Wang Shen, Ma Cong
Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
Biophys Rep. 2024 Feb 29;10(1):31-40. doi: 10.52601/bpr.2023.230017.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNAER) family proteins are the engines of most intra-cellular and exocytotic membrane fusion pathways (Jahn and Scheller 2006). Over the past two decades, liposome fusion has been proven to be a powerful tool to reconstruct physiological SNARE-mediated membrane fusion processes (Liu 2017). The reconstitution of the membrane fusion process not only provides direct evidence of the capability of the cognate SNARE complex in driving membrane fusion but also allows researchers to study the functional mechanisms of regulatory proteins in related pathways (Wickner and Rizo 2017). Heretofore, a variety of delicate methods for SNARE-mediated liposome fusion have been established (Bao 2018; Diao 2012; Duzgunes 2003; Gong 2015; Heo . 2021; Kiessling 2015; Kreye 2008; Kyoung 2013; Liu 2017; Scott 2003). Although technological advances have made reconstitution more physiologically relevant, increasingly elaborate experimental procedures, instruments, and data processing algorithms nevertheless hinder the non-experts from setting up basic SNARE-mediated liposome fusion assays. Here, we describe a low-cost, timesaving, and easy-to-handle protocol to set up a foundational SNARE-mediated liposome fusion assay based on our previous publications (Liu 2023; Wang and Ma 2022). The protocol can be readily adapted to assess various types of SNARE-mediated membrane fusion and the actions of fusion regulators by using appropriate alternative additives (.., proteins, macromolecules, chemicals, .). The total time required for one round of the assay is typically two days and could be extremely compressed into one day.
可溶性N - 乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）家族蛋白是大多数细胞内和胞吐膜融合途径的驱动因素（扬和谢勒，2006年）。在过去的二十年中，脂质体融合已被证明是重建生理SNARE介导的膜融合过程的有力工具（刘，2017年）。膜融合过程的重建不仅为同源SNARE复合体驱动膜融合的能力提供了直接证据，还使研究人员能够研究相关途径中调节蛋白的功能机制（维克纳和里佐，2017年）。迄今为止，已经建立了多种用于SNARE介导的脂质体融合的精细方法（鲍，2018年；刁，2012年；杜兹古内斯，2003年；龚，2015年；何，2021年；基斯林，2015年；克雷耶，2008年；金，2013年；刘，2017年；斯科特，2003年）。尽管技术进步使重建更符合生理学，但日益复杂的实验程序、仪器和数据处理算法仍然阻碍非专业人员建立基本的SNARE介导的脂质体融合测定。在此，我们基于我们之前的出版物（刘，2023年；王和马，2022年）描述了一种低成本、省时且易于操作的方案，以建立基础的SNARE介导的脂质体融合测定。该方案可以很容易地通过使用适当的替代添加剂（如蛋白质、大分子、化学物质等）来评估各种类型的SNARE介导的膜融合以及融合调节剂的作用。一轮测定所需的总时间通常为两天，并且可以极快地压缩到一天。
