Kainz K, Tadic J, Zimmermann A, Pendl T, Carmona-Gutierrez D, Ruckenstuhl C, Eisenberg T, Madeo F
Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria.
Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria; BioTechMed-Graz, Graz, Austria.
Methods Enzymol. 2017;588:367-394. doi: 10.1016/bs.mie.2016.09.086. Epub 2016 Nov 22.
Autophagy is a catabolic process that is crucial for cellular homeostasis and adaptive response to changing environments. Importantly, autophagy has been shown to be induced in many longevity-associated scenarios and to be required to maintain lifespan extension. Notably, autophagy is a highly conserved cellular process among eukaryotes, and the yeast Saccharomyces cerevisiae has become a universal model system for unraveling the molecular machinery underlying autophagic mechanisms. Here, we discuss different protocols to monitor survival and autophagy of yeast cells upon chronological aging. These include the use of propidium iodide to assess the loss of cell membrane integrity, as well as clonogenic assays to directly determine survival rates. Additionally, we describe methods to quantify autophagic flux, including the alkaline phosphatase activity or the GFP liberation assays, which measure the delivery of autophagosomal cargo to the vacuole. In sum, we have recapped established protocols used to evaluate a link between lifespan extension and autophagy in yeast.
自噬是一种分解代谢过程，对细胞内稳态和对不断变化的环境的适应性反应至关重要。重要的是，自噬已被证明在许多与长寿相关的情况下被诱导，并且是维持寿命延长所必需的。值得注意的是，自噬是真核生物中高度保守的细胞过程，酿酒酵母已成为揭示自噬机制背后分子机制的通用模型系统。在这里，我们讨论了监测酵母细胞在时序老化过程中的存活和自噬的不同方案。这些包括使用碘化丙啶评估细胞膜完整性的丧失，以及克隆形成试验直接确定存活率。此外，我们描述了量化自噬通量的方法，包括碱性磷酸酶活性或GFP释放试验，这些试验测量自噬体货物向液泡的递送。总之，我们总结了用于评估酵母寿命延长与自噬之间联系的既定方案。
