Fig 1.
Single-cell IF methods for identifying cells in G0, G1, S, G2, and M phases of the cell cycle.
(A) Cell-cycle signaling network depicting in red the proteins measured in this study. (B) Density scatter plot of EdU versus DNA content used to define G0/G1, early S, S, late S, and G2/M populations, as marked by the boxed populations. Note that y-axis units for Fig 1 B–D are in log base 10, such that, for example, a y-axis value of 2.8 = 102.8 = 631; y-axis units are arbitrary fluorescence units. (C) Density scatter plot of phospho-Rb-S807/811 versus DNA content. EdU-negative cells with approximately 2N DNA content can be subdivided into a G0/quiescent population with hypo-phosphorylated Rb and a G1 population with hyper-phosphorylated Rb, as marked by the boxed populations. (D) Density scatter plot of phospho-Histone H3 versus DNA content. EdU-negative cells with approximately 4N DNA content can be subdivided into a G2 population with no phospho-Histone H3 signal and a mitotic population that is phospho-Histone H3-positive, as marked by the boxed population. (E) Top: Cells were tracked by live-cell imaging for 24 hours using H2B-mTurquoise as a nuclear marker, while simultaneously monitoring CDK2 activity using DHB-mVenus. Cells were then fixed and stained with various antibodies against proteins of interest (POI). The IF image was precisely aligned to the time-lapse movie using a custom jitter correction algorithm so as to match each cell’s history with its IF signal; see Materials and methods. Bottom: Single-cell traces of CDK2 activity aligned computationally to the time of the last anaphase of the movie; see Materials and methods. Traces were computationally classified and manually verified as CDK2inc (blue), CDK2low (red), or CDK2emerge (green) based on CDK2 activity after mitosis: CDK2inc traces must remain ≥0.5 for all frames post-anaphase; CDK2low traces must remain <0.5 or all frames post-anaphase; CDK2emerge traces initially enter the CDK2low state and then emerge–these traces must remain <0.5 for at least 3 hours post-anaphase before rising at some point during the imaging period. The percentage of the total population in each category is indicated; error represents the standard deviation across 96 replicate wells. (F) Prolonged quiescent cells (purple) are defined as cells with CDK2 activity <0.6 for all 24 hours of imaging. These cells did not undergo mitosis. The percentage of the total population with this behavior is indicated; error represents the standard deviation across 96 replicate wells. (G) Single-cell traces of CDK2 activity for CDK2emerge cells are aligned computationally to the time of CDK2 activity buildup (the R-point; see Materials and methods). Heterogeneity in the time these cells spent in quiescence prior to emerging can be seen as variability in the timing of the drop in CDK2 activity that marks the previous mitosis. (H) Time-lapse imaging of CDK2 activity in asynchronous cells was followed by a 15-minute pulse of 10 μM EdU, fixation, and visualization of the EdU signal in the Cy5 channel. The Cy5 fluorescence image was precisely aligned to the time-lapse movie using a custom jitter correction algorithm. This enabled matching of the CDK2 activity trace of each cell to its EdU level at the end of the movie. EdU levels were then reconstructed as a function of time-since-anaphase for CDK2inc cells (blue dots) and CDK2low cells (red dots). Each dot represents a single cell. The level of the EdU signal in prolonged quiescent cells is plotted at the 24-hour mark (purple dots). Based on these data, we designate G1, S, and G2 phases of the cell cycle using bars above this plot and apply this classification to Figs 3 and 5. Number of cells plotted: 978. (I) Moving average through the blue, red, or purple points from (H). Error bars represent standard deviation. (J) EdU levels as a function of time since CDK2 activity buildup (R-point). Number of cells plotted: 174. All data are from asynchronous MCF10A cells. Abbreviations: APC/C, anaphase-promoting complex/cyclosome; CDK2, Cyclin-Dependent Kinase 2; IF, immunofluorescence; POI, proteins of interest; R-point, Restriction Point; Rb, retinoblastoma protein.
Fig 2.
Protein levels for asynchronous MCF10A cells in G0, G1, S, G2, and M phases of the cell cycle.
(A–H) Column 1: Density scatter of the indicated protein versus DNA content; data are pooled from 9 IF images from 1 representative well. Column 2: Contour plot of the indicated protein versus DNA content; contours are color-coded by cell-cycle phase according to the legend. Data are pooled from 9 IF images from 1 representative well. Column 3: Histogram (probability density) of the indicated protein for G0 cells (purple, defined as 2N DNA content, EdU-negative, and hypo-phosphorylated Rb) versus G1 cells (blue, defined as 2N DNA content, EdU-negative, and hyper-phosphorylated Rb). Two biological replicates are shown; each replicate represents 9 pooled IF images. Note that the y-axes for Column 1 and Column 2, and the x-axis for Column 3, are in log base 10; units are arbitrary fluorescence units. All signals are nuclear except where indicated. Also note that because of cell rounding during mitosis, IF signal intensities are artificially high in mitotic cells. Abbreviation: Cyto-CycB1, Cytoplasmic Cyclin B1 signal; IF, immunofluorescence; Rb, retinoblastoma protein.
Fig 3.
Protein dynamics for proliferating and spontaneously quiescent MCF10A cells.
(A–I) Column 1: Time-lapse imaging of CDK2 activity in asynchronous cells was followed by fixation and IF staining for the indicated protein. Protein signals were then reconstructed as a function of time-since-anaphase for CDK2inc cells (blue dots) and CDK2low cells (red dots), as in Fig 1H. The protein signal in prolonged quiescent cells is plotted at the 24-hour mark (purple dots). The approximate time spent in G1, S, or G2 is marked above the plots, based on EdU incorporation data from Fig 1H. Column 2: Moving average through the blue, red, or purple points from Column 1, as in Fig 1I. Error bars represent standard deviation. Column 3: Protein signal as a function of time since CDK2 activity buildup (R-point), as in Fig 1J. The y-axis units are arbitrary fluorescence units. Total number of cells plotted: (A) 1,949; (B) 433; (C) 1,549; (D) 1,729; (E) 2,596; (F) 1,043; (G) 1,885; (H) 2,117; (I) 1,300. The data for each antibody are pooled from 8 replicate wells (1 image per well). Abbreviations: CDK2, Cyclin-Dependent Kinase 2; IF, immunofluorescence; R-point, Restriction Point.
Fig 4.
Validation of antibody staining in serum-starved cells and validation of Cyclin D1 results by time-lapse imaging of mCitrine-Cyclin D1.
(A–B) Cells were serum starved for the indicated time and analyzed by western blotting (A) or IF (B). (B) Column 1: Quantification of western blots in (A). Column 2: Average IF signal of the indicated antibody signal across 3 replicate wells upon serum starvation for the amounts of time indicated in (A). Error bars represent standard deviation from 3 replicate wells. Column 3: Probability density of the indicated antibody signal at 0, 16, 48, 96, and 168 hours after serum withdrawal. (C) Time-lapse imaging of asynchronous mCitrine-Cyclin D1 knock-in MCF10A cells expressing H2B-mTurquoise and mCherry-tagged CDK2 sensor. Single cell traces of CDK2inc and CDK2low cells are colored blue and red, respectively, and computationally synchronized to anaphase onset. Upper panel: traces of CDK2 activity in control treatment. Middle panel: corresponding traces of mCitrine-Cyclin D1 level in control treatment. Lower panel: traces of mCitrine-Cyclin D1 level in cells that received 1.4 μM MLN4924 at 1-2 hours after anaphase onset. (D) Examples of single-cell traces of CDK2 activity and mCitrine-Cyclin D1 in 1 CDK2inc cell (top) and 1 CDK2low cell (bottom). In CDK2inc cells, mCitrine-Cyclin D1 levels are moderate in G1, fall in S phase, and increase again in G2, whereas mCitrine-Cyclin D1 levels begin to increase steadily shortly after mitosis in CDK2low cells. Orange arrows mark mitoses. Abbreviations: CDK2, Cyclin-Dependent Kinase 2; IF, immunofluorescence; R-point, Restriction Point.
Fig 5.
Data synthesis to generate maps of protein dynamics during cell-cycle progression and cell-cycle exit.
(A) Moving average traces from Fig 3 Column 2 were normalized such that the minimum signal experienced across CDK2inc and CDK2low data for a given protein was set to 0, and the maximum signal experienced across CDK2inc and CDK2low data for this protein was set to 1. The approximate time spent in G1, S, or G2 phases is marked above the plots, based on EdU incorporation data from Fig 1H. Group 1 (top row): protein signals that are “off” in quiescent cells. Group 2 (bottom row): protein signals that change dynamically over time in quiescent cells. (B) Schematized representation of the data from (A).