CNRS, Biotechnology and Cell Signaling, Ecole Supérieure de Biotechnologie de Strasbourg, Illkirch, 67412 Strasbourg University/Laboratory of Excellence Medalis, 67000 Strasbourg, France.
Institut National de la Santé et de la Recherche Médicale, Centre de Recherche des Cordeliers, Sorbonne Université, Université de Paris, 75006 Paris, France.
Cells. 2020 May 25;9(5):1321. doi: 10.3390/cells9051321.
Tremendous efforts have been made these last decades to increase our knowledge of intracellular degradative systems, especially in the field of autophagy. The role of autophagy in the maintenance of cell homeostasis is well documented and the existence of defects in the autophagic machinery has been largely described in diseases and aging. Determining the alterations occurring in the many forms of autophagy that coexist in cells and tissues remains complicated, as this cellular process is highly dynamic in nature and can vary from organ to organ in the same individual. Although autophagy is extensively studied, its functioning in different tissues and its links with other biological processes is still poorly understood. Several assays have been developed to monitor autophagy activity in vitro, ex vivo, and in vivo, based on different markers, the use of various inhibitors and activators, and distinct techniques. This review emphasizes the methods applied to measure (macro-)autophagy in tissue samples and in vivo via a protein, which centrally intervenes in the autophagy pathway, the microtubule-associated protein 1A/1B-light chain 3 (MAP1LC3), which is the most widely used marker and the first identified to associate with autophagosomal structures. These approaches are presented and discussed in terms of pros and cons. Some recommendations are provided to improve the reliability of the interpretation of results.
几十年来，人们在提高对细胞内降解系统的认识方面做出了巨大努力，尤其是在自噬领域。自噬在维持细胞内稳态中的作用已经得到了很好的证明，并且在疾病和衰老中已经广泛描述了自噬机制的缺陷。确定细胞和组织中同时存在的多种形式的自噬所发生的变化仍然很复杂，因为这种细胞过程本质上是高度动态的，并且在同一个体的不同器官中可能会有所不同。尽管自噬已经得到了广泛的研究，但它在不同组织中的功能及其与其他生物学过程的联系仍知之甚少。已经开发了几种基于不同标志物的方法来在体外、离体和体内监测自噬活性，使用各种抑制剂和激活剂以及不同的技术。这篇综述强调了应用于通过一种蛋白质测量（大）自噬的方法，该蛋白质在自噬途径中起着核心作用，微管相关蛋白 1A/1B-轻链 3（MAP1LC3），这是最广泛使用的标志物，也是第一个与自噬体结构相关的标志物。这些方法根据优缺点进行了介绍和讨论。提供了一些建议来提高结果解释的可靠性。
