A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. If prey proteins can bind to bait protein, their interactions are demonstrated by localizing endosome of prey proteins. Here, I verify the poof-of-concept with FKBP-FRB interactions in response to rapamycin and confirmed Cdc42-WASP interactions as a case of constitutive P-P interactions. Moreover this method is capable to detect the interaction of protein-chemical inhibitor like DHFR and MTX, a popular anticancer drug. Because endosome localization seemed like total eclipse, this method was named as ECLPISE, Emerging CircLes of Induced Proteins at Specific Endosomes. Owing to simplicity of ECLIPSE, it is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. I visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. I observed that CaMKK1 and CaMKIIα bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
A new visualizing method was developed for monitoring protein-protein (P-P) interactions in live mammalian cells. P-P interactions are visualized by directing localization of a bait protein to endosomes. If prey proteins can bind to bait protein, their interactions are demonstrated by localizing endosome of prey proteins. Here, I verify the poof-of-concept with FKBP-FRB interactions in response to rapamycin and confirmed Cdc42-WASP interactions as a case of constitutive P-P interactions. Moreover this method is capable to detect the interaction of protein-chemical inhibitor like DHFR and MTX, a popular anticancer drug. Because endosome localization seemed like total eclipse, this method was named as ECLPISE, Emerging CircLes of Induced Proteins at Specific Endosomes. Owing to simplicity of ECLIPSE, it is sufficiently robust to analyze signal-dependent P-P interactions such as calcium-dependent protein interactions. I visualized interactions between activated calmodulin and calmodulin-binding proteins, and observed oscillatory interactions via time-lapse imaging. In addition, this new method can simultaneously monitor multiple P-P interactions in a single live cell, which allows comparison of interactions between several prey proteins and a single bait protein. I observed that CaMKK1 and CaMKIIα bind calmodulin with distinct binding affinities in live cell, which indicates that calcium signaling is fine-tuned by distinct activation patterns of CaM kinases. This method will enable investigation of cellular processes based on dynamic P-P interactions.
