Quantification of Giant Unilamellar Vesicle Fusion Products by High-Throughput Image Analysis

Author:

Caliari Adriano12ORCID,Hanczyc Martin M.2,Imai Masayuki3,Xu Jian1ORCID,Yomo Tetsuya1ORCID

Affiliation:

1. Laboratory of Biology and Information Science, School of Life Sciences, East China Normal University, Shanghai 200062, China

2. 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

3. Department of Physics, Graduate School of Science, Tohoku University, 6-3 Aramaki, Aoba, Sendai 980-8578, Japan

Abstract

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.

Funder

National Key R&D Program of China, Synthetic Biology Research

European Horizon 2020 project ACDC

Publisher

MDPI AG

Subject

Inorganic Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Computer Science Applications,Spectroscopy,Molecular Biology,General Medicine,Catalysis

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