ContactJ: Characterization of lipid droplet-mitochondrial contacts using fluorescence microscopy and image analysis

Abstract

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and together with mitochondria key regulators of cell bioenergetics. LDs communicate with mitochondria and other organelles forming “metabolic synapse” contacts to ensure that lipid supply occurs where and when necessary. Although transmission electron microscopy analysis allows an accurate and precise analysis of contacts, the characterization of a large number of cells and conditions can become a long-term process. In order to extend contact analysis to hundreds of cells and multiple conditions, we have combined confocal fluorescence microscopy with advanced image analysis methods. In this work, we have developed the ImageJ macro script ContactJ, a novel and straight image analysis method to identify and quantify contacts between LD and mitochondria in fluorescence microscopy images allowing the automatic analysis. This image analysis workflow combines colocalization and skeletonization methods, enabling the quantification of LD-mitochondria contacts together with a complete characterization of organelles and cellular parameters. The correlation and normalization of these parameters contribute to the complex description of cell behavior under different experimental energetic states. ContactJ is available here: https://doi.org/10.5281/zenodo.5810874

Insights Into the Biogenesis and Emerging Functions of Lipid Droplets From Unbiased Molecular Profiling Approaches

“…Biochemical enrichment and proteomic analyses of membranes associated with LDs recently identified the outer mitochondrial membrane MIGA2 as a tether between LDs and mitochondria, also interacting with the VAP-A/B ER protein; MIGA2-mediated tethering facilitates TG synthesis from non-lipid precursors, linking energy status in the cell to LD growth ( Freyre et al, 2019 ). Recently, routines based on skeletonization-aided mitochondrial segmentation and statistics-assisted colocalization analysis have been developed to accurately estimate LD-mitochondria apposition from standard confocal images in mammalian cells; these workflows allow for the description of these contacts in 3D, and constitute a potential platform to explore the regulation of LD-mitochondria contacts in metazoans ( Martn et al, 2022 ).…”