Live Cell Monitoring of Separase Activity, a Key Enzymatic Reaction for Chromosome Segregation, with Chimeric Fret-Based Molecular Sensor upon Cell Cycle Progression

Abstract

Separase is a key cysteine protease to separate sister chromatids through digestion of cohesion ring that inhibits chromosome segregation as trigger of metaphase–anaphase transition in eukaryotes and highly regulated its activity by binding with securin and cyclinB-CDK1 complex. Those bindings prevent proteolytic activity of separase until the onset of anaphase. Chromosome missegregation and aneuploidy are frequently observed in malignancies. However, there are some difficulties for biochemical examinations due to instability of separase in vitro and a few spatiotemporal resolution approaches monitoring live separase activity throughout mitotic processes. Here we have developed FRET-based molecular sensors including GFP variants with separase cleavable sequences as donor and covalently attached fluorescent dye as acceptor molecules applicable to conventional live cell imaging and flow cytometric analysis because of efficient live cell uptake. We investigated performance of equivalent molecular sensors localized or not localized inside nucleus under cell cycle control using flow cytometry. Synchronized cell cycle progression rendered significant separase activity detections in both molecular sensors. We obtained consistent outcomes with localized molecular sensor introduction and cell cycle control by fluorescent microscopic observations. We thus established live cell separase activity monitoring systems specifically or statistically that could lead to elucidate separase properties in detail.