Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3
Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3
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DOI：
10.4161/auto.4451
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发表时间：
2007-09-01
期刊：
影响因子：
13.3
通讯作者：
Yoshimori, Tamotsu
中科院分区：
文献类型：
--
作者：
Kimura, Shunsuke;Noda, Takeshi;Yoshimori, Tamotsu
在自噬过程中，自噬体经历了一个成熟的过程，包括与包封体和溶酶体的多次融合，为自噬体内部提供酸性环境和消化功能。在这里，我们发现单体红色荧光蛋白和LC 3的融合蛋白，最广泛使用的自噬体标记，表现出与GFP-LC 3完全不同的定位模式。GFP-LC 3由于溶酶体酸性和降解条件而失去荧光，但mRFP-LC 3不会，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，我们设计了一种新的方法来解剖自噬体的成熟过程。mRFP-GFP串联荧光标记的LC 3（tfLC 3）在与溶酶体融合之前显示GFP和mRFP信号，并且随后仅显示mRFP信号。使用这种方法，我们提供的证据表明，显性负性形式的Rab 7的过表达阻止了自噬体与溶酶体的融合，表明Rab 7参与了这一步骤。这种方法将是通用的自噬体成熟过程的分析。
During the process of autophogy, autophagosomes undergo a maturation process consisting of multiple fusions with enclosomes 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.