Cancer Research UK Scotland Centre, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XR, UK.
MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK.
Cells. 2023 Apr 11;12(8):1134. doi: 10.3390/cells12081134.
Autophagy is an intracellular lysosomal degradation pathway by which cytoplasmic cargoes are removed to maintain cellular homeostasis. Monitoring autophagy flux is crucial to understand the autophagy process and its biological significance. However, assays to measure autophagy flux are either complex, low throughput or not sensitive enough for reliable quantitative results. Recently, ER-phagy has emerged as a physiologically relevant pathway to maintain ER homeostasis but the process is poorly understood, highlighting the need for tools to monitor ER-phagy flux. In this study, we validate the use of the signal-retaining autophagy indicator (SRAI), a fixable fluorescent probe recently generated and described to detect mitophagy, as a versatile, sensitive and convenient probe for monitoring ER-phagy. This includes the study of either general selective degradation of the endoplasmic reticulum (ER-phagy) or individual forms of ER-phagy involving specific cargo receptors (e.g., FAM134B, FAM134C, TEX264 and CCPG1). Crucially, we present a detailed protocol for the quantification of autophagic flux using automated microscopy and high throughput analysis. Overall, this probe provides a reliable and convenient tool for the measurement of ER-phagy.
自噬是一种细胞内溶酶体降解途径，通过该途径可以清除细胞质货物以维持细胞内稳态。监测自噬通量对于理解自噬过程及其生物学意义至关重要。然而，用于测量自噬通量的测定法要么复杂、高通量低，要么不够灵敏，无法获得可靠的定量结果。最近，内质网自噬作为维持内质网稳态的一种生理相关途径而出现，但该过程知之甚少，这突出了需要有工具来监测内质网自噬通量。在这项研究中，我们验证了使用信号保留自噬指示剂 (SRAI) 的合理性，SRAI 是一种最近生成并描述用于检测线粒体自噬的可固定荧光探针，作为一种用于监测内质网自噬的通用、灵敏和方便的探针。这包括研究内质网的一般选择性降解（内质网自噬）或涉及特定货物受体的内质网自噬的个别形式（例如， FAM134B、FAM134C、TEX264 和 CCPG1）。至关重要的是，我们提出了一种使用自动化显微镜和高通量分析进行自噬通量定量的详细方案。总体而言，该探针为内质网自噬的测量提供了一种可靠且方便的工具。
