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• W2973995265 abstract "•CDK4/6 activity is continuously required to sustain Rb hyperphosphorylation in G1•Cyclin E/A-CDK can only sustain Rb hyperphosphorylation from the start of S•CDK4/6 activity following mitogen removal sustains Rb hyperphosphorylation•Transient hysteresis in CDK4/6 activity underlies stochastic passage of R Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point. Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point. Regulation of cell-cycle entry is critical for the growth, repair, and maintenance of mammalian tissues. Mitogen-stimulated cells can enter the cell cycle by exiting quiescence, or G0, to enter G1 phase before replicating their DNA in S phase and undergoing cell division in mitosis. Early work in mammalian tissue culture led to the concept of a mammalian cell-cycle restriction point, a point in time in G1 phase when cells transition from mitogen dependence to mitogen independence and commit to completing the cell cycle (Pardee, 1974Pardee A.B. A restriction point for control of normal animal cell proliferation.Proc. Natl. Acad. Sci. U S A. 1974; 71: 1286-1290Crossref PubMed Scopus (1059) Google Scholar, Zetterberg and Larsson, 1991Zetterberg A. Larsson O. Coordination between cell growth and cell cycle transit in animal cells.Cold Spring Harb. Symp. Quant. Biol. 1991; 56: 137-147Crossref PubMed Scopus (46) Google Scholar). The ability of cells to complete S phase, once initiated, protects against incomplete DNA replication and is thought to play an important role in maintaining genome stability (Henley and Dick, 2012Henley S.A. Dick F.A. The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle.Cell Div. 2012; 7: 10Crossref PubMed Scopus (183) Google Scholar, Matson and Cook, 2017Matson J.P. Cook J.G. Cell cycle proliferation decisions: the impact of single cell analyses.FEBS J. 2017; 284: 362-375Crossref PubMed Scopus (88) Google Scholar). Cell-cycle progression depends on the inactivation of the retinoblastoma protein Rb, which critically inhibits the transcription factor E2F (Fisher, 2016Fisher R.P. Getting to S: CDK functions and targets on the path to cell-cycle commitment.F1000Res. 2016; 5: 2374Crossref PubMed Scopus (23) Google Scholar, Malumbres and Barbacid, 2009Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.Nat. Rev. Cancer. 2009; 9: 153-166Crossref PubMed Scopus (2642) Google Scholar, Matson and Cook, 2017Matson J.P. Cook J.G. Cell cycle proliferation decisions: the impact of single cell analyses.FEBS J. 2017; 284: 362-375Crossref PubMed Scopus (88) Google Scholar, Sage et al., 2003Sage J. Miller A.L. Pérez-Mancera P.A. Wysocki J.M. Jacks T. Acute mutation of retinoblastoma gene function is sufficient for cell cycle re-entry.Nature. 2003; 424: 223-228Crossref PubMed Scopus (444) Google Scholar). Our laboratory has previously demonstrated that the inactivation of APC/CCdh1 at the G1/S transition is bistable with respect to stress (Cappell et al., 2016Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Cappell et al., 2018Cappell S.D. Mark K.G. Garbett D. Pack L.R. Rape M. Meyer T. EMI1 switches from being a substrate to an inhibitor of APC/CCDH1 to start the cell cycle.Nature. 2018; 558: 313-317Crossref PubMed Scopus (61) Google Scholar), but how the regulation of Rb exhibits memory with respect to mitogens remains an open question of fundamental importance. The ability of Rb to bind E2F is regulated by cyclin-dependent kinase (CDK) activity. Full phosphorylation of Rb (termed hyperphosphorylation) liberates E2F transcription factors and allows target gene expression. Mitogens induce the expression of cyclin D, the activating subunit of CDK4 and its close paralog CDK6 (hereafter CDK4/6), and CDK4/6 has been hypothesized to partially phosphorylate Rb, resulting in partial E2F activation. In turn, E2F activity induces the expression of cyclin E to activate CDK2. Finally, CDK2 has been proposed to complete the hyperphosphorylation of Rb in a self-sustaining positive feedback loop (Harbour et al., 1999Harbour J.W. Luo R.X. Dei Santi A. Postigo A.A. Dean D.C. Cdk phosphorylation triggers sequential intramolecular interactions that progressively block Rb functions as cells move through G1.Cell. 1999; 98: 859-869Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar, Lundberg and Weinberg, 1998Lundberg A.S. Weinberg R.A. Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-CDK complexes.Mol. Cell. Biol. 1998; 18: 753-761Crossref PubMed Scopus (855) Google Scholar, Merrick et al., 2011Merrick K.A. Wohlbold L. Zhang C. Allen J.J. Horiuchi D. Huskey N.E. Goga A. Shokat K.M. Fisher R.P. Switching Cdk2 on or off with small molecules to reveal requirements in human cell proliferation.Mol. Cell. 2011; 42: 624-636Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). As mitogen removal has been shown to result in the loss of cyclin D1 expression (Matsushime et al., 1991Matsushime H. Roussel M.F. Ashmun R.A. Sherr C.J. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle.Cell. 1991; 65: 701-713Abstract Full Text PDF PubMed Scopus (989) Google Scholar), the CDK2-Rb feedback loop has been proposed to trigger a bistable switch that mediates irreversible Rb hyperphosphorylation, E2F activation, and CDK2 activation as a plausible mechanism to explain passage of the restriction point in G1 (Fisher, 2016Fisher R.P. Getting to S: CDK functions and targets on the path to cell-cycle commitment.F1000Res. 2016; 5: 2374Crossref PubMed Scopus (23) Google Scholar, Matson and Cook, 2017Matson J.P. Cook J.G. Cell cycle proliferation decisions: the impact of single cell analyses.FEBS J. 2017; 284: 362-375Crossref PubMed Scopus (88) Google Scholar). Although additional mechanisms of bistability have been proposed for sustaining E2F activity, including positive feedback from E2F autoregulation and Skp2 autoinduction (Johnson et al., 1994Johnson D.G. Ohtani K. Nevins J.R. Autoregulatory control of E2F1 expression in response to positive and negative regulators of cell cycle progression.Genes Dev. 1994; 8: 1514-1525Crossref PubMed Scopus (452) Google Scholar, Yung et al., 2007Yung Y. Walker J.L. Roberts J.M. Assoian R.K. A Skp2 autoinduction loop and restriction point control.J. Cell Biol. 2007; 178: 741-747Crossref PubMed Scopus (40) Google Scholar), irreversible Rb hyperphosphorylation would theoretically suffice to mediate irreversible E2F activity, and as such, the CDK2-Rb feedback loop remains the primary model explaining sustained Rb hyperphosphorylation and inactivation following the removal of mitogens. Nevertheless, reports conflict on the relationship between CDK2 and the restriction point (Ekholm et al., 2001Ekholm S.V. Zickert P. Reed S.I. Zetterberg A. Accumulation of cyclin E is not a prerequisite for passage through the restriction point.Mol. Cell. Biol. 2001; 21: 3256-3265Crossref PubMed Scopus (84) Google Scholar, Hitomi et al., 2006Hitomi M. Yang K. Guo Y. Fretthold J. Harwalkar J. Stacey D.W. p27Kip1 and cyclin dependent kinase 2 regulate passage through the restriction point.Cell Cycle. 2006; 5: 2281-2289Crossref PubMed Scopus (30) Google Scholar, Schwarz et al., 2018Schwarz C. Johnson A. Kõivomägi M. Zatulovskiy E. Kravitz C.J. Doncic A. Skotheim J.M. A precise Cdk activity threshold determines passage through the restriction point.Mol. Cell. 2018; 69: 253-264.e5Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), and the relationship between Rb, CDK4/6, CDK2, and the restriction point remain to be elucidated. Several studies showed that cell-cycle signaling pathways exhibit significant plasticity, calling for refined working models. In particular, studies demonstrated substantial redundancy among CDK1, CDK2, and CDK3 in binding either E- or A-type cyclins (Aleem et al., 2005Aleem E. Kiyokawa H. Kaldis P. Cdc2-cyclin E complexes regulate the G1/S phase transition.Nat. Cell Biol. 2005; 7: 831-836Crossref PubMed Scopus (314) Google Scholar, Connell-Crowley et al., 1998Connell-Crowley L. Elledge S.J. Harper J.W. G1 cyclin-dependent kinases are sufficient to initiate DNA synthesis in quiescent human fibroblasts.Curr. Biol. 1998; 8: 65-68Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, Kalaszczynska et al., 2009Kalaszczynska I. Geng Y. Iino T. Mizuno S. Choi Y. Kondratiuk I. Silver D.P. Wolgemuth D.J. Akashi K. Sicinski P. Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells.Cell. 2009; 138: 352-365Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), while E- and A-type cyclins are also functionally redundant for DNA replication (Geng et al., 2003Geng Y. Yu Q. Sicinska E. Das M. Schneider J.E. Bhattacharya S. Rideout W.M. Bronson R.T. Gardner H. Sicinski P. Cyclin E ablation in the mouse.Cell. 2003; 114: 431-443Abstract Full Text Full Text PDF PubMed Scopus (571) Google Scholar, Kalaszczynska et al., 2009Kalaszczynska I. Geng Y. Iino T. Mizuno S. Choi Y. Kondratiuk I. Silver D.P. Wolgemuth D.J. Akashi K. Sicinski P. Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells.Cell. 2009; 138: 352-365Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Nevertheless, cyclin A is normally degraded in G1 phase (Cappell et al., 2016Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Peters, 2006Peters J.-M. The anaphase promoting complex/cyclosome: a machine designed to destroy.Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Crossref PubMed Scopus (1009) Google Scholar), cyclin E and A preferentially bind CDK2 over CDK1 (Merrick et al., 2008Merrick K.A. Larochelle S. Zhang C. Allen J.J. Shokat K.M. Fisher R.P. Distinct activation pathways confer cyclin-binding specificity on Cdk1 and Cdk2 in human cells.Mol. Cell. 2008; 32: 662-672Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and CDK3 expression is thought to be very low in most cells (Meyerson et al., 1992Meyerson M. Enders G.H. Wu C.L. Su L.K. Gorka C. Nelson C. Harlow E. Tsai L.H. A family of human cdc2-related protein kinases.EMBO J. 1992; 11: 2909-2917Crossref PubMed Scopus (785) Google Scholar). This argues that cyclin E-CDK2 is typically the main activity in G1 promoting the start of S phase. Nevertheless, cyclin E and A likely cooperate to activate CDK1, CDK2, and possibly CDK3 after G1. Additionally, as D-type cyclins have been shown to bind and activate CDK2 toward substrates traditionally associated with D-type cyclins but not E- or A-type cyclins (Malumbres et al., 2004Malumbres M. Sotillo R. Santamaría D. Galán J. Cerezo A. Ortega S. Dubus P. Barbacid M. Mammalian cells cycle without the D-type cyclin-dependent kinases Cdk4 and Cdk6.Cell. 2004; 118: 493-504Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar), the relevant activity of the holoenzymes generated by cyclin E/A and CDK1/2/3 are perhaps best expressed as a function of the cyclins, and we will use the terms “cyclin E/A-CDK” and “CDK4/6” to describe the collective activities of these two types of active holoenzymes. Here we investigate the regulation of Rb by CDK4/6 and cyclin E/A-CDK to elucidate the molecular basis for the restriction point. We find that cyclin E/A-CDK activity can indeed maintain Rb hyperphosphorylation following acute inhibition of CDK4/6, but only starting at the onset of S phase. Surprisingly, we show that CDK4/6 activity is required throughout G1 for cells to sustain Rb hyperphosphorylation and enter S phase, while cyclin E/A-CDK activity is dispensable for hyperphosphorylating Rb in G1. Consistently, cells crossing the restriction point continue to require CDK4/6 to sustain Rb hyperphosphorylation and enter the cell cycle. A rationale for the original cyclin E-CDK2-Rb feedback model was the observed rapid degradation of cyclin D1 following the loss of mitogens. However, we find that loss of cyclin D1 expression and CDK4/6 activity following mitogen removal in G1 is slow enough to sustain Rb hyperphosphorylation until S-phase entry in most, but not all cells. CDK4/6 activity following mitogen removal thus exhibits short-term memory, or transient hysteresis, with respect to mitogens, and Rb hyperphosphorylation in G1 does not require bistability or irreversibility previously associated with the restriction point. Consistently, we observe that mitogen removal in cells with hyperphosphorylated Rb and activated cyclin E/A-CDK results in a fraction of these cells eventually losing CDK4/6 activity, Rb hyperphosphorylation, and the ability to enter S phase. We thus conclude that the restriction point in G1 represents a probabilistic concept whereby, following mitogen removal, variable persistence in CDK4/6 activity enables some but not all cells to enter S phase, at which point cyclin E/A-CDK activity stably maintains Rb hyperphosphorylation to complete the cell cycle. Thus, CDK4/6 activity has a central role in controlling Rb hyperphosphorylation throughout G1, highlighting the relevance of altered CDK4/6 activity in proliferative pathologies such as cancer and aging. The model that positive feedback from cyclin E/A-CDK to Rb underlies an irreversible switch predicts that cyclin E/A-CDK activity can maintain Rb hyperphosphorylation independent of mitogens and CDK4/6 activity. This model can be translated into the testable prediction that a threshold activity level exists above which cyclin E/A-CDK activity can sustain Rb hyperphosphorylation upon acute inhibition of CDK4/6 (Figure 1A). Rb hyperphosphorylation has traditionally been detected using western blot. To analyze the effects of CDK4/6 inhibition on Rb hyperphosphorylation, we synchronized MCF-10A cells in quiescence by mitogen starvation, after which we stimulated cells with mitogens for different durations (hereafter mitogen release). To acutely inhibit CDK4/6, we added the CDK4/6-specific inhibitor palbociclib (Chen et al., 2016Chen P. Lee N.V. Hu W. Xu M. Ferre R.A. Lam H. Bergqvist S. Solowiej J. Diehl W. He Y.-A. et al.Spectrum and degree of CDK drug interactions predicts clinical performance.Mol. Cancer Ther. 2016; 15: 2273-2281Crossref PubMed Scopus (212) Google Scholar, Jorda et al., 2018Jorda R. Hendrychová D. Voller J. Řezníčková E. Gucky T. Krystof V. How selective are pharmacological inhibitors of cell cycle-regulating cyclin-dependent kinases?.J. Med. Chem. 2018; 61: 9105-9120Crossref PubMed Scopus (48) Google Scholar, O’Leary et al., 2016O’Leary B. Finn R.S. Turner N.C. Treating cancer with selective CDK4/6 inhibitors.Nat. Rev. Clin. Oncol. 2016; 13: 417-430Crossref PubMed Scopus (635) Google Scholar) for just 15 min prior to harvesting for western blot analysis (Figure 1B). Here we observed that Rb hyperphosphorylation was achieved both 12 and 18 h after mitogen release, but acute CDK4/6 inhibition abolished hyperphosphorylation only for the 12 h condition. These results suggest that cells with hyperphosphorylated Rb do not necessarily have enough cyclin E/A-CDK activity to stably maintain the hyperphosphorylated state of Rb following acute CDK4/6 inhibition. To assess the levels of cyclin E/A-CDK activity reached at the 12 and 18 h time points, we transduced MCF-10A cells with a previously characterized reporter that measures cyclin E/A-CDK activity and is believed to mostly reflect cyclin E-CDK2 activity before S phase in unperturbed conditions (Barr et al., 2016Barr A.R. Heldt F.S. Zhang T. Bakal C. Novák B. A dynamical framework for the all-or-none G1/S transition.Cell Syst. 2016; 2: 27-37Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Daigh et al., 2018Daigh L.H. Liu C. Chung M. Cimprich K.A. Meyer T. Stochastic endogenous replication stress causes ATR-triggered fluctuations in CDK2 activity that dynamically adjust global DNA synthesis rates.Cell Syst. 2018; 7: 17-27.e3Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, Gu et al., 2004Gu J. Xia X. Yan P. Liu H. Podust V.N. Reynolds A.B. Fanning E. Cell cycle-dependent regulation of a human DNA helicase that localizes in DNA damage foci.Mol. Biol. Cell. 2004; 15: 3320-3332Crossref PubMed Scopus (47) Google Scholar, Hahn et al., 2009Hahn A.T. Jones J.T. Meyer T. Quantitative analysis of cell cycle phase durations and PC12 differentiation using fluorescent biosensors.Cell Cycle. 2009; 8: 1044-1052Crossref PubMed Scopus (93) Google Scholar, Schwarz et al., 2018Schwarz C. Johnson A. Kõivomägi M. Zatulovskiy E. Kravitz C.J. Doncic A. Skotheim J.M. A precise Cdk activity threshold determines passage through the restriction point.Mol. Cell. 2018; 69: 253-264.e5Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Spencer et al., 2013Spencer S.L. Cappell S.D. Tsai F.C. Overton K.W. Wang C.L. Meyer T. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.Cell. 2013; 155: 369-383Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar) (Figure 1C). Consistent with previous results (Spencer et al., 2013Spencer S.L. Cappell S.D. Tsai F.C. Overton K.W. Wang C.L. Meyer T. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.Cell. 2013; 155: 369-383Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), we observed that cells synchronized by mitogen starvation exhibit significant heterogeneity in the times at which cyclin E/A-CDK is activated, with a percentage of cells having activated cyclin E/A-CDK for a few hours by the 12 h time point and a larger percentage of cells having activated cyclin E/A-CDK for a longer period of time by the 18 h time point (Figure 1D). The cell-to-cell heterogeneity in cyclin E/A-CDK activation thus obscures the precise relationship between cyclin E/A-CDK and Rb hyperphosphorylation in bulk-cell analysis, arguing that single-cell analysis is needed to determine this relationship. To overcome the issue of cell-to-cell heterogeneity in cyclin E/A-CDK activity, we sought to infer the hyperphosphorylated form of Rb by measuring Rb phosphorylation at the single-cell level. Such an analysis is challenging, as there are 15 putative CDK phosphorylation sites in Rb (Lee et al., 1998Lee J.O. Russo A.A. Pavletich N.P. Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7.Nature. 1998; 391: 859-865Crossref PubMed Scopus (377) Google Scholar), and different Rb molecules in the same cell may be phosphorylated at different sites. Narasimha et al., 2014Narasimha A.M. Kaulich M. Shapiro G.S. Choi Y.J. Sicinski P. Dowdy S.F. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.eLife. 2014; 3: e02872Crossref Google Scholar demonstrated that Rb exists primarily in an unphosphorylated, stochastically monophosphorylated, or hyperphosphorylated form, with no evidence of intermediate or partially phosphorylated isoforms under unperturbed conditions. We therefore reasoned that antibodies against a specific phosphorylation site on Rb could be exploited to infer Rb hyperphosphorylation using quantitative single-cell analysis. This approach assumes that a cell in which most of the Rb protein is hyperphosphorylated has each specific CDK phosphorylation site phosphorylated on most copies of Rb, whereas a cell with monophosphorylated Rb has the same residue phosphorylated on only a small fraction of the Rb proteins, and a cell with unphosphorylated Rb has the same residue phosphorylated on almost none of the Rb proteins in the cell (Figure 2A). Using immunofluorescence to stain individual cells with phospho-specific Rb antibodies, one would therefore predict that cells with hyperphosphorylated Rb would show a much stronger signal than cells with unphosphorylated or monophosphorylated Rb. We tested a phospho-antibody against Rb(p-S807/S811) as a potential single-cell marker for distinguishing cells with hyperphosphorylated Rb, on the basis of previous studies showing a statistical correlation between Rb(p-S807/S811) and E2F target gene expression (Cappell et al., 2016Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, Yang et al., 2017Yang H.W. Chung M. Kudo T. Meyer T. Competing memories of mitogen and p53 signalling control cell-cycle entry.Nature. 2017; 549: 404-408Crossref PubMed Scopus (128) Google Scholar). We simultaneously stained cycling MCF-10A cells with small interfering RNA (siRNA)-validated antibodies against Rb(p-S807/S811) as well as against total Rb (regardless of phospho-status) to calculate a phosphorylation signal that is normalized against the Rb expression level in each cell (Figures 2B, S1A, and S1B). Using this ratiometric analysis, we observed a highly bimodal signal, raising the question of whether the upper peak of the normalized Rb(p-S807/S811) signal exclusively reflects the hyperphosphorylated state, or whether it also includes cells in the monophosphorylated state. We thus performed several critical tests to determine whether this single-cell normalized Rb(p-S807/S811) signal can be used to specifically infer whether a cell has most of its Rb hyperphosphorylated. To test whether, at the protein level, the Rb(p-S807/S811) antibody detects hyperphosphorylated and not hypophosphorylated Rb, we performed western blot analysis on cycling MCF-10A cells with antibodies against both Rb(p-S807/S811) and Rb, and we monitored the two antibodies using multiplex fluorescent antibody detection (Figure 2C). Here we observed similar levels of Rb in both bands but much higher Rb(p-S807/S811) signal in the upper band. This result is consistent with the upper peak of the single-cell normalized Rb(p-S807/S811) signal specifically reflecting cells with hyperphosphorylated Rb. To determine whether, in intact cells, the upper peak of the single-cell normalized Rb(p-S807/S811) signal excludes cells with primarily monophosphorylated Rb, we used iterative indirect immunofluorescence imaging (Gut et al., 2018Gut G. Herrmann M.D. Pelkmans L. Multiplexed protein maps link subcellular organization to cellular states.Science. 2018; 361: eaar7042Crossref PubMed Scopus (171) Google Scholar) to sequentially stain cells with antibodies against Rb(p-S807/S811) as well as three other CDK phosphorylation sites, and we further stained cells with an antibody against Rb to normalize the phospho-signals (Figure 2D; method controls in Figures S1C–S1E). Here we observed that cells in the upper peak of the normalized Rb(p-S807/S811) signal were also in the upper peak of the normalized Rb(p-T373), Rb(p-S608), and Rb(p-S780) signals, consistent with the interpretation that the high normalized Rb(p-S807/S811) signal excludes cells with primarily monophosphorylated Rb. The bimodal distribution of the normalized Rb(p-S807/S811) signal suggests that individual cycling cells do not typically exhibit hyperphosphorylation of an intermediate fraction of Rb at steady state, and instead most Rb proteins in a single cell are either hyperphosphorylated or not. To additionally test whether the initial establishment of Rb hyperphosphorylation in cells exiting quiescence might occur in a graded fashion, we fixed and stained mitogen-released cells at different time points for normalized Rb(p-S807/S811), and we additionally incubated the cells with 5-ethynyl-2′-deoxyuridine (EdU) prior to fixation to identify cells actively replicating their DNA in S phase (Figure 2E). Here we observed a bimodal distribution of normalized Rb(p-S807/S811) from the earliest observed establishment of the signal, and the upper peak of this signal matched the upper peak observed in S phase, when maximal Rb hyperphosphorylation occurs, suggesting that the initial establishment of Rb hyperphosphorylation also occurs in an all-or-none manner. Together, these data are consistent with the interpretation that high normalized Rb(p-S807/S811) signal excludes cells with monophosphorylated Rb and specifically reflects a state where most of the Rb protein in a cell is hyperphosphorylated. The inactivation of Rb has previously been shown to be associated with reduced binding of Rb to the nuclear compartment (Mittnacht and Weinberg, 1991Mittnacht S. Weinberg R.A. G1/S phosphorylation of the retinoblastoma protein is associated with an altered affinity for the nuclear compartment.Cell. 1991; 65: 381-393Abstract Full Text PDF PubMed Scopus (271) Google Scholar). Concretely, in situ extraction of soluble proteins with a low-salt buffer results in the loss of hyperphosphorylated Rb and the retainment of nucleus-bound hypophosphorylated Rb. To test for a correlation between normalized Rb(p-S807/S811) and reduced nuclear binding, we performed in situ extraction and standard fixation in parallel experiments on mitogen-released cells (Figure 2F). Here we observed that the percentage of cells with low nuclear binding closely matched the percentage of cells with high normalized Rb(p-S807/S811) (we arbitrarily used a threshold of 0.5, though this percentage was relatively stable from 0.3 to 0.6; Figure S1F). These results are consistent with high normalized Rb(p-S807/S811) signal reflecting the hyperphosphorylated and inactive state of Rb. Taken together, these different measurements and controls suggest that in MCF-10A cells, a high normalized Rb(p-S807/S811) signal can be used to infer that most of the Rb proteins in an individual cell are hyperphosphorylated. Having established a means to infer Rb hyperphosphorylation at the single-cell level, we investigated the relationship between cyclin E/A-CDK activity and Rb hyperphosphorylation upon acute inhibition of CDK4/6. We first monitored cyclin E/A-CDK activity in cycling MCF-10A cells before treating cells with CDK4/6i for only 15 min, followed by fixing and staining for normalized Rb(p-S807/S811) (Figure 3A). Under control conditions, we observed low or high Rb(p-S807/S811) signals already at the lowest levels of cyclin E/A-CDK activity that could not be distinguished from background. Any measurable increase in cyclin E/A-CDK activity level was paralleled by an exclusively high Rb(p-S807/S811) signal. However, after acute CDK4/6 inhibition, there was a sharp transition, with cells maintaining high Rb(p-S807/S811) only when the activity of cyclin E/A-CDK exceeded a level of approximately 0.8. These data suggest that a threshold level of cyclin E/A-CDK activity must be reached for cells to maintain Rb hyperphosphorylation following acute CDK4/6 inhibition. We next investigated whether the sharp transition in the ability of cyclin E/A-CDK to maintain Rb hyperphosphorylation corresponds to a specific point in the cell cycle. Here we noted that the level of cyclin E/A-CDK activity at which the transition occurs corresponds closely to the point at which DNA replication has previously been reported to begin (Spencer et al., 2013Spencer S.L. Cappell S.D. Tsai F.C. Overton K.W. Wang C.L. Meyer T. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit.Cell. 2013; 155: 369-383Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar), and we confirmed these results by incubating cycling cells with EdU prior to fixation (Figure 3B). By including analysis of DNA content, as reflected by Hoechst staining, cells can be gated for G1, S," @default.
• W2973995265 created "2019-09-26" @default.
• W2973995265 creator A5007196747 @default.
• W2973995265 creator A5013677980 @default.
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• W2973995265 date "2019-11-01" @default.
• W2973995265 modified "2023-10-11" @default.
• W2973995265 title "Transient Hysteresis in CDK4/6 Activity Underlies Passage of the Restriction Point in G1" @default.
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