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1.
Proc Natl Acad Sci U S A ; 119(18): e2115638119, 2022 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-35476521

RESUMO

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.


Assuntos
Proteínas de Ciclo Celular , Ciclinas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Ciclinas/genética , Ciclinas/metabolismo , Células-Tronco/metabolismo
2.
STAR Protoc ; 4(1): 102008, 2023 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-36640368

RESUMO

The ability of stem cells to activate and differentiate is critical for maintaining the regenerative capacity of skeletal muscle. Here, we detail steps for specific quantification and isolation of primary human fibro-adipogenic progenitors and skeletal muscle stem cells using fluorescence-activated cell sorting. We describe important phenotypic traits such as time to enter the cell cycle and assessment of cell differentiation for the isolated cell populations. The technique has been applied on tissue obtained from surgery and needle biopsies. For complete details on the use and execution of this protocol, please refer to Farup et al. (2021).1.


Assuntos
Adipogenia , Células-Tronco , Humanos , Citometria de Fluxo/métodos , Diferenciação Celular/fisiologia , Músculo Esquelético
3.
Cells ; 9(3)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-32121096

RESUMO

Mitochondrial dysfunction has been implicated as a central mechanism in the metabolic myopathy accompanying critical limb ischemia (CLI). However, whether mitochondrial dysfunction is directly related to lower extremity ischemia and the structural and molecular mechanisms underpinning mitochondrial dysfunction in CLI patients is not understood. Here, we aimed to study whether mitochondrial dysfunction is a distinctive characteristic of CLI myopathy by assessing mitochondrial respiration in gastrocnemius muscle from 14 CLI patients (65.3 ± 7.8 y) and 15 matched control patients (CON) with a similar comorbidity risk profile and medication regimen but without peripheral ischemia (67.4 ± 7.4 y). Furthermore, we studied potential structural and molecular mechanisms of mitochondrial dysfunction by measuring total, sub-population, and fiber-type-specific mitochondrial volumetric content and cristae density with transmission electron microscopy and by assessing mitophagy and fission/fusion-related protein expression. Finally, we asked whether commonly used biomarkers of mitochondrial content are valid in patients with cardiovascular disease. CLI patients exhibited inferior mitochondrial respiration compared to CON. This respiratory deficit was not related to lower whole-muscle mitochondrial content or cristae density. However, stratification for fiber types revealed ultrastructural mitochondrial alterations in CLI patients compared to CON. CLI patients exhibited an altered expression of mitophagy-related proteins but not fission/fusion-related proteins compared to CON. Citrate synthase, cytochrome c oxidase subunit IV (COXIV), and 3-hydroxyacyl-CoA dehydrogenase (ß-HAD) could not predict mitochondrial content. Mitochondrial dysfunction is a distinctive characteristic of CLI myopathy and is not related to altered organelle content or cristae density. Our results link this intrinsic mitochondrial deficit to dysregulation of the mitochondrial quality control system, which has implications for the development of therapeutic strategies.


Assuntos
Extremidades/irrigação sanguínea , Isquemia/complicações , Mitocôndrias Musculares/metabolismo , Mitocôndrias Musculares/patologia , Doenças Musculares/complicações , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Idoso , Biomarcadores/metabolismo , Respiração Celular , Extremidades/patologia , Feminino , Humanos , Masculino , Mitocôndrias Musculares/ultraestrutura , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo
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