A Precise Cdk Activity Threshold Determines Passage through the Restriction Point

Abstract

At the restriction point (R), mammalian cells irreversibly commit to divide. R has been viewed as a point in G1 that is passed when growth factor signaling initiates a positive feedback loop of Cdk activity. However, recent studies have cast doubt on this model by claiming R occurs prior to positive feedback activation in G1 or even before completion of the previous cell cycle. Here we reconcile these results and show that whereas many commonly used cell lines do not exhibit a G1 R, primary fibroblasts have a G1 R that is defined by a precise Cdk activity threshold and the activation of cell-cycle-dependent transcription. A simple threshold model, based solely on Cdk activity, predicted with more than 95% accuracy whether individual cells had passed R. That a single measurement accurately predicted cell fate shows that the state of complex regulatory networks can be assessed using a few critical protein activities.

Keywords: Cdk2; G1/S; HDHB; restriction point.

Figure 1. Primary fibroblasts, but not cell lines, exhibit a serum-dependent R

(A) Experiment schematic: cells were grown and imaged for two days in medium with 10% serum. Then, serum was removed. (B–G) Birth times relative to the time of serum removal and cell cycle durations were measured for (B) MRC5-hTERT cells (N = 206), (C) RPE1-hTERT cells (N = 43), (D) T98G cells (N = 124), (E) WI-38 cells (N = 171), (F) primary human foreskin fibroblasts (HFF; N = 93), and (G) primary human lung fibroblasts (HLF; N = 376). Horizontal dashed lines denote cell cycle durations greater than 50 hours. Black dots indicate cells that did not divide again. Blue dots indicate cells born into serum-containing medium that went on to divide. Red dots indicate cells born into serum-free medium that went on to divide. The percentage of cells born into serum-free medium that went on to divide is indicated.

Figure 2. The timing of primary fibroblast passage through R is variable

(A) Fraction of HLFs that go on to divide following serum removal as a function of cell age at the time of serum removal (N = 268). (B) Logistic regression estimate of the fraction of post-R HLFs from (A) as a function of cell age. Dashed lines represent 95% CI based on bootstrap analysis of the data (see Methods). (C) Cell cycle durations of individual cells plotted as a function of cell age at the time of serum removal (N = 268). Yellow region highlights cells with ages ≥3 and <10 hours at the time of serum removal. (D) Cell cycle durations of individual cells plotted as a function of cell age at the time of medium replacement (replotted from Figure S1E; N = 120). (E) Fraction of cells within the yellow regions from (C) and (D) that fail to divide following serum removal or medium replacement (serum removal: 72/122 = 59%; medium replacement: 15/39 = 38%). Chi-square test p-value = 0.025. (F) Cell cycle durations of individual cells plotted as a function of cell age at the time of serum removal (replotted from Martinsson et al. (2005), Figure 1B; N = 172). (G) Cell cycle durations of individual cells plotted as a function of cell age at the time of medium replace (replotted from Martinsson et al. (2005), Figure 1A; N = 103). (H) Fraction of cells within the yellow regions from (F) and (G) that fail to divide following serum removal or medium replacement (serum removal: 30/99 = 30%; medium replacement: 10/56 = 18%). Chi-square test p-value = 0.089.

Figure 3. A Cdk activity threshold accurately predicts passage through R

(A) Schematic of mammalian G1/S regulatory network. (B) Schematic of HDHB-EGFP Cdk activity sensor. As it is increasingly phosphorylated over the course of the cell cycle, it translocates from the nucleus to the cytoplasm. (C) Example single-cell traces of Cdk activity. Cdk activity was measured as the ratio of median cytoplasmic to median nuclear HDHB-EGFP fluorescence. (D) Fluorescence images from the traces measured in (C) at 30 minute time points. Scale bar = 30μm. (E) Traces from a lineage of cells exposed to a medium replace control at time 0. At the end of the experiment, contact inhibition prevents further divisions. (F) Traces from a lineage of cells exposed to serum removal at time 0. Cells with high Cdk activity at the time of serum removal (the top two rows at time 0) go on to divide, while cells with low Cdk activity at that time arrest (the bottom six rows at time 0). (G) Each row is a Cdk activity trace for an individual cell (N = 102). Rows are ordered by Cdk activity at the time of serum removal. Cells that divide after serum removal are marked with a black square to the right of the trace. Note that all cells that did not divide were tracked at least 24 hours following serum removal, but were not necessarily segmented for that entire period. (H) A commitment plot (as in Figure 1G) in which each cell from (G) is colored based on its Cdk activity at the time of serum removal using the same scale as in panels (E–G). (I) Logistic regression estimating the probability a cell was committed to division as a function of its Cdk activity at the time of serum removal. The Cdk activity threshold of 0.84 was calculated from the midpoint of the logistic regression (95% confidence interval from bootstrap analysis: 0.76 to 0.93). Inset: accuracy of threshold models based on cell age or Cdk activity (see Methods).

Figure 4. Cell cycle-dependent activation of the E2F1 promoter coincides with R, which occurs one to two hours before S phase

(A) Schematic of E2F1pr-EGFP-PEST fluorescent reporter. (B) Example traces from a cell expressing E2F1pr-EGFP-PEST (blue). Dashed lines denote E2F1 transcriptional activation which is calculated as the maximum of the second derivative of a spline fit of the trace (red; see Methods). (C) Fluorescence images from the traces measured in (B) at 30 minute time points. Scale bar = 30μm. (D) Box plots summarizing the distributions of the times from cell birth to the Cdk activity commitment threshold (N = 30), activation of E2F1 transcription (N = 25), or S phase (N = 52) from populations of cells expressing one of the following constructs: HDHB-EGFP, E2F1pr-EGFP-PEST, or geminin-GFP. Also a box plot summarizing the distribution of S phase entry times minus Cdk activation times from cells expressing both HDHB-mCherry and geminin-GFP. Red bars indicated medians, blue boxes indicate 25^th and 75^th percentiles (for Cdk activation, both the median and the 75^th percentile are 7 hours), and bars indicate the most extreme values. (E) Example traces from a cell expressing HDHB-mCherry and geminin-GFP. Cdk activation time is the first time point > 0.84 followed by another time point > 0.84. S phase entry time is determined by fitting the data to two lines and identifying the intersection.

Figure 5. Cells commit at a lower Cdk activity threshold in response to MEK inhibition

(A) Experiment schematic: asynchronous HLFs expressing HDHB-EGFP were treated with 500 nM MEK inhibitor (PD0325901) while grown in constant 10% serum. (B) Each row is a Cdk activity trace for an individual cell (N = 66). Rows are ordered by Cdk activity at the time of MEK inhibitor addition. Cells that divide after serum removal are marked with a black square to the right of the trace. Note that all cells that did not divide were tracked at least 24 hours following MEK inhibition, but were not necessarily segmented for that entire period. (C) A commitment plot (as in Figure 3H) in which each cell is colored based on its Cdk activity at the time of MEK inhibitor addition using the same scale as in (B). (D) Logistic regression estimating the probability a cell was committed to division as a function of its Cdk activity at the time of MEK inhibitor addition. The Cdk activity threshold of 0.68 was calculated from the midpoint of the logistic regression (95% confidence interval from bootstrap analysis: 0.59 to 0.79). The Cdk activity threshold for commitment in response to MEK inhibition is significantly lower than the threshold in response to serum removal (0.68 vs. 0.84; p<0.01).

Figure 6. HDHB-EGFP likely senses the activity of multiple Cdk/cyclin complexes

(A) Illustration of the hypothesis that multiple cyclin-Cdk complexes phosphorylate HDHB-EGFP. (B) In vitro kinase assays were performed with the indicated Cdk/cyclin complexes and each of three substrates: purified HDHB-EGFP, histone H1, or a C-terminal fragment of Rb (aa 771-928). Approximately equal amounts of each kinase complex were used except for Cdk4/cyclin D1 which was 10-fold in excess. After 16 minute reactions with [γ-32P] ATP, the results were visualized using autoradiography following SDS-PAGE. A quantification of HDHB-EGFP phosphorylation signal divided by RbC signal and normalized to the Cdk2-cyclin E1 signal is shown in the lower panel. (C) Probability of division of Cdk2^as RPEs treated with either 10 μM 3-MB-PP1 or DMSO. Cells were split into groups based on whether their Cdk activity at the time of treatment was below or above 0.7 (3-MB-PP1, low: N = 28; DMSO, low: N = 11; 3-MB-PP1, high: N = 14; DMSO, high: N = 8). Error bars represent 50% confidence intervals calculated with bootstrap analysis. (D) Averaged traces (± standard error of the mean) of unsynchronized Cdk2^as RPEs with high Cdk activity (>0.7) at the time of treatment. Cells were treated with DMSO (black; N = 23), 10 μM 3-MB-PP1 (blue; N = 45), or 10 μM EMD #217714 (red; N = 29). Only dividing cells were averaged from the DMSO and 3-MB-PP1 treatments. All EMD #217714 treated cells failed to divide. (E) HLFs expressing HDHB-mCherry were serum-starved for 72 hours. At 0, 10, 20, and 30 hours after serum addition, HDHB-mCherry expressing cells were imaged and then lysed. Above: Phosphorylated and unphosphorylated HDHB-mCherry from the lysates was resolved with Phos-tag SDS-PAGE and detected with anti-mCherry antibodies. Below: Cdk activity (cyt/nuc ratio from HDHB-mCherry images) was measured for cells from each time point (0h: N = 268; 10h: N = 269; 20h: N = 252; 30h: N = 222). (F) For each time point, the mean Cdk activity measured from the HDHB-mCherry images was plotted as a function of the ratio of phosphorylated to unphosphorylated HDHB-mCherry measured from the phos-tag gel.

Figure 7. A differential equation model of R is consistent with measured Cdk activity thresholds

(A) Schematic indicating molecular interactions included in the model adapted from another study (Yao et al., 2008) (see Supplemental Methods). MEK-dependent growth factor signals and a serum-independent E2F synthesis term were added (orange) to the interactions in the previously existing model (green). (B) Schematic for calculating the Cdk threshold necessary to pass R. We examined the effects of removing serum after different amounts of time to simulate the experiments in Figures 3F–G. This determines the Cdk activity threshold to pass R. Example traces show dynamics for cells that were just before R (red) and just after R (blue) at the time serum was removed. (C) Experimental and computational commitment thresholds in response to serum removal and MEK inhibition. Growth factor proliferative signaling in response to serum is decomposed into MEK-independent and MEK-dependent fractions. (D) Schematic of Cdk activity (cyt/nuc HDHB ratio) changes over the course of the cell cycle in primary human lung fibroblasts.