Kimura Shunsuke, Noda Takeshi, Yoshimori Tamotsu
Research Institute for Microbial Diseases, Osaka University, Osaka, Japan.
Autophagy. 2007 Sep-Oct;3(5):452-60. doi: 10.4161/auto.4451. Epub 2007 May 21.
During the process of autophagy, autophagosomes undergo a maturation process consisting of multiple fusions with endosomes and lysosomes, which provide an acidic environment and digestive function to the interior of the autophagosome. Here we found that a fusion protein of monomeric red-fluorescence protein and LC3, the most widely used marker for autophagosomes, exhibits a quite different localization pattern from that of GFP-LC3. GFP-LC3 loses fluorescence due to lysosomal acidic and degradative conditions but mRFP-LC3 does not, indicating that the latter can label the autophagic compartments both before and after fusion with lysosomes. Taking advantage of this property, we devised a novel method for dissecting the maturation process of autophagosomes. mRFP-GFP tandem fluorescent-tagged LC3 (tfLC3) showed a GFP and mRFP signal before the fusion with lysosomes, and exhibited only the mRFP signal subsequently. Using this method, we provided evidence that overexpression of a dominant negative form of Rab7 prevented the fusion of autophagosomes with lysosomes, suggesting that Rab7 is involved in this step. This method will be of general utility for analysis of the autophagosome maturation process.
在自噬过程中，自噬体经历一个成熟过程，该过程包括与内体和溶酶体的多次融合，这些细胞器为自噬体内部提供了酸性环境和消化功能。在此，我们发现单体红色荧光蛋白与自噬体最常用的标记物LC3的融合蛋白，其定位模式与GFP-LC3有很大不同。GFP-LC3在溶酶体酸性和降解条件下会失去荧光，但mRFP-LC3不会，这表明后者可以在与溶酶体融合之前和之后标记自噬区室。利用这一特性，我们设计了一种剖析自噬体成熟过程的新方法。mRFP-GFP串联荧光标记的LC3（tfLC3）在与溶酶体融合之前显示GFP和mRFP信号，随后仅显示mRFP信号。使用这种方法，我们提供了证据表明Rab7显性负性形式的过表达会阻止自噬体与溶酶体的融合，这表明Rab7参与了这一步骤。该方法将普遍用于分析自噬体成熟过程。
