A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level
J Cell Sci. 2016-11-25; 130(2): 512-520
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1. J Cell Sci. 2017 Jan 15;130(2):512-520. doi: 10.1242/jcs.195164. Epub 2016 Nov
A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell
Koh SB(1), Mascalchi P(2)(3), Rodriguez E(2), Lin Y(2)(4), Jodrell DI(2),
Richards FM(1), Lyons SK(2)(5).
(1)Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing
Centre, Robinson Way, Cambridge CB2 0RE, UK
(2)Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing
Centre, Robinson Way, Cambridge CB2 0RE, UK.
(3)Bordeaux Imaging Center, UMS 3420 CNRS-Université de Bordeaux-US4 INSERM, Pôle
d’imagerie photonique, Bordeaux F-33000, France.
(4)College of Life Sciences, Fujian Normal University, Fujian 350117, P. R.
(5)Cold Spring Harbor Laboratory, 1 Bungtown Road, New York 11724, US.
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.
© 2017. Published by The Company of Biologists Ltd.
PMID: 27888217 [Indexed for MEDLINE]