Assessment and Optimization of Autophagy Monitoring Methods in Arabidopsis Roots Indicate Direct Fusion of Autophagosomes with Vacuoles
Assessment and Optimization of Autophagy Monitoring Methods in Arabidopsis Roots Indicate Direct Fusion of Autophagosomes with Vacuoles
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DOI：
10.1093/pcp/pcu041
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发表时间：
2014-04-01
影响因子：
4.9
通讯作者：
Yoshimoto, Kohki
中科院分区：
文献类型：
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作者：
Merkulova, Ekaterina A.;Guiboileau, Anne;Yoshimoto, Kohki
自噬是一种降解途径，在遇到应激条件时或在特定的发育过程中回收细胞材料。为了更好地了解自噬的生理作用，合适的监测方法是非常重要的。在哺乳动物和酵母中，自噬的监测通常使用绿色荧光蛋白(GFP)-ATG8融合蛋白或嗜酸性染料，如单丹西卡维林(MDC)和溶血追踪红(LTR)。为了评估这些监测方法，我们在这里通过诱导拟南芥根部的自噬来检验这些系统，作为监测植物自噬的模型。在碳氮缺乏的条件下，GFP-ATG8标记的囊泡的数量和大小在几个小时后逐渐增加，然后逐渐减少到高于实验开始前的水平。我们还观察到，在Wortmannin处理后，GFP-ATG8标记的囊泡消失，Wortmannin是一种磷脂酰肌醇3-激酶抑制剂，被称为自噬抑制剂，表明GFP-ATG8转基因系构成了监测自噬的良好方法。将这些数据与MDC和LTR染色的植物进行了比较。在自噬诱导下，根中的小细胞器未见明显的MDC/LTR染色。最终只在根尖观察到一些小泡，但共定位实验以及自噬缺陷ATG突变体的实验提供了证据，证明这些结构位于液泡中，而不是明显的自噬小体和/或自溶酶体。因此，在利用MDC/LTR监测自噬时，应格外小心。此外，我们的观察强烈表明，在拟南芥根中，自噬小体直接与液泡融合。
Autophagy is a degradation pathway that recycles cell materials upon encountering stress conditions or during specific developmental processes. To better understand the physiological roles of autophagy, proper monitoring methods are very important. In mammals and yeast, monitoring of autophagy is often performed with a green fluorescent protein (GFP)-ATG8 fusion protein or with acidotropic dyes such as monodansylcadaverine (MDC) and LysoTracker Red (LTR). To evaluate these monitoring methods, here we examined these systems by inducing autophagy in Arabidopsis thaliana roots as a model for monitoring autophagy in planta. Under carbon- and nitrogen-starved conditions, the number and size of vesicles labeled by GFP-ATG8 was increased for several hours and then gradually decreased to a level higher than that observed before the start of the experiment. We also observed the disappearance of GFP-ATG8-labeled vesicles after treatment with wortmannin, a phosphatidylinositol 3-kinase inhibitor known as an autophagy inhibitor, showing that the GFP-ATG8 transgenic line constitutes an excellent method for monitoring autophagy. These data were compared with plants stained with MDC and LTR. There was no appreciable MDC/LTR staining of small organelles in the root under the induction of autophagy. Some vesicles were eventually observed in the root tip only, but co-localization experiments, as well as experiments with autophagy-deficient atg mutants, provided the evidence that these structures were located in the vacuole and were not manifestly autophagosomes and/or autolysosomes. Extreme caution should therefore be used when monitoring autophagy with the aid of MDC/LTR. Additionally, our observations strongly suggest that autophagosomes fuse directly to vacuoles in Arabidopsis roots.