Assessing Kinetics from Fixed Cells Reveals Activation of the Mitotic Entry Network at the S/G2 Transition

Abstract

During the cell cycle, DNA duplication in S phase must occur before a cell divides in mitosis. In the intervening G2 phase, mitotic inducers accumulate, which eventually leads to a switch-like rise in mitotic kinase activity that triggers mitotic entry. However, when and how activation of the signaling network that promotes the transition to mitosis occurs remains unclear. We have developed a system to reduce cell-cell variation and increase accuracy of fluorescence quantification in single cells. This allows us to use immunofluorescence of endogenous marker proteins to assess kinetics from fixed cells. We find that mitotic phosphorylations initially occur at the completion of S phase, showing that activation of the mitotic entry network does not depend on protein accumulation through G2. Our data show insights into how mitotic entry is linked to the completion of S phase and forms a quantitative resource for mathematical models of the human cell cycle.

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

“…1A). 6 We next established a U2OS 18 and an RPE (Fig. S1A) cell line encoding a Cyclin B1-eYFP fusion protein at the endogenous CCNB1 locus, which allowed us to directly monitor Cyclin B1 protein dynamics in single live cells.…”

“…As Cyclin B1-eYFP levels are a direct readout of cell cycle progression, we grew cells on fibronectincoated micropatterns, which enabled us to accurately quantify fluorescence in single U2OS or RPE cells over long periods of time. 18 Whereas on Western blot, U2OS cells reach a steady state of Cyclin B1 after DNA damage, Cyclin B1-eYFP levels vary dramatically over time in individual cells ( Fig. 5B and C).…”

“…18,31 Briefly, the open reading frame of eYFP lacking the start codon was inserted into the CCNB1 locus by adeno-associated virus-mediated homologous recombination. The targeting construct was designed to contain 959 bp of homology with the CCNB1 locus in the 5 0 of the STOP codon and 1253 bp 3 0 of the STOP codon.…”

Group IVA Cytosolic Phospholipase A2 Regulates the G2-to-M Transition by Modulating the Activity of Tumor Suppressor SIRT2

“…Compared to WT SIRT2, the S331E phosphomimetic mutation diminished binding to mitotic spindles and centrosomes, whereas the S331A phosphodeficient mutation significantly enhanced binding. Relative intensities of SIRT2 on mitotic spindles and centrosomes at prophase and prometaphase were quantified by ImageJ software on over 200 cells in each group, as described previously (26,39) (Fig. 8G, right panel).…”

“…For colocalization studies, scatter plots and Manders' coefficients were obtained using the ImageJ plug-in Intensity Correlation Analysis. Quantification of relative accumulation of SIRT2 at mitotic spindles and centrosomes was performed using ImageJ as previously described (26). Briefly, a mask was created for quantification of SIRT2 signal on the mitotic structures, centered on the maximum intensity of the signal (3 by 3 pixels).…”