Quantitative Mapping of Organelle Positioning in Cultured Cells Using Semi-Automated Image Analysis Pipeline

Subcellular architecture is tightly controlled and contributes to the maintenance of cell homeostasis. Organelles are regulated in size, shape, number, and position, all of which respond to changes in the extracellular environment. Lysosomes are of particular interest as they integrate various functions in the cells (nutrient sensing, metabolism, cell migration, and adhesion), serving as signaling hubs. Their function is tightly linked to their subcellular position, and deregulation of lysosome homeostasis leads to several diseases, including cancer. Therefore, methods allowing precise analysis of organelle subcellular distribution can aid in fundamental, diagnostic, and therapeutic approaches. Here, we provide a versatile image analysis pipeline using ImageJ and CellProfiler. This workflow allows for the quantification of subcellular lysosome distribution in living and fixed melanoma cells, and it is applicable to other subcellular compartments and to various cell types.