人造细胞是基于动态分隔系统。因此,膜结合系统的重塑,如巨大的单层囊泡,正在寻找超越生物学研究的应用,设计细胞模拟结构。随着人造细胞在合成生物学中的突出地位,巨大的单层囊泡融合正迅速成为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,具有不同的效率和选择性。迄今为止,囊泡融合的表征依赖于巨大囊泡的小样本,手动检查或通过荧光测定法检查小型和大型单层囊泡的悬浮液。现在,融合产物的检测和表征的自动化对于这些融合方案的筛选和优化是必要的。为此,我们基于荧光团在膜上和囊泡腔中的共定位实施了融合测定。通过图像分割以最少的用户输入在单个隔室中评估荧光共定位,允许将该技术应用于高通量筛选。检测后,可以总结和可视化囊泡荧光和形态特性的统计信息,通过不同荧光通道的相关系数评估每个对象的脂质和含量转移。使用这个工具,我们报告并描述了氯化钠对磷脂酰胆碱巨囊泡的意外融合活性。孵育20小时后,可以检测到大多数囊泡中的脂质转移,而含量交换仅在约8%的囊泡中发生额外的刺激。
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.