RESUMO
A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an 'alert' phase we term G(Alert). Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into G(Alert) and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-G(Alert) transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into G(Alert) possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into G(Alert) functions as an 'alerting' mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.
Assuntos
Ciclo Celular/fisiologia , Complexos Multiproteicos/metabolismo , Músculo Esquelético/citologia , Fase de Repouso do Ciclo Celular/fisiologia , Células Satélites de Músculo Esquelético/citologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Ciclo Celular/genética , Células Cultivadas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Complexos Multiproteicos/genética , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Regeneração/fisiologia , Fase de Repouso do Ciclo Celular/genética , Células Satélites de Músculo Esquelético/metabolismo , Serina-Treonina Quinases TOR/genéticaRESUMO
Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene approximately 3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Engenharia Genética/métodos , Higromicina B/análogos & derivados , Saccharomyces cerevisiae/genética , Sequência de Bases , Western Blotting , Cinamatos/farmacologia , Proteínas de Ligação a DNA/metabolismo , Farmacorresistência Fúngica/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Fluorescência Verde , Higromicina B/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mutação , Plasmídeos/genética , RNA Fúngico/genética , RNA Fúngico/metabolismo , Rad51 Recombinase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
Targeted nucleotide exchange (TNE) is a process in which an oligonucleotide bearing sequence complementarity aligns with the sequence of a target gene and directs the alteration of a single base. This technique can be used to repair a point mutation or mediate site-specific mutagenesis. A critical factor in the development of this approach centers around the elevation and stabilization of the frequencies with which these events occur. Here we describe a protocol for increasing the frequency of TNE in the true yeast, Saccharomyces cerevisiae, through the use of nonspecific, carrier oligonucleotides. These molecules, when added to the reaction, increase the TNE frequency up to 25-fold in some cases, perhaps by providing a molecular trap to bind factors, which may inactivate the specific targeting oligos.
Assuntos
Marcação de Genes/métodos , Oligodesoxirribonucleotídeos/farmacologia , Saccharomyces cerevisiae/genética , Sequência de Bases , Cromossomos Fúngicos/efeitos dos fármacos , Cromossomos Fúngicos/genética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição/métodos , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
Muscular dystrophies are a class of disorders that cause progressive muscle wasting. A major hurdle for discovering treatments for the muscular dystrophies is a lack of reliable assays to monitor disease progression in animal models. We have developed a novel mouse model to assess disease activity noninvasively in mice with muscular dystrophies. These mice express an inducible luciferase reporter gene in muscle stem cells. In dystrophic mice, muscle stem cells activate and proliferate in response to muscle degeneration, resulting in an increase in the level of luciferase expression, which can be monitored by noninvasive, bioluminescence imaging. We applied this noninvasive imaging to assess disease activity in a mouse model of the human disease limb girdle muscular dystrophy 2B (LGMD2B), caused by a mutation in the dysferlin gene. We monitored the natural history and disease progression in these dysferlin-deficient mice up to 18 months of age and were able to detect disease activity prior to the appearance of any overt disease manifestation by histopathological analyses. Disease activity was reflected by changes in luciferase activity over time, and disease burden was reflected by cumulative luciferase activity, which paralleled disease progression as determined by histopathological analysis. The ability to monitor disease activity noninvasively in mouse models of muscular dystrophy will be invaluable for the assessment of disease progression and the effectiveness of therapeutic interventions.