Detecting and Controlling Dye Effects in Single-Virus Fusion Experiments
Detecting and Controlling Dye Effects in Single-Virus Fusion Experiments
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DOI：
10.1016/j.bpj.2019.06.022
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发表时间：
2019-08-06
影响因子：
3.4
通讯作者：
Kasson, Peter M.
中科院分区：
文献类型：
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作者：
Rawle, Robert J.;Giraldo, Ana M. Villamil;Kasson, Peter M.
荧光染料去猝灭测定提供了一种强大且通用的方法来监测膜融合事件。它们已用于批量测定，用于测量活细胞中的单个事件，以及用于脂质体、病毒和细胞融合过程的融合动力学的详细分析；然而，所使用的染料也有可能干扰膜融合。在这里，使用流感膜融合的单病毒测量，我们表明荧光膜探针可以以染料和照明依赖性方式改变脂质混合的效率和动力学。 R18 是一种常用于监测膜间脂质混合的染料，特别容易受到这些影响，而德克萨斯红则不太敏感。 R18 进一步以光照依赖性方式与病毒蛋白发生光缀合，这与其病毒融合的失活相关。这些结果证明了荧光探针如何干扰生物活性的测量，并提供数据和确定最小干扰测量条件的方法。
Fluorescent dye-dequenching assays provide a powerful and versatile means to monitor membrane fusion events. They have been used in bulk assays, for measuring single events in live cells, and for detailed analysis of fusion kinetics for liposomal, viral, and cellular fusion processes; however, the dyes used also have the potential to perturb membrane fusion. Here, using single-virus measurements of influenza membrane fusion, we show that fluorescent membrane probes can alter both the efficiency and the kinetics of lipid mixing in a dye- and illumination-dependent manner. R18, a dye that is commonly used to monitor lipid mixing between membranes, is particularly prone to these effects, whereas Texas Red is somewhat less sensitive. R18 further undergoes photoconjugation to viral proteins in an illumination-dependent manner that correlates with its inactivation of viral fusion. These results demonstrate how fluorescent probes can perturb measurements of biological activity and provide both data and a method for determining minimally perturbative measurement conditions.