Mammalian cell growth dynamics in mitosis
- University College London, United Kingdom
- Massachusetts Institute of Technology, United States
Figures
Figure 1 with 2 supplements
Various animal cell types grow during mitosis and cytokinesis.
(a) Left, schematic of a suspended microchannel resonator (SMR). Single-cell buoyant mass is repeatedly measured as the cell flows back and forth through the vibrating cantilever. Right, at cell …
-
Figure 1—source data 1
L1210 buoyant mass measurement data.
- https://doi.org/10.7554/eLife.44700.005
Figure 1—figure supplement 1
Suspended microchannel resonator (SMR) setups and noise characterization.
(a) Left, schematic of automated fluid control strategy for continuous single-cell mass measurements. Steps in order: 1) A single cell (pink circle) flows left to right. Flow direction is depicted …
Figure 1—figure supplement 2
Detection of cell cycle transitions.
(a) A table summarizing cellular changes and corresponding signals measured in SMR. (*one refers to Son et al., 2015a; *two refers to Kang et al., 2019). Node deviation is an acoustics-based …
Figure 2 with 2 supplements
Cell mass accumulation persists through prophase, stops as cells approach metaphase-to-anaphase transition and recovers during late cytokinesis.
(a) Mass-normalized mass accumulation rate (MAR) of L1210 cells in late G2 and M-phase. G2/M and metaphase-to-anaphase transitions are indicated with dashed vertical lines. Typical durations of …
Figure 2—figure supplement 1
Mass accumulation rate (MAR) analysis.
(a) Schematic of obtaining the average MAR/mass for late G2, early mitosis and cytokinesis. The following steps have been performed in order. First, metaphase-to-anaphase transition (green star) and …
Figure 2—figure supplement 2
MAR in different growth conditions and cell types.
(a) Mass-normalized MAR of L1210 cells in late G2 and M-phase when grown under indicated FBS and glucose conditions. For comparison, in Figure 2a (as well as all other figures), L1210 cells were …
Figure 3 with 1 supplement
Mitotic protein synthesis dynamics are consistent with mass accumulation dynamics.
(a) Top, schematic of the protocol for quantifying mitotic protein synthesis rates using O-propargyl-puromycin (OPP). G2 synchronization was achieved by double thymidine block followed by RO-3306 …
Figure 3—figure supplement 1
Single-cell protein synthesis assays in mitotic cells.
(a) Top left, schematic indicating the approximate cell cycle stages separated by Cyclin B1 and phospho-Histone H3 (Ser10) antibody labeling. Bottom, representative FACS scatter plots indicating the …
Figure 4
Mitotic arrests result in growth inhibition independently of the mechanism of arrest.
(a) Mass-normalized MAR of 5 µM STLC or 1 mg/ml Nocodazole treated L1210 cells in late G2 and mitosis. Dashed vertical line indicates the approximate mitotic entry. Solid dark lines indicate the …
Figure 5 with 2 supplements
Cells in metaphase and anaphase display stage-specific mass accumulation regulation independently of cell elongation.
(a) Representative L1210 cell phase contrast (grey) and mAG-hGeminin cell cycle reporter (green) images in control (n = 9 cells) and 200 nM Tozasertib (n = 6 cells) treated cells. The degradation of …
Figure 5—figure supplement 1
Mitotic cell swelling affects MAR, but not protein synthesis in early mitosis.
(a) Mass-normalized MAR of control (blue) and 10 µM EIPA (pink) treated L1210 cells. Dashed vertical lines indicate the approximate mitotic entry and metaphase-to-anaphase transition. Solid dark …
Figure 5—figure supplement 2
The low MAR in metaphase and anaphase are not explained by cell elongation or time spent in mitosis.
(a) Examples of individual 200 nM Tozasertib treated L1210 mass-normalized MAR traces. Note that although MAR remains positive around metaphase-to-anaphase transition, the absence of cytokinesis in …
Figure 6 with 4 supplements
CDK1 drives mitotic growth through 4E-BP1 and cap-dependent protein synthesis.
(a) Schematic of growth regulation pathways. Chemical and genetic inhibitors (red), kinases (yellow) and the measured downstream consequences (green) are shown. 1NM-PP1-mediated inhibition of CDK1 …
Figure 6—figure supplement 1
4E-BP1 is phosphorylated in mitosis.
(a) L1210 cell levels of phosphorylated 4E-BP1 (Thr37/46) in mitosis and G2. Antibody isotype control is shown as a specificity control. (n = 5–6 separate cultures). p-Values obtained using ANOVA …
Figure 6—figure supplement 2
mTOR is active in mitosis but is not required for mitotic protein synthesis.
(a) L1210 cell levels of phosphorylated S6RP (Ser235/236) in mitosis and G2. Antibody isotype control is shown as a specificity control. (n = 5–6 separate cultures). (b) L1210 cell levels of …
Figure 6—figure supplement 3
Kinase inhibitors which have MELK kinase as an off-target do not reduce mitotic protein synthesis.
(a) Protein synthesis rates of G2 (blue) and mitotic (red) L1210 cells after 3 hr treatment with 1 µM SNS-032 (CDK2 inhibitor) or 50 nM Tozasertib (Aurora kinase inhibitor), or 5 hr treatment with 1 …
Figure 6—figure supplement 4
shRNA-mediated knockdown of 4E-BP1 in L1210 cells.
(a) Histogram of 4E-BP1 protein levels analyzed by immunostaining and flow cytometry in L1210 cells stably expressing the indicated shRNAs. (b) Quantifications of data in panel (a). n = 3 separate …
Figure 7 with 3 supplements
CDK1-driven mitotic protein synthesis supports daughter cell growth.
(a) MAR during indicated stages of cell cycle in control, 1 µM RO-3306, 30 nM OTSSP167 or 100 nM cycloheximide (CHX) treated L1210 cells. The MAR values were normalized to control mean at each …
Figure 7—figure supplement 1
Correlations between mitotic and G1 cell MAR.
Correlation plot between mother cell MAR in early mitosis (30 min section prior to M/A transition) and daughter cell MAR in early G1 (first 30 min after abscission) for control, 1 µM RO-3306 and 50 …
Figure 7—figure supplement 2
Daughter cell growth rate measurement workflow.
(a) Workflow for measuring MAR, protein synthesis and proliferation rate of G1 cells that have passed through mitosis under control or drug perturbed conditions. Mitotic RO-3306 treatments were …
Figure 7—figure supplement 3
Daughter cells protein synthesis rates in G1 following mitotic growth inhibitions.
Protein synthesis rates of G1 L1210 cells from control (normal mitosis) and from cells that have undergone mitosis in the presence of 1 µM RO-3306 or from cells that have been arrested to mitosis …
Author response image 1
Cell size histograms of control (blue), RO-3306 (red) and STLC (yellow) treated L1210 cells 50 h after release from G2 (or mitotic for STLC) arrest.
Author response image 2
Comparison of cell growth in early mitosis, cytokinesis and early G1 after indicated drug treatments.
Normalized mass accumulation rate is displayed on top, while total mass accumulated is displayed on the bottom. Both formats of data presentation lead to the same conclusion.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (M. musculus) | L1210 | ATCC | Cat#CCL-219; RRID:CVCL_0382 | |
| Cell line (M. musculus) | L1210 - FUCCI | Other | Generated in a previous study (Son et al., 2012), Nature Methods), cells originate from ATCC (Cat#CCL-219). | |
| Cell line (M. musculus) | L1210 - ECACC | ECACC | Cat#87092804 | |
| Cell line (M. musculus) | Fl5.12 | Other | Kindly provided by laboratory of Prof. Matthew Vander Heiden from MIT | |
| Cell line (M. musculus) | BaF3 | RIKEN BioResource Center | Cat#RCB4476 | |
| Cell line (G. gallus) | DT40-CDK1as | Other | Kindly provided by laboratory of Prof. Bill Earnshaw from University of Edinburgh | |
| Cell line (H. sapiens) | S-HeLa | Other | Kindly provided by laboratory of Kevin Elias from Brigham And Women's Hospital | |
| Transfected construct (M. musculus) | Scrambled shRNA | VectorBuilder | Cat#LVS (VB151023-10034) | Refers to a lentiviral construct used to transfect and express the indicated shRNA. |
| Transfected construct (M. musculus) | 4E-BP1 shRNA #1 | VectorBuilder | Cat#LVS (VB181217-1124dqm)-C | Refers to a lentiviral construct used to transfect and express the indicated shRNA. |
| Transfected construct (M. musculus) | 4E-BP1 shRNA #2 | VectorBuilder | Cat#LVS (VB181217-1125ypy)-C | Refers to a lentiviral construct used to transfect and express the indicated shRNA. |
| Biological sample (H. sapiens) | Unpurified buffy coat for isolation of T-cells | Research Blood Components | NA | |
| Antibody | Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit monoclonal Ab (Alexa Fluor 647 Conjugate) | Cell Signaling Technology | Cat#3458; RRID:AB_10694086 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Phospho-Histone H3 (Ser10) (D2C8) XP Rabbit monoclonal Ab (Alexa Fluor 488 Conjugate) | Cell Signaling Technology | Cat#3465; RRID:AB_10695860 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Cyclin B1 (V152) Mouse monoclonal Ab (Alexa Fluor 488 Conjugate) | Cell Signaling Technology | Cat#4112; RRID:AB_491024 | Dilution 1/50 in PBS supplemented with 5% BSA |
| Antibody | Phospho-4E-BP1 (Thr37/46) (236B4) Rabbit monoclonal Ab (PE Conjugate) | Cell Signaling Technology | Cat#7547; RRID:AB_10949897 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Phospho-S6 Ribosomal Protein (Ser235/236) (D57.2.2E) XP Rabbit monoclonal Ab (Alexa Fluor 647 Conjugate) | Cell Signaling Technology | Cat#4851; RRID:AB_10695457 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Rabbit (DA1E) monoclonal Ab IgG XP Isotype Control (PE Conjugate) | Cell Signaling Technology | Cat#5742; RRID:AB_10694219 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | α-Tubulin (11H10) Rabbit monoclonal Ab (Alexa Fluor 488 Conjugate) | Cell Signaling Technology | Cat#5063; RRID:AB_10694858 | Dilution 1/200 in PBS supplemented with 5% BSA |
| Antibody | Cyclin A Mouse monoclonal Ab (H-3) (FITC Conjugate) | Santa Cruz Biotechnology | Cat#sc-271645; RRID:AB_10707658 | Dilution 1/25 in PBS supplemented with 5% BSA |
| Antibody | 4E-BP1 (53H11) Rabbit monoclonal Ab (PE Conjugate) | Cell Signaling Technology | Cat#34470 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Phospho-4EBP1 (Thr37, Thr46) Rabbit monoclonal Ab (4EB1T37T46-A5) | Thermo Fisher Scientific | Cat#MA5-27999; RRID:AB_2745012 | Dilution 1/100 in PBS supplemented with 5% BSA |
| Antibody | Goat polyclonal anti-Rabbit IgG (H + L) Cross-Adsorbed Secondary Antibody (Alexa Fluor 568 Conjugate) | Thermo Fisher Scientific | Cat#A-11011; RRID:AB_143157 | Dilution 1/1000 in PBS supplemented with 5% BSA |
| Commercial assay or kit | Click-iT Plus OPP Alexa Fluor 594 Protein Synthesis Assay Kit | Thermo Fisher Scientific | Cat#C10457 | |
| Commercial assay or kit | T-cell isolation kit | Miltenyi Biotec | Cat#130-096-495 | |
| Commercial assay or kit | Lambda Protein Phosphatase | New England Biolabs | Cat#P0753S | |
| Chemical compound, drug | O-propargyl-puromycin (OPP) | Jena Bioscience | Cat#NU-931–5; CAS:1416561-90-4 | |
| Chemical compound, drug | S-trityl-l-cysteine (STLC) | Sigma-Aldrich | Cat#164739; CAS:2799-07-7 | |
| Chemical compound, drug | Nocodazole | Sigma-Aldrich | Cat#M1404; CAS:31430-18-9 | |
| Chemical compound, drug | MG132 | Sigma-Aldrich | Cat#474787; CAS:133407-82-6 | |
| Chemical compound, drug | proTAME | Thermo Fisher Scientific | Cat#I44001M; CAS:1362911-19-0 | |
| Chemical compound, drug | Vinblastine | Cayman Chemical | Cat#11762; CAS:143-67-9 | |
| Chemical compound, drug | Vincristine | Cayman Chemical | Cat#11764; CAS:2068-78-2 | |
| Chemical compound, drug | TORIN-1 | Tocris Bioscience | Cat#4247; CAS: 1222998-36-8 | |
| Chemical compound, drug | RO-3306 | Cayman Chemical | Cat#15149; CAS:872573-93-8 | We observed the chemical loses its activity within a month when stored in −20°C. All experiments were done using a stock under 2 weeks old. |
| Chemical compound, drug | OTSSP167 (OTS) | Cayman Chemical | Cat#16873; CAS:1431698-10-0 | |
| Chemical compound, drug | 1NM-PP1 | Sigma-Aldrich | Cat#529581; CAS:221244-14-0 | |
| Chemical compound, drug | 4EGI-1 | Cayman Chemical | Cat#15362; CAS:315706-13-9 | |
| Chemical compound, drug | Cycloheximide (CHX) | Cayman Chemical | Cat#14126; CAS:66-81-9 | |
| Chemical compound, drug | Defactinib | Cayman Chemical | Cat#17737; CAS:1073154-85-4 | |
| Chemical compound, drug | PF-3758309 | Cayman Chemical | Cat#19186; CAS:898044-15-0 | |
| Chemical compound, drug | Nintedanib | Cayman Chemical | Cat#11022; CAS:656247-17-5 | |
| Chemical compound, drug | SNS-032 | Cayman Chemical | Cat#17904; CAS:345627-80-7 | |
| Chemical compound, drug | Tozasertib | Cayman Chemical | Cat#13600; CAS:639089-54-6 | Alternative Names : MK 0457, VX 680 |
| Chemical compound, drug | GSK 626616 | R and D Systems | Cat#6638; CAS:1025821-33-3 | |
| Chemical compound, drug | EIPA | Sigma-Aldrich | Cat#A3085; CAS:1154-25-2 | |
| Chemical compound, drug | (-)-Blebbistatin | Sigma-Aldrich | Cat#B0560; CAS:856925-71-8 | |
| Software, algorithm | MATLAB R2014b | MathWorks | Used to analyze the SMR raw data and generate data plots. | |
| Software, algorithm | OriginPro 2019 | OriginLab | Used to perform statistical analyses and generate data plots. | |
| Software, algorithm | Mass accumulation rate analysis code | This paper | Used to analyze the SMR data. Code can be found attached to this manuscript. |
Additional files
-
Source code 1
Mass accumulation rate analysis code.
- https://doi.org/10.7554/eLife.44700.024
-
Transparent reporting form
- https://doi.org/10.7554/eLife.44700.025
