Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length
- The Hospital for Sick Children, Canada
- University of Toronto, Canada
- Novartis Institutes for BioMedical Research, United States
- University of Michigan, United States
- University of Washington, United States
- Carnegie Mellon University, United States
- Harvard Medical School, United States
Figures
Figure 1 with 5 supplements
Results from a small molecule screen implicate the p38 MAPK pathway in the coordination of cell size and progression through G1.
(A) A flowchart of the chemical screen demonstrating the major steps. (B) Raw data of cell size and cell cycle stage measured from a single control well in the screen. The scatter plot represents …
-
Figure 1—source data 1
The screen metadata used to identify on-axis and off-axis outliers.
- https://doi.org/10.7554/eLife.26947.009
-
Figure 1—source data 2
The analysis script to visualize on-axis and off-axis outliers using Figure 1—source data 1.
- https://doi.org/10.7554/eLife.26947.010
-
Figure 1—source code 1
The Matlab script used to perform the target enrichment analysis.
- https://doi.org/10.7554/eLife.26947.011
Figure 1—figure supplement 1
Percentage of the target class coverage of the MOA Box compounds.
https://doi.org/10.7554/eLife.26947.004
Figure 1—figure supplement 2
Cells from each well were partitioned, according to the three cell cycle indicators (DNA, Geminin, Cdt1), into discrete cell cycle stages.
The scatterplot illustrates a control well (DMSO treated). Each point in the scatterplot is an individual cell. The DNA axis is normalized (1 = 2N, 2 = 4N). The Geminin and Cdt1 axes are in log …
Figure 1—figure supplement 3
Identification of on-axis and off-axis outliers.
The outliers of the screen (as exemplified in Figure 1B) were identified by thresholding (5% significance) the Mahalanobis distance. The outliers were then classified into on-axis or off-axis based …
Figure 1—figure supplement 4
Ranked p-values from the enrichment analysis of compounds that increase cell size variability (by Fisher’s exact test).
Components from the p38 pathway (highlighted) were highly enriched. Specifically, MK2/MAPKAPK2, a direct downstream substrate of p38 is the top-ranking genes that associate with increased cell size …
Figure 1—figure supplement 5
The average z-score of cell size variability corresponding to each of the different target proteins.
To estimate the cell size variability that results from inhibition of a specific protein, z-scores were averaged from all screen compounds targeting that protein. An average cell size variability …
Figure 2 with 4 supplements
Pharmacological inhibition of the p38 MAPK pathway disrupts the coordination of cell size and G1 length.
(A) Quantifying the coordination of cell size and G1 length. Samples of unsynchronized cells were treated with increasing concentrations of rapamycin (a rapamycin concentration series: 0, 0.03, 0.3, …
-
Figure 2—source data 1
Measurements of cell size and cell cycle stages from the chemical inhibitor experiments as shown in Figure 2D, Figure 2—figure supplements 2 and 3.
- https://doi.org/10.7554/eLife.26947.017
Figure 2—figure supplement 1
Western-blot of cell lysates from conditions shown in Figure 2C confirms the chemical inhibitors are efficient towards inhibiting corresponding MAPKs pathway.
Cells were treated with indicated inhibitors for 24 hr before collecting lysates. Anisomycin was added to select wells 1 hr prior to making lysates, to activate MAPK pathways. All inhibitors were …
Figure 2—figure supplement 2
The negative correlation between cell size and proportion of cells in early G1 is perturbed or weakened under p38 inhibition.
Measurements collected in the same experiment as Figure 2C. (A) Scatterplot comparing cells of negative control (DMSO) with cells under p38 inhibition (treated with indicated inhibitor and …
Figure 2—figure supplement 3
Inhibitors of p38 display dose-dependent influence in the coordination of cell size and G1 length.
The p38 inhibitors and three higher concentrations shown here are also included in Figure 2D and Figure 2—figure supplement 2. (A) Cells treated with only rapamycin concentration series (blue) …
Figure 2—figure supplement 4
Quantification of p-p38, p-CREB and p27 in conditions shown Figure 2C.
The bar plot and error bar display mean and SEM across three replicate Western-blot experiments. Treatment of rapamycin or Torin-2 increases both p-p38 and p-CREB, confirming that activity in the …
Figure 3 with 3 supplements
Inhibition of p38 weakens the coordination of cell size and G1 length at a single-cell level.
(A) Live cells subject to p38 inhibition (SB203580) or to mTORC1 inhibition (rapamycin) were followed with time-lapse microscopy to monitor proliferation over a period of 50 hr. mTOR inhibition …
-
Figure 3—source data 1
Estimation of cell cycle duration and growth rate from bulk measurements of fixed cell populations.
- https://doi.org/10.7554/eLife.26947.022
-
Figure 3—source data 2
Measurements of single-cell dynamics of cell size captured by live-cell imaging.
- https://doi.org/10.7554/eLife.26947.023
Figure 3—figure supplement 1
Cellular growth rate also negatively correlates with G1 or cell cycle duration.
(A–C) Scatterplots displaying relationship between average growth rate in G1 stage with G1 duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. (D–F) Scatterplots …
Figure 3—figure supplement 2
Cell size at birth is negatively correlates with cell cycle duration. p38 inhibition, but not mTORC1 inhibition weakens this correlation.
(A–C) Scatterplots displaying relationship between nuclear size at birth with cell cycle duration for individual cells in DMSO control, p38 inhibition and mTOR inhibition. The points with error bar …
Figure 3—figure supplement 3
Inhibition of p38 MAPK increases cell size variability (A), promote proliferation (B) by shortening G1 length (C) without significant effect in S/G2 duration (D and E) or cellular growth rate (F).
Measurements were performed with the same experiments as indicated in Figure 3B. Shown here is representative of three independent replicate experiments. The meta data and source code used to for …
Figure 4 with 2 supplements
Knockdown of p38 pathway components disturbs the negative correlation between cell size and proportion of cells in G1.
Cells were transfected with siRNA as indicated and subsequently assayed with a rapamycin concentration series (0, 0.03, 0.1, 0.3, 3 and 30 nM) as described in Figure 2A to assay the correlation of …
-
Figure 4—source data 1
Binding activity (Kd’s in nM) of the p38 inhibitors used in the study against each of the p38 isoforms.
Kd values in the table were extracted from Davis et al. (2011). As marked in that paper, blank fields indicate combinations that were tested, but for which binding was weak (Kd >10 μM), or not detected in a 10 μM primary screen.
- https://doi.org/10.7554/eLife.26947.027
-
Figure 4—source data 2
Measurements of cell size and cell cycle stage from the knockdown experiments as shown in Figure 4.
- https://doi.org/10.7554/eLife.26947.028
Figure 4—figure supplement 1
Western-blot of cell lysates from conditions shown in Figure 3 confirms efficiency of knockdown of MKKs (A) or p38 isoforms (B).
https://doi.org/10.7554/eLife.26947.025
Figure 4—figure supplement 2
Cells are still cycling upon the knockdown treatments.
Cell count (A) and DNA histogram (B) 2 days post siRNA transfection corresponding to each of the knockdown conditions. Cell number was quantified by imaging the central region of the wells (~50% …
Figure 5 with 5 supplements
The p38 MAPK pathway is selectively upregulated in small cells.
(A) Cells were treated with either 50 nM of Torin-2 or DMSO (control) for 20 hr, followed by drug wash-out and media replacement. Cells undergoing mTOR inhibition, on average, decrease in size and …
-
Figure 5—source data 1
Measurements of cell size and p38 KTR as shown in Figure 5C and Figure 5—figure supplement 4.
- https://doi.org/10.7554/eLife.26947.035
Figure 5—figure supplement 1
Cells are still cycling upon Torin-2 treatment.
Measurements were obtained by time-lapse microscopy with cells expressing fluorescent markers of H2B. Cells were imaged and counted every 15 min over 22 hr. Compound treatment were performed at time …
Figure 5—figure supplement 2
Representative images showing response of the p38 KTR to indicated treatments.
The p38 KTR functions by translocating to the cytoplasm once p38 is activated. Cells were imaged after a 1 day treatment with DMSO (control), 3 nM rapamycin or 5 μM SB203580 (p38 inhibitor), or a 30 …
Figure 5—figure supplement 3
Immunofluorescence images of cells stained with a phospho-p38 antibody after indicated treatments.
Cells treated with either DMSO (control), 30 nM rapamycin or 1 μM cycloheximide for 1 day, or with 25 ng/mL Anisomycin (a stimulator of the p38 pathway) for 30 min were fixed and imaged. While …
Figure 5—figure supplement 4
p38 activity negatively correlates with cell size in G1 but not S or G2 cells.
Measurements were obtained from the same experiment as indicated in Figure 5C. Cells express dual reporters of both p38 MAPK and JNK were treated with a concentration series of rapamycin, decreasing …
Figure 5—figure supplement 5
Average cell size measured by Coulter counter after the cells were released from the indicated compound treatment.
The measurements were collected in parallel with the experiments as shown in Figure 5D. Cells were treated with either 50 nM Torin-2 or 1 μM Cycloheximide for a period of 22 hr, after which the …
Figure 6 with 1 supplement
Inhibition of p38, but not ERK or JNK, represses recovery of size in cells released from mTOR inhibition.
(A) Workflow of the experiment. Cells were treated with 50 nM Torin-2 with or without the indicated MAPK inhibitors for 22 hr, and then released from Torin-2 while still being subject to the …
-
Figure 6—source data 1
Cell size dynamics after released from mTOR inhibition.
- https://doi.org/10.7554/eLife.26947.038
Figure 6—figure supplement 1
Recovery in cell size is delayed even after the p38 inhibitor was wash-out.
(A) Workflow of the experiment. Similarly as indicated in Figure 6, Cells were co-treated with both Torin-2 (50 nM) with or without the indicated MAPK inhibitor for 22 hr. The cells were then …
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.26947.039
