title: A quantitative FastFUCCI assay defines cell cycle dynamics at a 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 in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated 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 or M phase but eventually advanced through mitosis into the next 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 re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK siangboon.koh@cantab.net fran.richards@cruk.cam.ac.uk.
authors.forename: Siang-Boon
authors.identifier: 0000-0002-5494-3800
authors.initials: SB
authors.lastname: Koh
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.|Bordeaux Imaging Center, UMS 3420 CNRS-Université de Bordeaux-US4 INSERM, Pôle d'imagerie photonique, Bordeaux F-33000, France.
authors.forename: Patrice
authors.initials: P
authors.lastname: Mascalchi
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Esther
authors.initials: E
authors.lastname: Rodriguez
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.|College of Life Sciences, Fujian Normal University, Fujian 350117, P. R. China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Lin
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.
authors.forename: Duncan I
authors.initials: DI
authors.lastname: Jodrell
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK siangboon.koh@cantab.net fran.richards@cruk.cam.ac.uk.
authors.forename: Frances M
authors.initials: FM
authors.lastname: Richards
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK.|Cold Spring Harbor Laboratory, 1 Bungtown Road, New York 11724, US.
authors.forename: Scott K
authors.initials: SK
authors.lastname: Lyons
mesh_terms: D050256:Antimitotic Agents / Q000494:pharmacology; D001331:Automation; D001681:Biological Assay / Q000379:methods*; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D004261:DNA Replication / Q000187:drug effects; D005453:Fluorescence; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D008938:Mitosis / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology; D059010:Single-Cell Analysis / Q000379:methods*; D062211:Spatio-Temporal Analysis; D043823:Taxoids / Q000494:pharmacology; D059008:Time-Lapse Imaging; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D050256:Antimitotic Agents; D001952:Bridged-Ring Compounds; D043823:Taxoids; C080625:taxane; D017239:Paclitaxel
keywords: Automated microscopy; DNA replication origin; Drug synergy; FUCCI; Live-cell imaging; Taxane