450scc, adrenodoxin (Adx) and adrenodoxin reductase (AdR), initiates mammalian steroidogenesis, converting cholesterol to pregnenolone. The foot-and-mouth disease virus 2A-based method allows to express multiple proteins from a single transcript. We developed a 2A-based multicistronic system for the coexpression of three bovine CHL system proteins as the self-processing polyprotein pCoxIV-P450scc-2A-Adx-2A-AdR-GFP (pCoxIV-CHL-GFP), with a cleavable N-terminal mitochondrial targeting presequence. HEK293T cells transfected with plasmid, containing complementary DNA (cDNA) for pCoxIV-CHL-GFP, efficiently performed the expression of P450scc-2A, targeted to mitochondria, and Adx-2A, AdR-GFP and the fusion protein Adx-2A-AdR-GFP, which were predominantly localized in the cytosol. Despite the spatial separation of expressed P450scc and redox partners, the transfected HEK293T cells were able to convert the steroid substrates of cytochrome P450scc to pregnenolone, whereas control HEK293T cells were not catalytically active. The presence of 2А peptide residue on the C-terminus of P450scc did not preclude its enzymatic activity. HEK293T cells transfected with a vector directing the synthesis of only P450scc-2A demonstrated cytochrome P450scc activity comparable to that of cells expressing all three CHL system components, and to that of nature steroidogenic cells. Thus, the P450scc activity detected in cells transfected with both constructed plasmids was the result of the effective functional coupling of the bovine cytochrome P450scc and endogenous mitochondrial electron transport proteins of HEK293T cells. The produced pregnenolone did not undergo further conversion to progesterone, which indicates the absence of catalytically active 3ß-hydroxysteroid dehydrogenase. Therefore, HEK293T cells may be suitable for the expression of steroidogenic enzymes and the study of their characteristics.


Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Mitocôndrias/metabolismo , 3-Hidroxiesteroide Desidrogenases/metabolismo , Adrenodoxina/metabolismo , Western Blotting , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Cromatografia Líquida de Alta Pressão , Ferredoxina-NADP Redutase , Citometria de Fluxo , Células HEK293 , Humanos , Microscopia de Fluorescência , Plasmídeos/genética , Pregnenolona/metabolismo
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