Signal-Retaining Autophagy Indicator as a Quantitative Imaging Method for ER-Phagy

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Autophagy is a key cellular process for homeostasis, involving lysosomal degradation of cytoplasmic components.
• Measuring autophagy flux is vital but current assays are often complex or lack sensitivity.
• Endoplasmic reticulum-selective autophagy (ER-phagy) is crucial for ER homeostasis but remains poorly understood.

Purpose of the Study:

• To validate the signal-retaining autophagy indicator (SRAI) probe for monitoring ER-phagy flux.
• To assess SRAI's utility for studying general and cargo receptor-specific ER-phagy.
• To establish a high-throughput method for quantifying ER-phagy.

Main Methods:

• Validation of the fixable fluorescent probe SRAI for ER-phagy detection.
• Application of SRAI to study ER-phagy involving cargo receptors FAM134B, FAM134C, TEX264, and CCPG1.
• Development of a protocol for automated microscopy and high-throughput analysis of autophagic flux.

Main Results:

• SRAI is confirmed as a versatile, sensitive, and convenient probe for ER-phagy.
• The probe enables monitoring of both general ER-phagy and specific ER-phagy pathways.
• A detailed protocol for automated, high-throughput quantification of ER-phagy flux was established.

Conclusions:

• SRAI provides a reliable and convenient tool for measuring ER-phagy.
• This probe facilitates deeper understanding of ER homeostasis and related cellular processes.
• The developed high-throughput method advances ER-phagy research.