Intrinsic checkpoint deficiency during cell cycle re-entry from quiescence

Jacob Peter Matson; Amy M. House; Gavin D. Grant; Huaitong Wu; Joanna Perez; Jeanette Gowen Cook

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) . CDK2 phosphorylates the reporter beginning in late G1 phase to induce export from the nucleus to the cytoplasm. The higher the ratio of cytoplasmic/nuclear signal, the higher the kinase activity. We imaged RPE1 cells expressing all three biosensors synchronized in G0 and released into the cell cycle for 72 hours, capturing an image every 10 minutes, starting 6.5 hours after release. PCNA-mTurq2 was present throughout the cell cycle and become punctate during S phase (Fig. 6A ). Cdc6-mVenus was not present in G0 due to APC Cdh1 -mediated degradation. Nuclear Cdc6 first appeared in G1 and increased until S phase at which point it was lost from the nuclei and accumulated in the cytoplasm instead. After mitosis, Cdc6 was degraded in early G1 phase, then nuclear Cdc6 increased again later in G1 (Fig. 6A ). The CDK reporter (DHB-mCherry) was nuclear in G0 (low CDK activity) and gradually became cytoplasmic beginning in late G1 (high CDK activity) (Fig. 6A) . We tracked 50 cells through the first and second cell cycles after G0 release. Fig. 6B shows an individual cell trace for nuclear Cdc6 in which the first full cell cycle took nearly 35 hours whereas the second cycle took only 20 hours mostly from the difference in G1 length.