A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes
- European Molecular Biology Laboratory, Germany
Figures
Figure 1 with 1 supplement
Network contraction is characterized by a single rate.
(A) Maximum z-projections of selected time points through the nuclear region of live starfish oocytes expressing H2B-mCherry (cyan) to label chromosomes during actin-driven chromosome congression. …
Figure 1—figure supplement 1
Contraction is independent of anchoring at the animal pole.
(A) Oocytes were injected with phalloidin-AlexaFluor 568 to label the initial contracting network ~ 2 min after the start of NEBD. Prior to injection, oocytes were centrifuged (bottom panel) in …
Figure 2
Concomitant with network contraction new filaments polymerize along its boundary.
(A) A pulse of phalloidin-AlexaFluor 568 (red) was injected into the nuclear region ~2 min after NEBD in an oocyte expressing 3mEGFP-UtrCH (grays) to label the population of F-actin present at the …
Figure 3 with 1 supplement
Forces in the network, rather than pushing by polymerization generate contractility.
(A) Schematics illustrating the expected response to ablation in case of a ‘pushing’ mechanism vs. active contraction of the network. (B) Selected sum-intensity z-projections through the nuclear …
Figure 3—figure supplement 1
Effects of laser ablation are independent of the direction of the cut.
Selected sum-intensity z-projections through the nuclear region of live oocytes expressing 3mCherry-UtrCH. One frame just before and frames after 3D ablation perpendicular to the animal-vegetal axis …
Figure 4 with 1 supplement
Network contraction is correlated with its disassembly.
(A) Quantification of F-actin mass by measuring 3mEGFP- or 3mCherry-UtrCH intensities in the region corresponding to the old network (orange circle). Right: scheme illustrating extrapolation of the …
Figure 4—figure supplement 1
F-actin densities in the old network.
Quantification of F-actin density (mean fluorescence intensity) in the old network in different experiments: (A) in untreated control oocytes (related to Figure 4); (B) in oocytes injected with …
Figure 5 with 1 supplement
Non-muscle myosin II perturbations do not affect the rate of contraction.
(A) Maximum-intensity z-projection through the nuclear region of oocytes expressing H2B-mCherry (cyan), either incubated for 3 hr with blebbistatin (300 μM), for 1 hr with ML-7 (100 μM) or Y-27632 …
Figure 5—figure supplement 1
Myosin Vb tail overexpression does not affect the rate of contraction.
(A) Selected frames from a time lapse of confocal sections through the nuclear region of live oocyte injected with DiIC16(3) to label endomembranes showing the absence of membranous structures in …
Figure 6 with 1 supplement
Stabilization of filaments slows disassembly and contraction rate.
(A) Maximum-intensity z projections of the nuclear region of oocytes expressing H2B-mCherry (cyan) and injected with different amounts of recombinant UtrCH or PBS as control. Dashed circles …
Figure 6—figure supplement 1
The rates of disassembly and contraction are coupled, and predicted by disassembly-driven model.
(A) Model predictions for contraction rate, α0 as a function of the depolymerization rate k0 are compared to experimental data derived from oocytes injected with varying amounts of UtrCH. Top: 10 …
Figure 7 with 1 supplement
Enhancing disassembly speeds up contraction.
(A) Selected frames from a time lapse of confocal sections through the nuclear region of live oocytes expressing mEGFP3-UtrCH (gray). Oocytes were treated with either Latrunculin A (2.5 μM) or a …
Figure 7—figure supplement 1
Enhancing filament disassembly by Latrunculin A leads to chromosome loss during contraction.
Maximum z-projections of selected time points through the nuclear region of oocytes expressing mEGFP3-UtrCH (gray) and injected with H1-AlexaFluor 568 to visualize chromosomes (cyan). Latrunculin A …
Figure 8 with 1 supplement
In silico reconstruction of a disassembly-driven contractile system.
(A) Top: schematic representation of the mechanism by a hypothetical end-tracking cross-linker serving as ‘depolymerization harnessing factor’. Bottom: zoom on two filaments linked by such …
Figure 8—figure supplement 1
Network contraction is independent of the rate of filament production at the boundary.
(A) Selected snapshots of Cytosim simulations for different mechanisms of contraction. Actin filaments are shown in red. (B) Plot of the network radius over time for each simulation shown in (A). (C)…
Figure 9
Inhibiting formin FH2 domain activity slows contraction.
(A) Maximum-intensity z-projection through the nuclear region of oocytes expressing H2B-mCherry (cyan), either incubated with DMSO or SMIFH2 (50 μM). Right: pseudocolored time projection of …
Videos
Video 1
Pulse labeling of the contracting F-actin network.
Oocyte expressing 3mEGFP-UtrCH (gray) was injected with phalloidin-AlexaFluor 568 to label the population of F-actin present at the time of injection. Scale bar: 20 μm.
Video 2
The response of the F-actin network to 3D laser ablation.
3D laser ablation was performed in oocytes expressing mCherry3-UtrCH without (left) or with recombinant UtrCH-AlexaFluor 568 nm (right) injection. Scale bar: 20 μm.
Video 3
Contraction of the F-actin network accelerates upon acute treatment with Latrucunlin A.
Oocytes expressing 3mEGFP-UtrCH to visualize F-actin were acutely treated either with DMSO (left) or Latrunculin A (right). Scale bar: 20 μm.
Video 4
Cytosim simulations of motor- and disassembly-driven mechanisms to generate contraction.
Simulations show production of filaments (gray) while initially present filaments (red) are contracting to transport chromosomes (cyan).
Tables
Table 1
Dimensionless viscoelastic parameters for untreated oocytes.
https://doi.org/10.7554/eLife.31469.011| Parameter name | Symbol | Model M | Model D |
|---|---|---|---|
| Elasticity | 2.1264 | 1.4778 | |
| Contractility | 1.1961 | 1.3082 | |
| Adhesion to boundary | ϵ | 0.6232 | 0.7838 |
| Strain relaxation factor | 9.5898 | 2.3754 | |
| Elastic power law exponent | 2 | 1 | |
| Contractile power law exponent | m | 2 | 1 |
| Viscous power law exponent | 1 | 1 |
Table 2
Disassembly rates k0 (min−1) and dispersion of F-actin length distributions.
https://doi.org/10.7554/eLife.31469.016| UtrCH-stabilized | Lat A-treated* | SMIFH2-treated | ||||||
|---|---|---|---|---|---|---|---|---|
| Control | Low | Medium | High | Control | Lat A | Control | SMIFH2 | |
| k0 | 0.162 | 0.126 | 0.073 | 0.048 | 0.0819 | 0.5106 | 0.1995 | 0.1650 |
| 0.677 | 0.889 | 6.946 | 709.4 | 0.0671 | 14.66 | 1.555 | 1.540 | |
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*The solvent, DMSO had an effect on the viscolelastic parameters even in controls (Supplementary file 3).
Table 3
Contraction rates (min−1 ± S.D.) in response to increasing stabilization (UtrCH).
https://doi.org/10.7554/eLife.31469.017| UtrCH injection | ||||
|---|---|---|---|---|
| Control | Low | Medium | High | |
| Experiment | 0.0831 ± 0.012 | 0.0612 ± 0.011 | 0.0280 ± 0.006 | 0.0164 ± 0.005 |
| Model M | 0.0818 | 0.1821 | 0.2063 | 0.2133 |
| Model D | 0.0825 | 0.0586 | 0.0226 | 0.0117 |
Table 4
Contraction rates (min−1 ± S.D.) in response to SMIFH2 treatment.
https://doi.org/10.7554/eLife.31469.025| Control | SMIFH2 | |
|---|---|---|
| Experiment | 0.086 ± 0.006 | 0.073 ± 0.007 |
| Model M | 0.0859 | 0.1139 |
| Model D | 0.0848 | 0.0675 |
Key resources table
| Reagent type or resource | Designation | Source or reference |
|---|---|---|
| Biological sample | ||
| Patiria miniata | Patiria miniata | https://scbiomarine.com/ |
| Transfected construct | ||
| MRLC (Patiria miniata) | MRLC-mEGFP | doi:10.1038/s41467-017-00979-6 |
| H2B (human) | H2B-mCherry, H2B-3mEGFP | doi:10.1038/nmeth876 |
| Utrophin CH domain (human) | mEGFP3-UtrCH, 3mCherry-UtrCH | doi:10.1002/cm.20226 |
| myosinVb tail domain (mouse) | myosinVb-Tail | doi:10.1038/ncb2802 |
| Peptide, recombinant protein | ||
| Histone H1 (calf) | H1 | Merck |
| Utrophin CH domain (human) | UtrCH | doi:10.1002/cm.20226 |
| Commercial assay or kit | ||
| AmpliCap-Max T7 High Yield Message Maker | AmpliCap-Max T7 High Yield Message Maker | CellScript |
| Poly(A) tailing kit | Poly(A) tailing kit | CellScript |
| Gel filtration column PD-10 | Gel filtration | GE Healthcare |
| Ni-NTA resin | Ni-NTA resin | Qiagen |
| Vivaspin column 10,000 MW | Vivaspin column | Sartorius |
| Alexa Fluor 568 succinimidyl ester | Alexa Fluor 568 succinimidyl ester | Invitrogen |
| Alexa Fluor 488 succinimidyl ester | Alexa Fluor 488 succinimidyl ester | Invitrogen |
| Chemicals, drugs | ||
| DiIC16(3) | DiI | Invitrogen |
| 1-methyladenine | 1-MA | ACROS organics |
| Phalloidin-AlexaFluor 568 | Phalloidin-AlexaFluor 568 | Invitrogen |
| LatrunculinA | Lat A | Abcam |
| SMIFH2 | SMIFH2 | Tocris |
| Y-27632 | Y-27632 | Enzo Life Sciences |
| ML-7 | ML-7 | Tocris |
| Blebbistatin | BB | Abcam |
| Software, algorithm | ||
| Matlab | Matlab | Mathworks |
| Cytosim | Cytosim | doi:10.1088/1367-2630/9/11/427 |
Additional files
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Supplementary file 1
Theory of the viscoelastic gel model for F-actin network.
- https://doi.org/10.7554/eLife.31469.026
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Supplementary file 2
Dimensionless viscoelastic parameters for UtrCH injections.
- https://doi.org/10.7554/eLife.31469.027
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Supplementary file 3
Dimensionless viscoelastic parameters for Latrunculin A treatments.
- https://doi.org/10.7554/eLife.31469.028
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Supplementary file 4
Dimensionless viscoelastic parameters for SMIFH2 treatments.
- https://doi.org/10.7554/eLife.31469.029
-
Supplementary file 5
Cytosim configuration file to simulate a contracting F-actin network.
- https://doi.org/10.7554/eLife.31469.030
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Supplementary file 6
Matlab scripts used to analyze chromosome tracks.
- https://doi.org/10.7554/eLife.31469.031
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Transparent reporting form
- https://doi.org/10.7554/eLife.31469.032
