Laboratory of Biology and Information Science, School of Life Sciences, East China Normal University, Shanghai 200062, China.
Laboratory for Artificial Biology, Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, Polo Scientifico e Tecnologico Fabio Ferrari, Polo B, Via Sommarive 9, 38123 Povo, Italy.
Int J Mol Sci. 2023 May 4;24(9):8241. doi: 10.3390/ijms24098241.
Artificial cells are based on dynamic compartmentalized systems. Thus, remodeling of membrane-bound systems, such as giant unilamellar vesicles, is finding applications beyond biological studies, to engineer cell-mimicking structures. Giant unilamellar vesicle fusion is rapidly becoming an essential experimental step as artificial cells gain prominence in synthetic biology. Several techniques have been developed to accomplish this step, with varying efficiency and selectivity. To date, characterization of vesicle fusion has relied on small samples of giant vesicles, examined either manually or by fluorometric assays on suspensions of small and large unilamellar vesicles. Automation of the detection and characterization of fusion products is now necessary for the screening and optimization of these fusion protocols. To this end, we implemented a fusion assay based on fluorophore colocalization on the membranes and in the lumen of vesicles. Fluorescence colocalization was evaluated within single compartments by image segmentation with minimal user input, allowing the application of the technique to high-throughput screenings. After detection, statistical information on vesicle fluorescence and morphological properties can be summarized and visualized, assessing lipid and content transfer for each object by the correlation coefficient of different fluorescence channels. Using this tool, we report and characterize the unexpected fusogenic activity of sodium chloride on phosphatidylcholine giant vesicles. Lipid transfer in most of the vesicles could be detected after 20 h of incubation, while content exchange only occurred with additional stimuli in around 8% of vesicles.
人工细胞基于动态分隔系统。因此，膜结合系统的重塑，如巨大的单层囊泡，除了在生物学研究之外，还发现了在工程细胞模拟结构方面的应用。随着人工细胞在合成生物学中的重要性日益增加，巨大的单层囊泡融合正在迅速成为一个必不可少的实验步骤。已经开发了几种技术来完成这一步骤，其效率和选择性各不相同。迄今为止，囊泡融合的表征依赖于巨囊泡的小样本，要么手动检查，要么通过悬浮在小和大单层囊泡中的荧光测定法进行检查。为了筛选和优化这些融合方案，现在需要对融合产物的检测和表征进行自动化。为此，我们实施了一种基于荧光团在膜和囊泡腔室内共定位的融合测定法。通过最小化用户输入的图像分割，对单个隔室中的荧光共定位进行评估，从而允许将该技术应用于高通量筛选。在检测之后，可以对囊泡荧光和形态特性的统计信息进行总结和可视化，通过不同荧光通道的相关系数评估每个对象的脂质和内容转移。使用这个工具，我们报告并描述了氯化钠对磷脂酰胆碱巨大囊泡的意外融合活性。在孵育 20 小时后，可以检测到大多数囊泡中的脂质转移，而只有在大约 8%的囊泡中，通过额外的刺激才会发生内容交换。
