The structure of the human cell cycle

Abstract

Understanding the organization of the cell cycle has been a longstanding goal in cell biology. We combined time-lapse microscopy, highly multiplexed single-cell imaging of 48 core cell cycle proteins, and manifold learning to render a visualization of the human cell cycle. This data-driven approach revealed the comprehensive "structure" of the cell cycle: a continuum of molecular states that cells occupy as they transition from one cell division to the next, or as they enter or exit cell cycle arrest. Paradoxically, progression deeper into cell cycle arrest was accompanied by increases in proliferative effectors such as CDKs and cyclins, which can drive cell cycle re-entry by overcoming p21 induction. The structure also revealed the molecular trajectories into senescence and the unique combination of molecular features that define this irreversibly arrested state. This approach will enable the comparison of alternative cell cycles during development, in response to environmental perturbation and in disease. A record of this paper's transparent peer review process is included in the supplemental information.

Keywords: cell cycle; machine learning; manifold learning; quiescence; senescence; single-cell imaging.

Figure 1.. Probing the structure of the cell cycle.

(A) Schematic of the experimental approach. (B) Distributions of cell cycle phase and age (time since mitosis) of all cells annotated by time-lapse imaging. (C-D) Cell cycle phase (C) and age (D) annotations/predictions (see Methods) of 8850 individual cells mapped onto the structure. Representative images of mitotic cells and their locations on the structure are shown in (C). (E-F) Distribution of RB phosphorylation (phospho/total nuclear intensity) in individual cells (E) and mapped on the structure (F). (G) Proliferative (G1/S/G2/M) and arrest (G0) trajectories through the five canonical phases. Scale bars = 10 μm.

Figure 2.. Measuring the rate of molecular change during the cell cycle.

(A) Diffusion pseudotime (DPT) is mapped onto the cell cycle structure. (B-C) Comparisons of molecular age (pseudotime) and actual cell cycle age along the proliferative (B) and arrest (C) trajectories. Individual cells are colored by cell cycle phase. Mitotic cells are excluded.

Figure 3.. Visualizing the mechanisms of the G1/S transition.

(A) Mechanistic model of the core events regulating progress through G1 and the transition to S. (B-I) Median nuclear intensities of cyclin D1 (B), E2F1 (C), cyclin E (D), Cdh1 (E), cyclin A (F) and p21 (I), DNA content (copy number) (G), and variability in nuclear PCNA intensity (H) are mapped onto cells in the proliferative trajectory (left panels) and plotted against cell cycle age (right panels). Population medians in time courses indicated by solid grey lines and individual cells are colored by cell cycle phase (G1: blue, S: orange, G2: green). Non-cycling (G0) cells (phospho/total RB < 1.6) are shown in grey on the structure and are excluded from time courses.

Figure 4.. Cell cycle exit and re-entry from arrest (G0).

(A) Divergence of cell cycle trajectories into G1 or G0 following cell division and subsequent cell cycle re-entry from G0. Age of newborn cells (<3h since mitosis) is mapped onto the structure. (B-I) The z-normalized nuclear intensity ratio of cyclin D1:p21 (B) and median nuclear expression of cyclin D1 (C), p21 (D), CDK4 (E), cyclin E (F), RB (G), E2F1 (H) and Cdt1 (I) are mapped onto G0/G1 cells. Note the dotted circle representing cell cycle re-entry in F, H and I. Non-G0/G1 cells are shown in grey. (J) List of features with the highest predictive power in a random forest regression model trained to predict arrest duration. (K) Representative single-cell traces of p21-mTq2 (green) and cyclin D1-YFP expression (blue) aligned at anaphase. The dotted line represents cell cycle re-entry as indicated by CDK2 reactivation (an increase in the cytoplasmic:nuclear ratio of DHB-mCherry, not shown). (L) p21 expression at cell cycle exit (1h post-mitosis) versus re-entry (at time of CDK2 reactivation) in individual cells. Statistical significance was determined using a Student’s paired t test. (M) Median nuclear intensities of cyclin D1 and p21 measured by time-lapse imaging at cell cycle re-entry. Individual cells are colored by their duration of arrest. N = 117 cells for panels L and M.

Figure 5.. Molecular signature of cellular senescence.

(A) Graph-based clustering of arrested cells mapped onto the cell cycle structure. Senescent cells are found in cluster 4. (B) List of features with the highest predictive power in a random forest model trained to identify senescent cells (cluster 4) from the rest of the population. (C-D) Cell area (C) and ratio of cytoplasmic area:DNA content (D) mapped onto arrested cells. (E-H) Validated features of senescent cells mapped onto the arrest trajectory of the cell cycle structure (left panels), single-cell distributions of log2-normalized feature intensity in non-senescent (β-galactosidase (β-gal)-negative, green) and senescent (β-gal-positive, orange) cells (middle panels), and representative images of feature and β-gal staining of senescent cells (right panels). All senescent features shown here (and in Figure S6) showed significant differences in feature intensity between non-senescent and senescent populations (p<0.01 using an unpaired Welch t test with unequal variance). (I-K) DNA content (I) and nuclear median intensity of phospho-H2AX (J) and p21 (K) mapped onto the structure. Scale bar = 40 μm. Non-arrested cells (phospho/total RB > 1.6) are shown in grey on cell cycle structures.

Figure 6.. Annotated structure of the human cell cycle.

Dotted lines indicate proliferative and arrest trajectories, as well as exit and re-entry trajectories to/from the proliferative cell cycle. The molecular changes that accompany cell cycle exit/re-entry are also shown, as well as the molecular signature of cellular senescence.