Quantitative FastFUCCI assay defines cell cycle dynamics at single-cell level

Abstract

The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput and quantitative robustness. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells, using an all-in-one expression cassette FastFUCCI. The approach obviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2-M but eventually advanced through mitosis into interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently reentered S/G2. Accordingly, we showed evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrated the utility of the FastFUCCI assay in quantitating spatiotemporal dynamics and identified its potential in preclinical drug development.