The degradation of proteins during the cell cycle is important for triggering cell cycle transitions. The non-reversible degradation also allows a uni-directionality of the cell cycle. We use fluorescently marked proteins to monitor the relative stability of protein of interests (POI) at different stages of the cell cycle. Fluorecent values and cell cycle stage of individual cells are determined using flow cytometry as outlined in the diagram.
RPS-Assay
The main readout of the relative protein stability is the ration of flourescent values between a reference fluorescent protein (e.g. GFP) that is not degraded in a cell cycle dependent manner and the flourescently labeled (e.g. Cherry-labelled) protein of interest.
1) Tissue culture cells (S2R+ Drosophila cells) are transfected with plasmids that encode the two different fluorescent proteins.
2) The use of T2A sequences allows the stoichiometric expression of the two fluorescently labelled proteins
3) Fluorescent values as well as DNA content is recorded for indiviual cells using flow cytometry
4) Medium expression levels are selected (to avoid strong overexpression artefacts)
5) The fluorescent ratios are calculated
6) DNA-content allows simultenously a rough classification into different cell cycle stages
7) An example for a CHE-tagged Cyclin B Construct for which each cells analyzed (represented by a block dot) the DNA-content and the CHE/GFP ratios are combined
8) Box blot diagram of CHE/GFP rations of selected cell cycle stages
9) Box blot diagram of the median CHE/GFP ratios obtain after biological replicates on different days
The result in this case illustrates the low relative stability of Cyclin B during G1 phase and its stabilization during S and G2-phase. The low stability of Cyclin B in G1 is caused by APC/C mediated ubiqutitination of CyclinB and its subsequent degradation in the proteasome.