Koh, S. B., Mascalchi, P., Rodriguez, E., Lin, Y., Jodrell, D. I., Richards, F. M., Lyons, S. K. (January 2017) A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level. J Cell Sci, 130 (2). pp. 512-520. ISSN 0021-9533
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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.
| Item Type: | Paper |
|---|---|
| Uncontrolled Keywords: | Automated microscopy DNA replication origin Drug synergy Fucci Live-cell imaging Taxane |
| Subjects: | bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > cell cycle Investigative techniques and equipment > microscopy |
| CSHL Authors: | |
| Communities: | CSHL Cancer Center Program > Cancer Genetics CSHL labs > Lyons lab CSHL Cancer Center Program > Cellular Communication in Cancer Program |
| Depositing User: | Matt Covey |
| Date: | 15 January 2017 |
| Date Deposited: | 16 Feb 2017 20:33 |
| Last Modified: | 26 Oct 2020 15:58 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/34133 |
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