ExoJ: an ImageJ2/Fiji plugin for automated spatiotemporal detection of exocytosis

AbstractExocytosis is a dynamic physiological process that enables the release of biomolecules to the surrounding environment via the fusion of membrane compartments to the plasma membrane. Defects in exocytosis could compromise essential biological functions so that it is crucial to elucidate the fundamentals of the exocytosis mechanisms. The development of pH-sensitive optical reporters alongside Total Internal Reflection Fluorescence (TIRF) microscopy has greatly expanded the knowledge of exocytosis, allowing the assessment of individual vesicle exocytosis events on a single-cell basis. The amount of data generated by live-cell imaging approaches poses the need of an analytical tool to identify and quantify the dynamics of exocytic events with high accuracy from fluorescent time series. Manual annotation represents a time-consuming task, and prone to selection biases and human operational errors.In this work, we report the development of ExoJ, an ImageJ2/Fiji-based plugin to automate the detection of exocytic events in single-cell studies. The pipeline of ExoJ entirely relies on a set of user-customizable parameters to identify and further quantify features of exocytic events such as the intensity, the apparent size and the duration of the process. ExoJ proposes a user-friendly interface with multiple optional tools to visualize features for individual exocytic events and/or the whole cell population events. To illustrate the capacity of the plugin, we recorded exocytic events reported by several tetraspanins (TSPANs) and vesicular soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (v-SNAREs) proteins, coupled to pHluorin. Using ExoJ, we characterize the dynamics of the exocytic event population reported by these two families of proteins. We showcase a similar fusion intensity but significantly distinct dynamics among TSPAN subpopulation. In particular, TSPAN CD9-pHluorin subpopulation displays a fast and similar dynamics to v-SNARE populations.ExoJ is a user-friendly plugin to detect exocytic events in a supervised manner. We designed ExoJ to be fully user-controllable at each step of the pipeline. We argue the user-oriented plugin is a useful tool to search, monitor and quantify the dynamic aspect of content release during exocytosis.