Relating individual cell division events to single-cell ERK and Akt activity time courses

Abstract

Biochemical correlates of stochastic single-cell fates have been elusive, even for the well-studied mammalian cell cycle. We monitored single-cell dynamics of the ERK and Akt pathways, critical cell cycle progression hubs and anti-cancer drug targets, and paired them to division events in the same single cells using the non-transformed MCF10A epithelial line. Following growth factor treatment, in cells that divide both ERK and Akt activities are significantly higher within the S-G2 time window (~ 8.5-40 h). Such differences were much smaller in the pre-S-phase, restriction point window which is traditionally associated with ERK and Akt activity dependence, suggesting unappreciated roles for ERK and Akt in S through G2. Simple metrics of central tendency in this time window are associated with subsequent cell division fates. ERK activity was more strongly associated with division fates than Akt activity, suggesting Akt activity dynamics may contribute less to the decision driving cell division in this context. We also find that ERK and Akt activities are less correlated with each other in cells that divide. Network reconstruction experiments demonstrated that this correlation behavior was likely not due to crosstalk, as ERK and Akt do not interact in this context, in contrast to other transformed cell types. Overall, our findings support roles for ERK and Akt activity throughout the cell cycle as opposed to just before the restriction point, and suggest ERK activity dynamics may be more important than Akt activity dynamics for driving cell division in this non-transformed context.

Figure 1

Evaluating the association of cell division with univariate single cell ERK or Akt dynamics. In these experiments, cells were either expressing the ERK or the Akt KTR. (A) Cell treatment workflow for pairing single cell KTR dynamics to cell division. ERK or Akt KTR expressing MCF10A cells were seeded, allowed to attach overnight, and then serum and growth factor starved. Following starvation, baseline images were acquired, cells were treated with EGF and insulin, and then imaged every 15 min for 48 h. Images were quantified using the analysis pipeline described in the “Methods”. (B, C) Quantified ERK or Akt KTR dynamics paired to division events for EGF and Insulin doses that match those used in culture medium (B) or tenfold less (C). Single cell traces of dividing (blue) and non-dividing (red) cells are shown with thin lines, and population median (per time point) is shown with thick lines. (D) Left, representative single cell trace of ERK KTR for a dividing (blue) or non-dividing (red) cell. Median ERK activity within the 8.5–40 h interval for each cell becomes a single dot in the boxplots. (E) notBoxplots for single cell median ERK or Akt activity within the 8.5–40 h interval for EGF and Insulin doses that match those used in culture medium (top) or tenfold less (bottom). p-values for the right-tailed rank-sum test were calculated at the 95% confidence interval. D, dividing; ND, non-dividing.

Figure 2

Evaluating the association of cell division with paired, bivariate single-cell ERK and Akt dynamics. In these experiments, cells were expressing both the ERK and Akt KTR simultaneously. (A) Quantified ERK and Akt dynamics for EGF and Insulin doses that match those used in culture medium (top) or ten-fold less (bottom). Single cell traces of dividing (blue) and non-dividing (red) cells are shown with thin lines, and population median (per time point) is shown with thick lines. (B) Scatter plot of ERK vs. Akt KTR median activity in the 8.5–40 h window from 150 randomly sampled cells. Each dot is a single cell. Dividing cells are blue and non-dividing cells are red. Left and right are high and low growth factor concentrations, respectively. (C) Statistical significance of logistic regression coefficients for either ERK or Akt median activity (8.5–40 h), with regression coefficients shown in parentheses above the respective bars (uncertainty is standard error).

Figure 3

Investigating properties of the ERK and Akt network. (A) Single cell ERK and Akt activity plotted across all time points within the 8.5–40 h interval for dividing and non-dividing cells. These cells expressed both the ERK and Akt KTR. Pearson correlation coefficient, along with the number of cell-time datapoint combinations are indicated. Uncertainty in the correlation coefficients is calculated as described in “Methods”. (B) Cell treatment workflow for network reconstruction in the “acute” regime. Single ERK or Akt KTR expressing MCF10A cells were seeded, allowed to attach overnight, and then serum and growth factor starved. Following starvation, inhibitor was added (PD: PD0325091; MK: MK2206), baseline images were acquired, cells were treated with EGF and insulin, and then imaged every 15 min for 12 h. Images were quantified using the analysis pipeline described in the “Methods”. (C) Quantified ERK and Akt activity dynamics in the acute regime. Solid lines are population median (per time point), and shaded areas denote the standard deviation across cells. (D) Cell treatment workflow for network reconstruction in the “chronic” regime. Dual ERK and Akt KTR expressing MCF10A cells were seeded, allowed to attach overnight, and then serum and growth factor starved. Following starvation, EGF and insulin were added, baseline images were acquired, cells were treated with inhibitors, and then imaged every 6.5 min for the remaining ~ hour. Images were quantified using the analysis pipeline described in the “Methods”. (E) Quantified ERK and Akt activity dynamics in the chronic regime. Solid lines are population median (per time point), and shaded areas denote the standard deviation.