In situ Investigation of dynamic protein-protein interactions in live cells via Magnetic Co-concentration (MCoC)

University Of California, San Francisco, San Francisco CA

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Abstract

ABSTRACT Monitoring dynamic protein-protein interactions (PPIs) in situ has been a topic of interest due to the importance of PPIs in cell signaling regulation and implications in therapeutic interventions. In this proposal, we introduce a new PPI method based on nanotechnology-assisted magnetic co-concentration (MCo-C). The key component of this technology that enables this PPI investigation method is the ability of the nanoprobe-microtweezer system to magnetically co-concentrate the targeted protein and its interacting partners, enabling significant amplification of weak and transient PPI signals to be detectable with robust signals. MCo-C can induce signal amplifications more than 100 times (in vitro; >10 times for live cells) and hence promises to identify weak PPIs that are not detectable with existing PPI methods. Additionally, this method does not involve any harsh post- treatments (e.g. cell lysis, chemical membrane crosslinking) and hence allows in situ real-time monitoring of dynamic PPIs in live cells during the course of cell signaling. To demonstrate the capacity of MCo-C, we will first carry out PPI assays in in vitro model reaction systems using DNA duplex formation/dissociation. Once the system is optimized, as initial studies, we propose to investigate PPIs of Notch with potential interaction partners, including Notch (homodimerization), cis-ligand (e.g. Dll4), and ?-catenin. Ultimately, we aim to provide a platform technology for the systematic investigation of PPIs, accelerating our understanding of the dynamic regulation of interactome networks in cell signaling.