ABSTRACT
Exocytosis 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. Understanding its mechanisms is crucial, as defects can compromise essential biological functions. The development of pH-sensitive optical reporters alongside fluorescence microscopy enables the assessment of individual vesicle exocytosis events at the cellular level. Manual annotation represents, however, a time-consuming task that is prone to selection biases and human operational errors. Here, we introduce ExoJ, an automated plugin based on Fiji/ImageJ2 software. ExoJ identifies user-defined genuine populations of exocytosis events, recording quantitative features including intensity, apparent size and duration. We designed ExoJ to be fully user-configurable, making it suitable for studying distinct forms of vesicle exocytosis regardless of the imaging quality. Our plugin demonstrates its capabilities by showcasing distinct exocytic dynamics among tetraspanins and vesicular SNARE protein reporters. Assessment of performance on synthetic data shows that ExoJ is a robust tool that is capable of correctly identifying exocytosis events independently of signal-to-noise ratio conditions. We propose ExoJ as a standard solution for future comparative and quantitative studies of exocytosis.
Footnotes
Author contributions
Conceptualization: J.L., L.D., P.B.; Methodology: J.L., F.J.V., L.D., P.B.; Software: J.L., P.B.; Validation: F.J.V.; L.D.; Formal analysis: P.B.; J.L.; Investigation: F.J.V., L.D., P.B.; Resources: J.L., F.J.V., T.G., L.D.; Data curation: J.L., P.B.; Writing - original draft: J.L., P.B.; Writing - review & editing: J.L., F.J.V., G.v.N., T.G., L.D., P.B.; Visualization: J.L., P.B.; Supervision: G.v.N., L.D., P.B.; Project administration: P.B.; Funding acquisition: G.v.N., L.D.
Funding
This work was supported by Agence Nationale de la Recherche (ANR; ANR-19-HBPR-0003, ANR-19-CE16-0012) and FLAGship European Research Area network (FLAG-ERA network; Sensei grant 19-CE16-0012-01) to L.D., by a European Molecular Biology Organization grant (EMBO ALTF 1383-2014), a Fondation ARC pour la Recherche sur le Cancer fellowship (PGA1RF20190208474) to F.J.V., a Fondation pour la Recherche Médicale grant (AJE20160635884) to G.v.N, and an Institut National Du Cancer grant (INCA no. 2019-125 PLBIO19-059) and ANR (ANR-20-CE18-0026-01) to F.J.V. and G.v.N.
Data availability
The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request. Time series including the one used in this report as well as five simulated movies are available in the Zenodo repository: https://doi.org/10.5281/zenodo.6610894 and https://doi.org/10.5281/zenodo.7595198. The latest version of ExoJ is available at https://www.project-exoj.com. and source code for ExoJ and for the generation of simulated movies is available at https://github.com/zs6e/excytosis-analyzer-plugin.
Special Issue
This article is part of the Special Issue ‘Imaging Cell Architecture and Dynamics’, guest edited by Lucy Collinson and Guillaume Jacquemet. See related articles at https://journals.biologists.com/jcs/issue/137/20.
