Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy
Development of LC3/GABARAP sensors containing a LIR and a hydrophobic domain to monitor autophagy
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DOI：
10.15252/embj.201696315
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发表时间：
2017-04-13
期刊：
影响因子：
11.4
通讯作者：
Lee, Jin-A
中科院分区：
文献类型：
--
作者：
Lee, You-Kyung;Jun, Yong-Woo;Lee, Jin-A
巨自噬可以大量降解溶酶体中的胞浆成分。GFP/RFP-LC3/GABARAP的过表达通常用于监测自噬小体，这是自噬的一个标志，尽管它们的过度表达存在伪迹。在这里，我们开发了新的传感器，利用LC3相互作用区(LIR)和短疏水结构域(HYD)在自噬体处检测内源性LC3/GABARAP蛋白。在含有34个已知LC3结合蛋白的LIR基序的Hyd-LIR-GFP传感器中，使用TP53INP2的LIR基序的Hyd-LIR(TP)-GFP传感器可以检测到所有LC3/GABARAPs阳性的自噬小体。然而，Hyd-LIR(TP)-GFP以LIR依赖的方式优先定位于GABARAP/GABARAPL1阳性自噬小体。相反，使用来自FYCO1的LIR基序的hyd-LIR(FY)-GFP特异性地检测到LC3A/B阳性的自噬小体。Hyd-Lir(TP)-GFP和hyd-Lir(FY)-GFP在内源LC3/GABARAP存在的情况下有效地定位于自噬小体，而不影响自噬通量。这两个传感器在诱导有丝分裂时也能有效地定位到MitoTracker阳性的受损线粒体。Hyd-Lir(TP)-GFP允许在自噬诱导时对动态自噬小体进行活体成像。因此，这些新型的自噬传感器可广泛应用于自噬研究。
Macroautophagy allows for bulk degradation of cytosolic components in lysosomes. Overexpression of GFP/RFP-LC3/GABARAP is commonly used to monitor autophagosomes, a hallmark of autophagy, despite artifacts related to their overexpression. Here, we developed new sensors that detect endogenous LC3/GABARAP proteins at the autophagosome using an LC3-interacting region (LIR) and a short hydrophobic domain (HyD). Among HyD-LIR-GFP sensors harboring LIR motifs of 34 known LC3-binding proteins, HyD-LIR(TP)-GFP using the LIR motif from TP53INP2 allowed detection of all LC3/GABARAPs-positive autophagosomes. However, HyD-LIR(TP)-GFP preferentially localized to GABARAP/GABARAPL1-positive autophagosomes in a LIR-dependent manner. In contrast, HyD-LIR(Fy)-GFP using the LIR motif from FYCO1 specifically detected LC3A/B-positive autophagosomes. HyD-LIR (TP)-GFP and HyD-LIR(Fy)-GFP efficiently localized to autophagosomes in the presence of endogenous LC3/GABARAP levels and without affecting autophagic flux. Both sensors also efficiently localized to MitoTracker-positive damaged mitochondria upon mitophagy induction. HyD-LIR(TP)-GFP allowed live-imaging of dynamic autophagosomes upon autophagy induction. These novel autophagosome sensors can thus be widely used in autophagy research.