Dynamics extracted from fixed cells reveal feedback linking cell growth to cell cycle - PubMed (original) (raw)

Dynamics extracted from fixed cells reveal feedback linking cell growth to cell cycle

Ran Kafri et al. Nature. 2013.

Abstract

Biologists have long been concerned about what constrains variation in cell size, but progress in this field has been slow and stymied by experimental limitations. Here we describe a new method, ergodic rate analysis (ERA), that uses single-cell measurements of fixed steady-state populations to accurately infer the rates of molecular events, including rates of cell growth. ERA exploits the fact that the number of cells in a particular state is related to the average transit time through that state. With this method, it is possible to calculate full time trajectories of any feature that can be labelled in fixed cells, for example levels of phosphoproteins or total cellular mass. Using ERA we find evidence for a size-discriminatory process at the G1/S transition that acts to decrease cell-to-cell size variation.

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Figures

Figure 1

Figure 1

Dynamic information from static data using ERA. (A) Levels of DNA (DAPI) and Geminin (mAG-hGem) in an unsynchronized, exponential population of HeLa cells. The black contour lines denote the distribution, f, of cells within the DNA/Geminin plane. Also shown is the average cell cycle trajectory, I, (red). The different densities of cells in boxes A and B result from the different rates at which cells traverse those regions. (B) The transformation of the cell cycle axis, I, to a real time axis. For convenience we chose 100 units of I to represent a complete path from the beginning of G1 to the end of G2. Comparison of ERA with direct dynamical measurements (C-D). (C) Accumulation of Geminin (mAG-hGem) was followed in 10 individual live cells by time lapse microscopy (blue lines) and compared to the dynamics of mAG-hGem calculated from fixed cells using ERA (red). (D) DNA replication rate was measured by exposing cells to a 20 min. pulse of EdU (5-ethynyl-2′-deoxyuridine, Invitrogen) prior to fixation. Average levels of EdU fluorescence per cell (indicating rate of DNA replication) are plotted (blue) as a function of DAPI, giving a measure of the mean rate of DNA replication as a function of passage through S. For comparison, ERA was used to calculate DNA replication rates directly from fixed cells not treated with EdU (red).

Figure 2

Figure 2

Calculation of growth as a function of cell cycle progression using ERA. (A) Cells were stained and imaged for DNA content (DAPI), the geminin degron (mAG-hGem) and for protein content (succinimidyl ester-Alexa 647 (SE-A647)). (B) Growth during cell cycle progression, computed by ERA. (C) Comparison of SE-A647 fluorescence intensity with quantitative phase microscopy measurements in single cells. Each data point represents a single cell measured for both protein content (SE-647) and phase retardation (QPM). (D) A scatter plot for the levels of DNA (DAPI) Geminin (mAG-hGem) and cell size (SE-A647) in an unsynchronized population of HeLa cells. Also shown is the projection of the 2-dimensional distribution of Geminin and DNA overlaid with I. (E) The 3-dimensional probability distribution from the data shown in 2D (F, G) Growth rate versus time for HeLa (F) and RPE1 (G) calculated by ERA. Confidence intervals are shown as described in Supplementary material.

Figure 3

Figure 3

Rate of cell growth as a function of size and cell cycle. Size discrimination at the G1/S transition. (A) Calculation of growth rate as a function of size for a defined interval, ΔI, on the cell cycle axis, I. To calculate feedback regulation as a function of cell size, the size distributions of cells entering and leaving ΔI are estimated from the sizes of cells on the boundary of ΔI. (B) Plots of growth-rate vs. cell-size at late G1 ( I = 20 ), the G1/S transition ( I = 40) and late S-phase ( I = 80 ). (C,D) The slope, φ, of growth rate vs. cell size plotted as a function of I, for (C), HeLa cells and (D), RPE1 cells. The red horizontal line positioned at φ = 0 is added to emphasize the distinction between positive and negative slopes (positive and negative feedbacks). Blue vertical lines demarcate borders between different stages of cell cycle. Confidence intervals are in gray.

Figure 4

Figure 4

Cell size variability (full width/half maximum) as a function of cell cycle time for HeLa cells incubated for 1hr with (A) 0.001 v/v Dimethylsulfoxide; (B); 1uM cycloheximide; (C ); 10uM G132; M (D) 100nM rapamycin. Confidence intervals are shown in gray.

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