Palmisano Nicholas J, Meléndez Alicia
Department of Biology, Queens College-CUNY, Flushing, New York 11367; The Graduate Center, The City University of New York, New York 10016.
Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot086496. doi: 10.1101/pdb.prot086496.
In yeast and mammalian cells, the autophagy protein Atg8/LC3 (microtubule-associated proteins 1A/1B light chain 3B encoded by MAP1LC3B) has been the marker of choice to detect double-membraned autophagosomes that are produced during the process of autophagy. A lipid-conjugated form of Atg8/LC3B is localized to the inner and outer membrane of the early-forming structure known as the phagophore. During maturation of autophagosomes, Atg8/LC3 bound to the inner autophagosome membrane remains in situ as the autophagosomes fuse with lysosomes. The nematode Caenorhabditis elegans is thought to conduct a similar process, meaning that tagging the nematode ortholog of Atg8/LC3-known as LGG-1-with a fluorophore has become a widely accepted method to visualize autophagosomes. Under normal growth conditions, GFP-modified LGG-1 displays a diffuse expression pattern throughout a variety of tissues, whereas, when under conditions that induce autophagy, the GFP::LGG-1 tag labels positive punctate structures, and its overall level of expression increases. Here, we present a protocol for using fluorescent reporters of LGG-1 coupled to GFP to monitor autophagosomes in vivo. We also discuss the use of alternative fluorescent markers and the possible utility of the LGG-1 paralog LGG-2.
在酵母和哺乳动物细胞中，自噬蛋白Atg8/LC3（由MAP1LC3B编码的微管相关蛋白1A/1B轻链3B）一直是检测自噬过程中产生的双膜自噬体的首选标志物。Atg8/LC3B的脂质共轭形式定位于早期形成结构（即吞噬泡）的内膜和外膜。在自噬体成熟过程中，当自噬体与溶酶体融合时，与自噬体内膜结合的Atg8/LC3会原位保留。线虫秀丽隐杆线虫被认为进行类似的过程，这意味着用荧光团标记Atg8/LC3的线虫直系同源物（即LGG-1）已成为可视化自噬体的广泛接受的方法。在正常生长条件下，绿色荧光蛋白（GFP）修饰的LGG-1在各种组织中呈现弥散性表达模式，而在诱导自噬的条件下，GFP::LGG-1标签标记阳性点状结构，且其整体表达水平增加。在此，我们展示了一种使用与GFP偶联的LGG-1荧光报告基因在体内监测自噬体的方案。我们还讨论了替代荧光标记物的使用以及LGG-1旁系同源物LGG-2的可能用途。
