Abstract
To elucidate the immediate effects (pharmacological action or toxicity) of drugs on cells, it is effective to capture the spatiotemporal dynamics of multiple signaling proteins as a network. Our novel analysis method, the PLOM-CON (Protein localization and modification-based covariation network) analysis, allows for the visualization of the changes in protein quantity, localization, and quality (such as post-translational modifications), in response to specific stimuli, as a network. This is achieved by quantification of immunofluorescence images and by generating a network that consists of edges that connects the proteins having a strong temporal correlation in feature quantities. In this study, we obtained covariation networks for ~50 proteins in insulin-stimulated rat liver H4IIEC3 cells. We discovered that Akt, a central molecule in insulin signaling, and its phosphorylated form p-Akt (Ser473) served as the hub of the network. Our analysis also revealed that the proteins involved in glycogen synthesis such as p-GSK3β showed temporal correlation mainly at the actin domain, a transiently formed structure in response to insulin. By inhibiting actin domain formation, we found that glycogen synthesis was also inhibited, which suggested that the actin domain is the site in which proteins accumulate to regulate glycogen synthesis in response to insulin signaling. In addition, the networks generated under conditions in which actin domain formation was inhibited by CK666 treatment (which recapitulates diabetic hepatocyte conditions) showed a significant change compared to the network in control conditions. Thus, PLOM-CON analysis not only aids in uncovering location-dependent protein function but also provides a means of verifying cellular state and drug toxicity by sensitively capturing cellular changes in response to various stimuli.
