The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes
The dynamics of autophagy visualized in live cells - From autophagosome formation to fusion with endo/lysosomes
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DOI：
10.4161/auto.1.1.1495
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发表时间：
2005-04-01
期刊：
影响因子：
13.3
通讯作者：
Tolkovsky, Aviva M.
中科院分区：
文献类型：
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作者：
Bampton, Edward T. W.;Goemans, Christoph G.;Tolkovsky, Aviva M.
自噬与一系列疾病有关，因此备受关注。然而，由于缺乏合适的标记物，实时成像自噬受到了阻碍。我们比较了广泛用作自噬体标记物单丹磺酰尸胺和 Atg8 同源物 LC3 的潜力，通过荧光显微镜跟踪自噬，同时使用 EGFP-CD63 同时标记晚期内体和溶酶体。单丹磺酰尸胺仅标记酸性 CD63 阳性区室，以响应各种活细胞或固定后细胞中的一系列自噬诱导剂，在自噬体形成被禁用的 atg5(+/+) 和 atg5(-/-) MEF 中染色相同。单丹磺酰尸胺染色与 LysoTracker Red、LAMP-1 或 LAMP-2 的染色基本上没有区别。相比之下，60-90% 的 EGFP-LC3 阳性点状细胞器不与 LAMP-1/LAMP-2/CD63 共定位，并且单丹磺酰尸胺阴性，而与 LAMP-1/LAMP-2/CD63 共定位的 EGFP-LC3 点状细胞器也是单丹磺酰尸胺阳性。因此，单丹磺酰尸胺与酸性区室的其他标记物没有什么不同，并且它不能用于跟踪自噬体的形成。相比之下，mRFP-LC3 标记的自噬体与 EGFP-CD63 阳性内体和溶酶体的融合以及 EGFP-LC3 和 EGFP-CD63 阳性区室对 dsRed 标记线粒体的隔离可以实时可视化。此外，通过免疫印迹追踪并通过[H-3]乙醇胺标记验证的EGFP-LC3-I (45 kDa)到EGFP-LC3-II (43 kDo)的转变揭示了对自噬体稳态动力学的新见解，包括在细胞凋亡之前由凋亡诱导剂星形孢菌素快速激活自噬。使用荧光 LC3 和反荧光内体/溶酶体蛋白显然可以让整个自噬过程进行高保真度的活细胞成像。
Autophagy has been implicated in a range of disorders and hence is of major interest. However, imaging outophagy in real time has been hampered by lack of suitable markers. We have compared the potential of monodansylcadaverine, widely used as an autophagosomal marker, and the Atg8 homologue LC3, to follow outophagy by fluorescence microscopy whilst labelling late endosomes and lysosomes simultaneously using EGFP-CD63. Monodansylcadaverine labelled only acidic CD63-positive compartments in response to a range of autophagic inducers in various live or post-fixed cells, staining being identical in atg5(+/+) and atg5(-/-) MEFs in which autophagosome formation is disabled. Monodansylcadaverine staining was essentially indistinguishable from that of LysoTracker Red, LAMP-1 or LAMP-2. In contrast, 60-90% of EGFP-LC3-positive punctate organelles did not colocalise with LAMP-1/LAMP-2/CD63 and were monodansylcadaverine-negative while EGFP-LC3 puncta that did colocalise with LAMP-1/LAMP-2/CD63 were also monodansylcadaverine-positive. Hence monodansylcadaverine is no different from other markers of acidic compartments and it cannot be used to follow autophagosome formation. In contrast, fusion of mRFP-LC3-labelled autophagosomes with EGFP-CD63-positive endosomes and lysosomes and sequestration of dsRed-labelled mitochondria by EGFP-LC3- and EGFP-CD63-positive compartments could be visualized in real time. Moreover, transition of EGFP-LC3-I (45 kDa) to EGFP-LC3-II (43 kDo)-traced by immunoblotting and verified by [H-3]ethanolamine labelling-revealed novel insights into the dynamics of autophagosome homeostasis, including the rapid activation of autophagy by the apoptotic inducer staurosporine prior to apoptosis proper. Use of fluorescent LC3 and a counter-fluorescent endosomal/lysosomal protein clearly allows the entire autophagic process to be followed by live cell imaging with high fidelity.