Martin Sophie, Tazi Jamal
Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535;
Institut de Génétique Moléculaire de Montpellier, CNRS UMR 5535.
J Vis Exp. 2014 May 21(87):51197. doi: 10.3791/51197.
SGs can be visualized in cells by immunostaining of specific protein components or polyA+ mRNAs. SGs are highly dynamic and the study of their assembly and fate is important to understand the cellular response to stress. The deficiency in key factors of SGs like G3BP (RasGAP SH3 domain Binding Protein) leads to developmental defects in mice and alterations of the Central Nervous System. To study the dynamics of SGs in cells from an organism, one can culture primary cells and follow the localization of a transfected tagged component of SGs. We describe time-lapse experiment to observe G3BP1-containing SGs in Mouse Embryonic Fibroblasts (MEFs). This technique can also be used to study G3BP-containing SGs in live neurons, which is crucial as it was recently shown that these SGs are formed at the onset of neurodegenerative diseases like Alzheimer's disease. This approach can be adapted to any other cellular body and granule protein component, and performed with transgenic animals, allowing the live study of granules dynamics for example in the absence of a specific factor of these granules.
通过对特定蛋白质成分或多聚腺苷酸加尾（polyA+）mRNA进行免疫染色，可在细胞中观察到应激颗粒（SGs）。应激颗粒高度动态变化，研究其组装和命运对于理解细胞对压力的反应至关重要。应激颗粒关键因子如G3BP（RasGAP SH3结构域结合蛋白）的缺陷会导致小鼠发育缺陷和中枢神经系统改变。为了研究生物体细胞中应激颗粒的动态变化，可以培养原代细胞并追踪转染的应激颗粒标记成分的定位。我们描述了在小鼠胚胎成纤维细胞（MEFs）中观察含G3BP1应激颗粒的延时实验。该技术也可用于研究活神经元中含G3BP的应激颗粒，这至关重要，因为最近有研究表明这些应激颗粒在阿尔茨海默病等神经退行性疾病发作时形成。这种方法可适用于任何其他细胞体和颗粒蛋白成分，并可用于转基因动物，例如在缺乏这些颗粒的特定因子的情况下，对颗粒动态变化进行活体研究。
