Giant Unilamellar Vesicles Fusion By Ionic Strength Gradients And Its Quantification

Membrane remodelling is a foundation of cellular biology thanks to which cells can respond accurately to complex environmental stimuli.As artificial cell research gains momentum and the models built under this framework become more and more complex,an increased demand for in-vitro biomimetic membrane remodelling methods is emerging.One of the most fundamental remodelling events is membrane fusion and division.They are the cornerstone of intracellular vesicle trafficking and cell division,and they show many potential applications in bottom-up synthetic biology,to deliver contents to specific compartments or to achieve growth-division cycles.Researchers developed several protocols for membrane fusion and division in vitro over the years.A bottleneck currently hindering progress in the direction of efficient and repeatable fusion division cycles is the high experimental heterogeneity of vesicles samples paired with the lack of tools to extract statistically significant information from vesicle experiments.To address this,an automated analysis pipeline for quantification of lipid exchange,content exchange,vesicle fluorescence and morphology is here developed.An Image J macro that segments vesicles based on their membrane fluorescence was used to readily analyse imaging data from widely available microscopy setups.Statistical information and colocalization among pairs of fluorescence channels is performed with an R script.The new method is compared quantitatively with imaging flow cytometry,representing one of the most advanced and reliable techniques to quantify vesicle fusion currently available.The analysis pipeline is then applied to the study of aggregated vesicles by ionic strength gradients of sodium chloride,assessing relevant properties to increase lipid exchange and content exchange,using the information obtained from the statistical analysis to guide future experimental steps and optimization attempts.The development of flexible tools for automated and unbiased analysis of vesicles population can further their application into complex and dynamical artificial cell models.The tools presented in this work provide means for straightforward analysis of microscopy data containing any number of fluorescence channels for both contents and membrane markers,with the ability to assess their interaction through colocalization metrics.Since this analysis routine lays on a minimal set of assumptions with robust detection,it could prove useful to research groups interested in assessing fluorescence redistribution in vesicles population upon specific stimuli.