The molecular architecture of cell cycle arrest

Abstract

The cellular decision governing the transition between proliferative and arrested states is crucial to the development and function of every tissue. While the molecular mechanisms that regulate the proliferative cell cycle are well established, we know comparatively little about what happens to cells as they diverge into cell cycle arrest. We performed hyperplexed imaging of 47 cell cycle effectors to obtain a map of the molecular architecture that governs cell cycle exit and progression into reversible ("quiescent") and irreversible ("senescent") arrest states. Using this map, we found multiple points of divergence from the proliferative cell cycle; identified stress-specific states of arrest; and resolved the molecular mechanisms governing these fate decisions, which we validated by single-cell, time-lapse imaging. Notably, we found that cells can exit into senescence from either G1 or G2; however, both subpopulations converge onto a single senescent state with a G1-like molecular signature. Cells can escape from this "irreversible" arrest state through the upregulation of G1 cyclins. This map provides a more comprehensive understanding of the overall organization of cell proliferation and arrest.

Keywords: cell cycle; proliferation; quiescence; senescence; single-cell.

Figure 1. Mapping the architecture of cell cycle arrest

1. A

   Schematic of the experimental approach.

2. B

   Cell cycle map of unperturbed cells (N = 11,268 cells). Proliferative (G1/S/G2/M) and arrested (G0) cell cycle phases were predicted for each cell using a Gaussian‐mixture model and labeled on the map.

3. C–I

   (C) Diffusion pseudotime values, (D) phospho/total RB, (E) DNA content, (F) cyclin D1, (G) cyclin A, (H) cyclin B1 and (I) p21 of unperturbed cells are plotted on the map. Median nuclear values are shown for (D–I).

Figure 2. The arrest architecture of hypomitogenic stress

1. A

   Unified cell cycle map of unperturbed (gray) and serum‐starved cells (1 day: light blue, 7 days: dark blue, N = 3,007 cells). The proliferative cell cycle (dotted gray line) and the hypomitogenic arrest trajectory (black dotted line) are indicated on the map. Inset: Each treatment condition is shown individually on the unified map (other conditions are shown in lighter gray).

2. B–F

   (B) Diffusion pseudotime, (C) p21, (D) cyclin D1, (E) DNA content and (F) phospho/total RB of unperturbed (left panels) or serum‐starved cells (right panels) are plotted on the map.

3. G

   Time‐lapse imaging of cyclinD1‐mVenus intensity in unperturbed (control, green) and serum‐starved cells (blue). Cells were serum‐starved for at least 8 h prior to imaging. The solid line represents the population median and the shaded area indicates the 95% confidence interval. N = 105 control cells and N = 111 starved cells.

4. H

   Heatmap of feature intensity along the hypomitogenic arrest trajectory. Features were ordered by hierarchical clustering according to their dynamics along the arrest trajectory. Diffusion pseudotime values were binned and pseudotime values with < 15 cells were excluded from the visualization.

5. I

   Median nuclear p27 abundance in serum‐starved cells is plotted on the map.

Figure EV1. The cell cycle map of unperturbed RPE cells

1. Left: Distribution of nuclear intensity ratios of phospho/total RB in unperturbed RPE cells. A threshold value of 0.7 was used to label cells as arrested (low phospho/total RB) or actively cycling (high phospho/total RB). Right: Cycling and arrested labels are overlaid on the cell cycle map.

2. Cells from three technical replicates are labeled on the cell cycle map.

Figure 3. The arrest architecture of replication stress

1. A

   Unified cell cycle map of unperturbed (gray) and etoposide‐treated cells (1 μM; 1 day: light green, 2 days: green, 3 days: dark green, 4 days: darker green – see inset, N = 4,315 cells). The unperturbed cell cycle trajectory (dotted gray line) and two arrest trajectories (into G0_2C and G0_4C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).

2. B–E

   (B) Diffusion pseudotime, (C) DNA content, (D) phospho/total RB and (E) p21 of unperturbed (left panels) or etoposide‐treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.

3. F

   Time‐lapse imaging of CDK2 activity (DHB‐mCherry, gray) and p21‐YPet (green) intensity in etoposide‐treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 (“Mothers,” N = 32 cells) but arrest following cell division (“Daughters,” N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.

Figure EV2. Arrest trajectories following replication stress

1. A

   Cell cycle map arrest of unperturbed (gray) and etoposide‐treated cells (1 μM; 1 day: light green, 2 days: green, 3 days: dark green, 4 days: darker green). The unperturbed cell cycle (dotted gray line) and two arrest trajectories (into 2C and 4C, pink and purple, respectively) are indicated on the map.

2. B–E

   (B) Phospho‐H2AX, (C) phospho‐CHK1, (D) phospho‐p65 and (E) p53 of unperturbed (left panels) or etoposide‐treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown.

3. F, G

   Heatmap of feature intensity along the (F) 2C and (G) 4C arrest trajectories. Features were ordered by hierarchical clustering according to their dynamics along each arrest trajectory. Diffusion pseudotime values were binned and pseudotime values with < 15 cells were excluded from the visualization.

4. H

   Top: Nuclear phospho‐RB intensities of control and etoposide‐treated MCF10A cells (7 days, 1 μM). Dashed‐red box indicates arrested etoposide‐treated cells (phospho‐RB < 500 au). Bottom: Distribution of DNA content in arrested etoposide‐treated cells. Arrested MCF10A cells were observed with both 2C and 4C DNA content.

Figure EV3. The arrest architecture of oxidative stress

1. A

   Unified cell cycle map arrest of unperturbed (gray) and H₂O₂‐treated cells (200 μM; 1 day: light purple, 2 days: purple, 3 days: dark purple – see inset, N = 5,015 cells). The unperturbed cell cycle (dotted gray line) and two arrest trajectories (into G0_2C and G0_4C; black dotted lines) are indicated on the map. Inset: Each condition is shown individually on the map (other conditions are shown in lighter gray).

2. B–G

   (B) Phospho‐H2AX, (C) phospho‐CHK1, (D) p53, (E) p21, (F) phospho/total RB and (G) DNA content of unperturbed (left panels) or H₂O₂‐treated cells (right panels) are plotted on the arrest architecture. Median nuclear values are shown for B‐F.

3. H, I

   Distribution of (H) phospho‐H2AX and (I) phospho/total RB in individual cells following etoposide (1 μM) or H₂O₂ treatment (200 μM).

Figure 4. Replication stress promotes mitotic skipping and cellular senescence

1. A

   Arrest architecture of replicative stress. 2C and 4C arrest trajectories are shown, the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated. N = 4,315 cells.

2. B

   Progression of arrest states over time following etoposide treatment. Etoposide‐treated cells (upper panel: colored by condition, lower panel: colored by DNA content) populating 2C, 4C, 8C and senescent arrest states at each day of treatment (1–4 days).

3. C

   Distribution of senescence‐associated β‐galactosidase (SA‐βgal) activity in individual cells following etoposide treatment (1 μM, 0–4 days).

4. D–K

   (D) GSK3β, (E) phospho(Thr157)‐p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide‐treated cells are plotted on the map. Median nuclear values are shown in D‐I. Area indicated with a dotted line represents the senescent region of the map.

5. L–O

   (L) Cyclin A, (M) cyclin B1, (N) CDH1 and (O) SKP2 of etoposide‐treated cells are plotted on the map. Median nuclear values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.

Figure 5. Palbociclib‐ and etoposide‐induced arrest trajectories converge on a single state of cellular senescence

1. A

   Distribution of senescence‐associated β‐galactosidase (SA‐βgal) activity in individual cells following etoposide or palbociclib treatment (1 μM, 4/8 days). Control: N = 6,050 cells, etop 4 days: N = 409 cells, etop 8 days: N = 732 cells, palbo 4 days: N = 1837 cells, palbo 8 days: N = 1,677 cells. Statistical significance was determined using a one‐way analysis of variance (ANOVA) with Sidak's post hoc test (***P < 0.0001).

2. B

   Unified cell cycle map of the unperturbed (gray), palbociclib‐ (palbo, 1 μM, 4/8 days, blue) and etoposide‐ (etop,1 μM, 4/8 days, green) treated cells. Control: N = 10,499 cells, etop: N = 2,692 cells, palbo: N = 4,931 cells.

3. C–K

   (C) DNA content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL‐6, (H) IL‐8, (I) phospho‐p38, (J) phospho‐STAT3 and (K) phospho‐STAT5 are plotted on the map. Median nuclear values are shown.

Figure EV4. Replication stress induces mitotic skipping in RPE, MCF10A and U‐2 OS cells

1. A–L

   (A) RPE cells with ≥4C DNA content were selected using the intensity of nuclear Hoechst staining. Cyclin A versus cyclin D1 intensity was plotted for each cell and overlaid with (B) condition labels (control vs. etoposide), (C) phospho‐RB intensity and (D) senescence‐associated β‐galactosidase (SA‐βgal) activity. The same analysis as above was performed on (E–H) MCF10A and (I–L) U‐2 OS cells.

2. M

   Distribution of DNA content in control (blue) and etoposide‐treated (green, 7 days, 1 μM) MCF10A cells. The percent of polyploid cells with DNA content = 8C is shown for both conditions.

Figure 6. Sustained replication stress can induce polyploidy through mitotic skipping and endoreduplication

1. A

   Arrest architecture of replicative stress. The trajectory of endoreduplication following cell cycle arrest and mitotic skipping indicated.

2. B, C

   (B) CDT1 and (C) E2F1 are plotted on the map. Dotted line indicates the trajectory to endoreduplication. Median nuclear values are shown.

3. D

   Representative single‐cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment (1 μM) by time‐lapse imaging. CDK2 activity (DHB‐mCherry, blue), cell cycle phase (PCNA‐mTq2, S phase shown as gray shaded area) and p21‐YPet intensity (green) are plotted versus time of etoposide treatment.

4. E

   Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following siRNA‐mediated knockdown of cyclin D in control and etoposide‐treated cells. Boxplots show data from four independent replicates (gray circles).

5. F

   Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)‐induced upregulation of cyclin D1 in control and etoposide‐treated cells. Boxplots show data from six independent replicates (gray circles).

6. G

   Cyclin A abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)‐induced upregulation of cyclin A2 in control and etoposide‐treated cells. Boxplots show data from six independent replicates (gray circles).

Data information: Statistical significance in right panels of E‐G was determined using a two‐way analysis of variance (ANOVA) with Sidak's post hoc test (***P < 0.001, *P = 0.02). For panels (E–H), boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.

Figure EV5. The arrest architecture of palbociclib‐induced arrest

1. A

   Unified cell cycle map of unperturbed (control 2, gray) and palbociclib‐treated cells (gold) from a separate experiment, plotted with unperturbed (control 1: light blue, from original experiment), serum‐starved (blue) etoposide‐ (etop, green) and H₂O₂‐treated (magenta) cells from initial experiment. Data integration is described in Materials and Methods.

2. B–F

   (B) p27, (C) phospho‐p65, (D) phospho‐AKT, (E) phospho‐BCL2 and (F) SMAD2 are plotted on the map. Median nuclear values are shown. Dotted area indicates senescent region.

Figure 7. The overall architecture of cell cycle arrest

1. A, B

   Summary map of cell cycle arrest. Unperturbed (yellow), serum‐starved (blue), etoposide‐ (green) and H₂O₂‐treated (pink) cells are plotted on a (A) single map. (B) Each condition is shown individually on the map (other conditions are shown in lighter gray). Inferred trajectories of the proliferative cell cycle and all arrest trajectories are shown.

2. C

   Diffusion pseudotime is plotted onto the map of each condition.