Nuclei determine the spatial origin of mitotic waves
- Laboratory of Dynamics in Biological Systems, Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Belgium
- MeBioS - Biosensors Group, Department of Biosystems, KU Leuven, Belgium
- Department of Pharmaceutical Chemistry, United States
Figures
Box 1—figure 1
Spatial cell cycle coordination in early frog and fly embryos.
Box 2—figure 1
Reconstituting cell cycle oscillations using cell-free extracts.
Figure 1 with 6 supplements
Nuclei serve as pacemakers to organize mitotic waves.
(A) Mitotic waves (orange) in a kymograph of cell-free extract experiment in a 100 µm Teflon tube. Wave dynamics are shown for cell cycle 1–6. For each time point we reduced the data from two to one …
Figure 1—figure supplement 1
Methodology of image analysis of the experiments.
(A) Example of microscope image (top) and binarized image from ilastik (bottom), with in blue pixels recognized as background and orange the nuclei. (B) Intensity profile in blue and the filtered …
Figure 1—figure supplement 2
Analysis of the experiment in panel A, quantifying the time evolution of the number of nuclei, the nuclear size, the internuclear distance, the oscillation period, the intensity of the nuclei, and the observed wave speed.
Analysis of the experiment shown in Figure 1. We plotted as function of the cycle number: the number of nuclei (A), the nuclear size (B), the observed wave speed (C), the period of the oscillation (D…
Figure 1—figure supplement 3
Analysis of the spatial GFP-NLS intensity profile and the internuclear distances for multiple experiments.
Kymographs of the GFP-NLS intensity for eight additional experiments in tubes of 100 and 200 µm, with a corresponding analysis of the spatial GFP-NLS intensity profile and the internuclear …
Figure 1—figure supplement 4
Analysis of the spatial GFP-NLS and Hoechst intensity profile and the internuclear distances.
(A) Mitotic waves (orange) in a kymograph of cell-free extract experiment in a 200 µm Teflon tube, using the GFP-NLS reporter. (B) Same as A, but using DNA staining (Hoechst 33342). C-J show an …
Figure 1—video 1
Video of the cell-free extract experiment in panel A, B.
8.86 hr of experiment in 484 frames, scale bar is 200 µm.
Figure 1—video 2
Video of the cell-free extract experiment in panel C.
15.68 hr of experiment in 79 frames, scale bar is 200 µm.
Figure 2 with 4 supplements
Nuclear density and nuclear import strength control cell cycle period and mitotic wave speed.
(A,B) Wave speed (A) and cell cycle period (B) over time obtained for N = 19 analyzed 100 and 200 µm Teflon tube experiments using the GFP-NLS reporter. Results are pooled from 11 different …
Figure 2—figure supplement 1
Influence of nuclear density on cell cycle period.
Cell cycle period over time obtained for N = 27 analyzed 100 and 200 µm Teflon tube experiments using the GFP-NLS reporter. Results are pooled from 14 different cell-free extracts for four different …
Figure 2—figure supplement 2
Influence of Eg5 kinesin inhibitor on wave speed and cell cycle period.
Wave speed (A) and cell cycle period (B) over time obtained for N = 17 analyzed 200 µm Teflon tube experiments using the GFP-NLS reporter. Results are pooled from three different cell-free extracts …
Figure 2—video 1
Video of the cell-free extract experiment in panel D.
Mitotic waves in a 200 µm wide Teflon tube using a GFP-NLS reporter with ≈ 10 ng/µl of added purified DNA. 14.96 hr of experiment in 94 frames, scale bar is 200 µm.
Figure 2—video 2
Video of the cell-free extract experiment in panels E-G.
Mitotic waves in a 200 µm wide Teflon tube using a GFP-NLS reporter with 40 µM nuclear import inhibitor importazole. Nuclear concentration: ≈ 250 nuclei/µl. 19.49 hr of experiment in 189 frames, …
Figure 3 with 6 supplements
A model where nuclei spatially redistribute cell cycle regulators predicts the location of pacemaker regions.
(A) Schematic of the two phases of the model, interphase (import of regulators) and mitotic phase (diffusion). The cell cycle has a fixed period, which controls the periodic spatial redistribution …
Figure 3—figure supplement 1
Influence of the distance of the outer nuclei to the system boundary on the build-up of regulators at the boundary.
Influence of the distance of outer nuclei to the system boundary on the build-up of regulators at the boundary. (A) Same simulations as in Figure 3E, but continuously varying the distance of the …
Figure 3—figure supplement 2
Influence of system parameters on the build-up of regulators at the boundary.
Influence of system parameters on the build-up of regulators at the boundary. We define the boundary strength as the relative difference of the maximum (at the boundary) with respect to the …
Figure 3—figure supplement 3
Influence of deviations to a perfect nuclear pattern in 1D on the build-up of regulators at the boundary.
(A) Different nuclear positioning influences the concentration profile (blue). The average concentration profile of the control is shown in red for comparison. The black dots denote the positions of …
Figure 3—figure supplement 4
Influence of internuclear distance on the build-up of regulators at the boundary.
Influence of internuclear distance on the build-up of regulators at the boundary. Same simulations as in Figure 3C, but changing the internuclear distance from 150 µm (A) to 100 µm (B) to 80 µm. The …
Figure 3—figure supplement 5
Internuclear distance in tubes of varying width.
Distance analysis of the tube experiments shown in Video 2 of the paper. Tube widths are 100 µm (A), 200 µm (B) and 560 µm (C). From the binarized kymographs, the centers of the nuclei are detected. …
Figure 3—figure supplement 6
Influence of varying system widths in 2D on the build-up of regulators at the boundary.
Strength of the build-up of regulators at the boundary in 2D with increasing system width and number of rows of nuclei. This boundary strength is defined as the relative difference of the maximum …
Figure 4 with 2 supplements
Multiple pacemakers compete to define the direction of mitotic waves.
Time evolution of Equation (21) in Appendix 1 in one spatial dimension. The profile on the right is the time average of the intensity over one cell cycle period (). The intensity is normalized …
Figure 4—figure supplement 1
Competing pacemakers in known PDE models for cell cycle oscillations reproduce similar mitotic wave dynamics.
(A-F) show that models of different complexity are able to capture cell cycle oscillations. (A,D) Core components and interactions of the cell cycle oscillator model (CCO) and the FitzHugh-Nagumo …
Figure 4—figure supplement 2
Boundary-driven waves can exist in spatially-extended systems based on different types of oscillators.
Boundary-driven waves can exist in spatially-extended systems based on different types of oscillators. We study the dynamics of mitotic waves using the same numerical setup as in Figure 4—figure …
Figure 5 with 5 supplements
Wider systems lead to boundary-driven mitotic waves.
Fraction of experiments dominated by internally-driven waves (‘I’) and by boundary driven waves (‘B’), evaluated at the end of each of the imaged tubes of varying width and varying concentration …
Figure 5—figure supplement 1
Kymographs of mitotic waves in tubes of varying width.
Kymographs corresponding to the experiments shown in Video 2 for the tubes of 100 µm (A), 200 µm (B), and 560 µm (C) in diameter. Boundary-driven waves are indicated by blue lines, while mitotic …
Figure 5—figure supplement 2
Kymographs of mitotic waves in thick tubes (560 µm diameter).
Three representative experiments in the thickest tubes with a diameter of 560 µm (corresponding to the situation in Figure 5—figure supplement 1C). Kymographs of mitotic waves (see blue lines) are …
Figure 5—figure supplement 3
Analysis of all experiments, including those that did not cycle or did not show any wave dynamics.
We carried out 120 experiments in total, 89 with a concentration of ∼ 250 nuclei/µL extract and 31 with a concentration of ∼ 60 nuclei/µL extract. These data also included experiments that showed …
Figure 5—figure supplement 4
Robustness of image analysis of experiments.
GFP-NLS strength of internal peaks () vs. the GFP-NLS boundary strength () for , (A) and for , (B). Colors denote the type of observed mitotic waves: orange for boundary-driven waves, …
Figure 5—figure supplement 5
Wave speed and cell cycle period for varying tube width.
Wave speed (A,C) and cell cycle period (B,D) over time obtained for N=27 analyzed Teflon tube experiments using the GFP-NLS reporter. Results are pooled from 15 different cell-free extracts for ≈250 …
Videos
Video 1
Video of cell-free extract experiment in a 200 µm wide Teflon tube imaged in bright-field and using a fluorescent microtubule reporter (HiLyte Fluor 488). The experiment on the bottom (see also Figure 1C) has few nuclei (≈ 30 nuclei/µl), while no nuclei are added in the experiment on the top. In the presence of few nuclei, mitotic waves originate from those nuclei and propagate through the whole tube. In the absence of nuclei, no mitotic waves are observed to travel through the tube. Scale bar is 200 µm.
Video 2
Video of cell-free extract experiments in Teflon tubes of varying diameters (≈ 100, 200, 300 and 560 µm wide) and a thin droplet of ≈ 1 mm wide.
Imaging is done with the GFP-NLS reporter. Mitotic waves are found to originate from the boundary as the system becomes wider. Scale bar is 200 µm.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Xenopus laevis, male and female) | Xenopus laevis | Centre de Res- sources Biolo- giques Xénopes | RRID:XEP_Xla | |
| Recombinant DNA reagent | GFP-NLS | DOI: 10.1038/nature12321 | Construct provided by James Ferrell (Stanford Univ., USA) | |
| Peptide, recombinant protein | (fluorescent) microtubule reporter | Cytoskeleton, Inc | Cat. #: TL488M-B | |
| Commercial assay or kit | GenElute Mammalian Genomic DNA kit | Sigma-Aldrich | Cat. #: G1N70 | |
| Chemical compound, drug | Human chorionic gonadotropin | MSD Animal Health | CHORULON | |
| Chemical compound, drug | Pregnant mare’s serumgonadotropin | MSD Animal Health | FOLLIGON | |
| Chemical compound, drug | Calcium ionophore A23187 | Sigma-Aldrich | PubChem CID: 11957499; Cat. #: C7522 | |
| Chemical compound, drug | Leupeptin | Sigma-Aldrich | PubChem CID: 72429; Cat. #: L8511 | |
| Chemical compound, drug | Pepstatin | Sigma-Aldrich | PubChem CID: 5478883; Cat. #: P5318 | |
| Chemical compound, drug | Chymostatin | Sigma-Aldrich | PubChem CID: 443119; Cat. #: C7268 | |
| Chemical compound, drug | Cytochalasin B | Sigma-Aldrich | PubChem CID: 5311281; Cat. #: C6762 | |
| Chemical compound, drug | Proteinase K | Sigma-Aldrich | Cat. #: P2308 | |
| Chemical compound, drug | Importazole | Sigma-Aldrich | PubChem CID: 2949965; Cat. #: SML0341 | |
| Chemical compound, drug | S-Trityl-L-cysteine | Acros Organics | PubChem CID: 76044; Cat. #: 173010050 | |
| Software, algorithm | Fiji | http://fiji.sc/ | RRID:SCR_002285 | |
| Software, algorithm | Wolfram Mathematica | www.wolfram.com/mathematical | RRID:SCR_014448 | |
| Software, algorithm | Ilastik | www.ilastik.org | RRID:SCR_015246 | |
| Software, algorithm | Model for nuclear import | This paper, used for Figure 3 | Code on GitHub (Nolet, 2020) | |
| Software, algorithm | Model for nuclear import, frequency dependent | This paper, used for Figure 4 | Code on GitHub (Nolet, 2020) | |
| Other | Teflon tube | Cole-Parmer | Cat. #: 06417–11 | |
| Other | Hoechst 33342 | ImmunoChemistry technologies | RRID:AB_265113; Cat. #: 639 | (5 µg/mL) |
| Other | Leica TCS SPE confocal microscope | Leica Microsystems | RRID:SCR_002140 | |
| Other | Ultracentrifuge OPTIMA XPN - 90 | Beckman Coulter | RRID:SCR_018238; Cat. #: A94468 |
