Live imaging of apoptotic signaling flow using tunable combinatorial FRET-based bioprobes for cell population analysis of caspase cascades

Abstract

Understanding cellular signaling flow is required to comprehend living organisms. Various live cell imaging tools have been developed but challenges remain due to complex cross-talk between pathways and response heterogeneities among cells. We have focused on multiplex live cell imaging for statistical analysis to address the difficulties and developed simple multiple fluorescence imaging system to quantify cell signaling at single-cell resolution using Förster Resonance Energy Transfer (FRET)-based chimeric molecular sensors comprised of fluorescent proteins and dyes. The dye-fluorescent protein conjugate is robust for a wide selection of combinations, facilitating rearrangement for coordinating emission profile of molecular sensors to adjust for visualization conditions, target phenomena, and simultaneous use. As the molecular sensor could exhibit highly sensitive in detection for protease activity, we customized molecular sensor of caspase-9 and combine the established sensor for caspase-3 to validate the system by observation of caspase-9 and -3 dynamics simultaneously, key signaling flow of apoptosis. We found cumulative caspase-9 activity rather than reaction rate inversely regulated caspase-3 execution times for apoptotic cell death. Imaging-derived statistics were thus applied to discern the dominating aspects of apoptotic signaling unavailable by common live cell imaging and proteomics protein analysis. Adopted to various visualization targets, the technique can discriminate between rivalling explanations and should help unravel other protease involved signaling pathways.

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

“…We have established live cell monitoring systems for separase activity through microscopic observation and flow cytometric analysis that could allow quantitative assay for separase activation profiles during cell cycle with spatiotemporal resolution and statistic considerations owing to consistent outcomes from both approaches. Our monitoring systems could easily be applied to other cell types [42]. This could render new insight for separase behaviors in live cells under cell cycle arrest or progress conditions in detail.…”

“…We have designed a chimeric FRET-based molecular sensor that utilizes a green fluorescent protein (GFP) mutant (donor molecule) with separase-cleavable sequences followed by unique cysteine for chemical modification with a fluorescent organic dye (acceptor molecule) as a simple conjugate (Figure 1) [41]. We could easily replace the donor fluorescent protein, recognition sequences, and dye species to optimize the assay system [42]. Furthermore, the molecular sensor can efficiently be delivered into live cells via endocytotic pathways.…”