Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates.
Quantitative analysis of autophagic flux by confocal pH-imaging of autophagic intermediates.
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DOI：
10.1080/15548627.2015.1084455
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发表时间：
2015
期刊：
影响因子：
13.3
通讯作者：
De Spirito M
中科院分区：
文献类型：
--
作者：
Maulucci G;Chiarpotto M;Papi M;Samengo D;Pani G;De Spirito M
尽管已经开发了许多技术来监测自噬并探测其细胞功能，但这些方法无法足够详细地评估自噬过程，并且受到复杂的实验设置和/或系统误差的限制。在这里，我们开发了一种方法，通过使用探针 mRFP-GFP-LC3B 作为比例 pH 传感器，根据上下文对自噬中间体的数量和 pH 进行成像。该信息通过 AIPD（每个细胞自噬中间体数量的 pH 分布）进行功能表达。 AIPD 分析揭示了中间体如何以 4.5-6.5 范围内的连续 pH 分布为特征，因此可以用一组更复杂的状态来描述，而不是通常的双相状态（自噬体和自溶酶体）。 AIPD 形状和幅度对药物或环境状态引起的自噬途径的变化敏感，并允许通过检索自噬中间体的浓度来定量估计自噬通量。
Although numerous techniques have been developed to monitor autophagy and to probe its cellular functions, these methods cannot evaluate in sufficient detail the autophagy process, and suffer limitations from complex experimental setups and/or systematic errors. Here we developed a method to image, contextually, the number and pH of autophagic intermediates by using the probe mRFP-GFP-LC3B as a ratiometric pH sensor. This information is expressed functionally by AIPD, the pH distribution of the number of autophagic intermediates per cell. AIPD analysis reveals how intermediates are characterized by a continuous pH distribution, in the range 4.5–6.5, and therefore can be described by a more complex set of states rather than the usual biphasic one (autophagosomes and autolysosomes). AIPD shape and amplitude are sensitive to alterations in the autophagy pathway induced by drugs or environmental states, and allow a quantitative estimation of autophagic flux by retrieving the concentrations of autophagic intermediates.