Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within subcellular compartments and allows the interrogation of known molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images, limiting the type of information that they reveal. Here, we describe an approach, termed “conditional colocalization analysis,” for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We benchmark the approach and showcase its application to investigate receptor-downstream adaptor colocalization relationships in the context of functionally relevant plasma membrane locations. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.

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