A reporter cell system to monitor autophagy based on p62/SQSTM1
A reporter cell system to monitor autophagy based on p62/SQSTM1
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DOI：
10.4161/auto.6.6.12510
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发表时间：
2010-08-16
期刊：
影响因子：
13.3
通讯作者：
Bjorkoey, Geir
中科院分区：
文献类型：
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作者：
Larsen, Kenneth Bowitz;Lamark, Trond;Bjorkoey, Geir
大自噬（Macroautophagy，以下简称自噬）是一种将细胞溶质成分分离并转运至溶酶体进行降解的分解代谢途径。最近，已经鉴定了自噬受体，如p62/SQSTM 1和NBR 1，其将自噬货物与位于形成自噬膜上的ATG 8/MAP 1-LC 3/GABARAP家族成员物理连接。为了鉴定影响自噬的条件或化合物，需要有效报告自噬通量的细胞系统。在这里，我们描述了报告细胞系统的基础上诱导表达的GFP-p62，GFP-NBR 1或GFP-LC 3B。在启动子关闭后，通过活细胞的流式细胞术跟踪融合蛋白的降解。所有三种融合蛋白通过自噬以基础速率降解。令人惊讶的是，三种报告融合蛋白的基础降解速率不同。GFP-LC 3B是最稳定的蛋白质。GFP-NBR 1在基础条件下降解最有效，而GFP-p62的降解显示出对氨基酸饥饿的最强响应。发现GFP-p62在所测试的报告基因中表现最好。通过流式细胞术的自噬通量的单细胞分析允许估计异质细胞群体。使用显性负性ULK 1激酶的瞬时过表达和siRNA介导的LC 3B敲低来抑制GFP-p62的自噬降解，证明了这种方法的可行性。可诱导的GFP-p62细胞系统允许通过几种方法进行定量，并且将用于筛选影响自噬速率的化合物或条件。自噬的诱导剂可以使用丰富的培养基鉴定，而抑制剂在饥饿条件下鉴定。
Macroautophagy (hereafter referred to as autophagy) is a catabolic pathway to isolate and transport cytosolic components to the lysosome for degradation. Recently, autophagy receptors, like p62/SQSTM1 and NBR1, which physically link autophagic cargo to ATG8/MAP1-LC3/GABARAP family members located on the forming autophagic membranes, have been identified. To identify conditions or compounds that affect autophagy, cell systems that efficiently report on autophagic flux are required. Here we describe reporter cell systems based on induced expression of GFP-p62, GFP-NBR1 or GFP-LC3B. The degradation of the fusion proteins was followed after promoter shut-off by flow cytometry of live cells. All three fusion proteins were degraded at a basal rate by autophagy. Surprisingly, the basal degradation rate varied for the three reporter fusion proteins. GFP-LC3B was the most stable protein. GFP-NBR1 was most efficiently degraded under basal conditions while degradation of GFP-p62 displayed the strongest response to amino acid starvation. GFP-p62 was found to perform the best of the tested reporters. Single cell analysis of autophagic flux by flow cytometry allows estimates of heterogeneous cell populations. The feasibility of this approach was demonstrated using transient overexpression of a dominant negative ULK1 kinase and siRNA-mediated knockdown of LC3B to inhibit autophagic degradation of GFP-p62. The inducible GFP-p62 cell system allows quantification by several approaches and will be useful in screening for compounds or conditions that affect the rate of autophagy. Inducers of autophagy can be identified using rich medium whereas inhibitors are identified under starvation conditions.