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J Vis Exp. 2012 Jan 27;(59):e3410. doi: 10.3791/3410.

Monitoring kinase and phosphatase activities through the cell cycle by ratiometric FRET.

Author information

1
Department of Cell and Molecular Biology, Karolinska Institutet.

Abstract

Förster resonance energy transfer (FRET)-based reporters(1) allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA(2), PKB(3), PKC(4), PKD(5), ERK(6), JNK(7), Cdk(18), Aurora B(9) and Plk1(9). Given the modular design, additional probes are likely to emerge in the near future(10). Progression through the cell cycle is affected by stress signaling pathways( 11). Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress(12).Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle(8,9,13,14). Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

PMID:
22314640
PMCID:
PMC3462574
DOI:
10.3791/3410
[Indexed for MEDLINE]
Free PMC Article

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