Assessment of Non-canonical Functions of the Autophagy Proteins in LC3-Associated Phagocytosis and LC3-Associated Endocytosis.

Q4 Biochemistry, Genetics and Molecular Biology Methods in molecular biology Pub Date : 2024-07-25 DOI:10.1007/7651_2024_561

Luigi Mari, Emilio Boada-Romero, Zhenrui Li, Joelle Magné, Douglas R Green

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Abstract

The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.

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评估自噬蛋白在 LC3 相关吞噬和 LC3 相关内吞中的非规范功能

对自噬途径中的非规范功能的鉴定和表征揭示了复杂的细胞过程，包括LC3相关吞噬（LAP）和LC3相关内吞（LANDO）。这些现象在 ATG8 与单膜吞噬体和内体的结合中起着关键作用，揭示了自噬与细胞稳态之间的动态相互作用。在此，我们介绍了定性和定量评估 LAP 的详细方案，包括免疫荧光、流式细胞术和分离 LAP 体的 Western 印迹。此外，我们还概述了通过免疫荧光检测受体循环来评估 LANDO 的方案。本文介绍的方法可作为研究人员揭示 LAP 和 LANDO 复杂性的实用指南。本章通过提供分步骤的说明，以及对潜在挑战和优化策略的见解，旨在增强研究人员探索自噬蛋白这些非规范功能的能力。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics

CiteScore

2.00

自引率

0.00%

发文量

3536

期刊介绍： For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.