Accurate delineation of cell cycle phase transitions in living cells with PIP-FUCCI

Abstract

Cell cycle phase transitions are tightly orchestrated to ensure efficient cell cycle progression and genome stability. Interrogating these transitions is important for understanding both normal and pathological cell proliferation. By quantifying the dynamics of the popular FUCCI reporters relative to the transitions into and out of S phase, we found that their dynamics are substantially and variably offset from true S phase boundaries. To enhance detection of phase transitions, we generated a new reporter whose oscillations are directly coupled to DNA replication and combined it with the FUCCI APC/C reporter to create "PIP-FUCCI". The PIP degron fusion protein precisely marks the G1/S and S/G2 transitions; shows a rapid decrease in signal in response to large doses of DNA damage only during G1; and distinguishes cell type-specific and DNA damage source-dependent arrest phenotypes. We provide guidance to investigators in selecting appropriate fluorescent cell cycle reporters and new analysis strategies for delineating cell cycle transitions. ARTICLE HISTORY

Evidence that the human cell cycle is a series of uncoupled, memoryless phases

“…According to the many‐for‐all model, observation of coupling between cell cycle phases would imply that phase‐controlling factors are relatively more abundant in certain cell types. Third, we employ a more accurate method of measuring cell cycle phase durations based on appearance and disappearance of PCNA foci during S phase and validate these results with an orthogonal measurement of phase duration (Grant et al , ; Appendix Fig S2B and C; Chao et al , ; Grant et al , ). Previous studies employed the FUCCI reporter system to distinguish G1 and S‐G2‐M cells, but this system is known to give unclear cell cycle phase boundaries (Wilson et al , ; Grant et al , ).…”

“…Furthermore, the lack of correlation was not due to the sampling frequency chosen, as the correlation coefficients did not depend on the sampling frequency in the range that was used (Appendix Fig S6). To confirm these results, we validated the lack of correlation among cell cycle phases in an independent fluorescent reporter system (Grant et al, 2018;Appendix Fig S2B and C, and Table S1). A statistical power analysis revealed that our sample size would be adequate to detect significant correlations, if present (Materials and Methods).…”

“…Third, we employ a more accurate method of measuring cell cycle phase durations based on appearance and disappearance of PCNA foci during S phase and validate these results with an orthogonal measurement of phase duration (Grant et al, 2018;Appendix Fig S2B and C;Chao et al, 2017;Grant et al, 2018). Previous studies employed the FUCCI reporter system to distinguish G1 and S-G2-M cells, but this system is known to give unclear cell cycle phase boundaries (Wilson et al, 2016;Grant et al, 2018). Fourth, it is possible that previous studies analyzed a mixed population of cells (e.g., cells at different stages of differentiation or maturation) that could result in correlation between phase durations across the different cell types (Roccio et al, 2013).…”

Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

“…We used live cell imaging of fluorescently tagged protein biosensors to compare the length of available MCM loading time between the first and second cell cycles after G0. We imaged an RPE1 cell line stably expressing three fluorescent fusion proteins: 1) full length Cdc6 fused to mVenus (Segev et al, 2016), 2) Proliferating Cell Nuclear Antigen (PCNA) fused to mTurq2 to track cell nuclei and the borders of S phase (Burgess et al, 2012;Grant, Kedziora et al, 2018), and 3) a CDK kinase activity sensor fused to mCherry (Hahn et al, 2009). This kinase sensor was previously established to report CDK2 activity in G1 and early S phase and both CDK2 and CDK1 activity in S and G2 phase; it is not a direct reporter of CDK4/6 activity (Spencer et al, 2013;Schwarz et al, 2018).…”

“…The RPE1 cells with doxycycline inducible Cyclin E1 and the RPE1 cells with doxycycline inducible Cdt1 and stable Cdc6 WT or Cdc6-mut were described previously (Matson et al, 2017). The pLenti-PGK hygro PCNA-mTurq2 was described previously (Grant et al, 2018). The CSII-EF zeo DHB-mCherry CDK activity reporter was a gift Dr. Sabrina Spencer.…”