Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Activator–inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium

Abstract

Animal cell cytokinesis results from patterned activation of the small GTPase Rho, which directs assembly of actomyosin in the equatorial cortex. Cytokinesis is restricted to a portion of the cell cycle following anaphase onset in which the cortex is responsive to signals from the spindle. We show that shortly after anaphase onset oocytes and embryonic cells of frogs and echinoderms exhibit cortical waves of Rho activity and F-actin polymerization. The waves are modulated by cyclin-dependent kinase 1 (Cdk1) activity and require the Rho GEF (guanine nucleotide exchange factor), Ect2. Surprisingly, during wave propagation, although Rho activity elicits F-actin assembly, F-actin subsequently inactivates Rho. Experimental and modelling results show that waves represent excitable dynamics of a reaction–diffusion system with Rho as the activator and F-actin the inhibitor. We propose that cortical excitability explains fundamental features of cytokinesis including its cell cycle regulation.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Cortical waves of actin assembly and disassembly in activated frog eggs and embryos.
Figure 2: Cortical F-actin waves in activated frog eggs are Rho-dependent and are accompanied by Rho activity waves.
Figure 3: Rho activity waves in starfish oocytes and embryos.
Figure 4: F-actin assembly fronts directly follow, but overlap minimally with, Rho activity waves.
Figure 5: Analysis of Rho and F-actin dynamics reveals cortical excitability and spiral turbulence.
Figure 6: Cdk1 gates excitability.
Figure 7: Excitability does not require spindles but is locally modulated by microtubules.
Figure 8: Model of excitable dynamics predicts that Ect2 spatio-temporal distribution determines the pattern of Rho–actin cortical activity.

References

  1. Bement, W. M., Miller, A. L. & von Dassow, G. Rho GTPase activity zones and transient contractile arrays. Bioessays 28, 983–993 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Su, K.-C., Bement, W. M., Petronczki, M. & von Dassow, G. An astral simulacrum of the central spindle accounts for normal, spindle-less, and anucleate cytokinesis in echinoderm embryos. Mol. Biol. Cell 25, 4049–4062 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  3. Green, R. A., Paluch, E. & Oegema, K. Cytokinesis in animal cells. Annu. Rev. Cell Dev. Biol. 28, 29–58 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Rappaport, R. Cytokinesis in Animal Cells (Cambridge Univ. Press, 1996).

    Book  Google Scholar 

  5. Shuster, C. B. & Burgess, D. R. Transitions regulating the timing of cytokinesis in embryonic cells. Curr. Biol. 12, 854–858 (2002).

    Article  CAS  PubMed  Google Scholar 

  6. Canman, J. C., Hoffman, D. B. & Salmon, E. D. The role of pre- and post-anaphase microtubules in the cytokinesis phase of the cell cycle. Curr. Biol. 10, 611–614 (2000).

    Article  CAS  PubMed  Google Scholar 

  7. Miller, A. L. & Bement, W. M. Regulation of cytokinesis by Rho GTPase flux. Nat. Cell Biol. 11, 71–77 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Weiner, O. D., Marganski, W. A., Wu, L. F., Altschuler, S. J. & Kirschner, M. W. An actin-based wave generator organizes cell motility. PLoS Biol. 5, e221 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Burkel, B. M., von Dassow, G. & Bement, W. M. Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin. Cell Motil. Cytoskeleton 64, 822–832 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nat. Methods 5, 605–607 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Yoo, S. K. et al. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev. Cell 18, 226–236 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Straight, A. F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science 299, 1743–1747 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Su, K.-C., Takaki, T. & Petronczki, M. Targeting of the RhoGEF Ect2 to the equatorial membrane controls cleavage furrow formation during cytokinesis. Dev. Cell 21, 1104–1115 (2011).

    Article  CAS  PubMed  Google Scholar 

  14. Benink, H. A. & Bement, W. M. Concentric zones of active RhoA and Cdc42 around single cell wounds. J. Cell Biol. 168, 429–439 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Piekny, A. J. & Maddox, A. S. The myriad roles of Anillin during cytokinesis. Semin. Cell Dev. Biol. 21, 881–891 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Lechleiter, J., Girard, S., Peralta, E. & Clapham, D. Spiral calcium wave propagation and annihilation in Xenopus laevis oocytes. Science 252, 123–126 (1991).

    Article  CAS  PubMed  Google Scholar 

  17. Shibata, T., Nishikawa, M., Matsuoka, S. & Ueda, M. Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using quantitative image analysis. J. Cell Sci. 125, 5138–5150 (2012).

    Article  CAS  PubMed  Google Scholar 

  18. Arai, Y. et al. Self-organization of the phosphatidylinositol lipids signaling system for random cell migration. Proc. Natl Acad. Sci. USA 107, 12399–12404 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Allard, J. & Mogilner, A. Traveling waves in actin dynamics and cell motility. Curr. Opin. Cell Biol. 25, 107–115 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Goryachev, A. B. & Pokhilko, A. V. Dynamics of Cdc42 network embodies a Turing-type mechanism of yeast cell polarity. FEBS Lett. 582, 1437–1443 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Taniguchi, D. et al. Phase geometries of two-dimensional excitable waves govern self-organized morphodynamics of amoeboid cells. Proc. Natl Acad. Sci. USA 110, 5016–5021 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Winfree, A. T. Electrical turbulence in three-dimensional heart muscle. Science 266, 1003–1006 (1994).

    Article  CAS  PubMed  Google Scholar 

  23. Von Dassow, G., Verbrugghe, K. J., Miller, A. L., Sider, J. R. & Bement, W. M. Action at a distance during cytokinesis. J. Cell Biol. 187, 831–845 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Murray, A. W. & Kirschner, M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280 (1989).

    Article  CAS  PubMed  Google Scholar 

  25. Gray, N., Détivaud, L., Doerig, C. & Meijer, L. ATP-site directed inhibitors of cyclin-dependent kinases. Curr. Med. Chem. 6, 859–875 (1999).

    CAS  PubMed  Google Scholar 

  26. Bement, W. M., Benink, H. A. & von Dassow, G. A microtubule-dependent zone of active RhoA during cleavage plane specification. J. Cell Biol. 170, 91–101 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Rappaport, R. & Ebstein, R. P. Duration of stimulus and latent periods preceeding furrow formation in sand dollar eggs. J. Exp. Zool. 158, 373–382 (1965).

    Article  CAS  PubMed  Google Scholar 

  28. Rankin, S. & Kirschner, M. W. The surface contraction waves of Xenopus eggs reflect the metachronous cell-cycle state of the cytoplasm. Curr. Biol. 7, 451–454 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Lim, D. et al. The M-phase-promoting factor modulates the sensitivity of the Ca2+ stores to inositol 1,4,5-trisphosphate via the actin cytoskeleton. J. Biol. Chem. 278, 42505–42514 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Chang, J. B. & Ferrell, J. E. Mitotic trigger waves and the spatial coordination of the Xenopus cell cycle. Nature 500, 603–607 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pérez-Mongiovi, D., Chang, P. & Houliston, E. A propagated wave of MPF activation accompanies surface contraction waves at first mitosis in Xenopus. J. Cell Sci. 111, 385–393 (1998).

    PubMed  Google Scholar 

  32. Burkel, B. M., Benink, H. A., Vaughan, E. M., von Dassow, G. & Bement, W. M. A Rho GTPase signal treadmill backs a contractile array. Dev. Cell 23, 384–396 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Charras, G. T., Hu, C.-K., Coughlin, M. & Mitchison, T. J. Reassembly of contractile actin cortex in cell blebs. J. Cell Biol. 175, 477–490 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Berndt, J. D., Clay, M. R., Langenberg, T. & Halloran, M. C. Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo. Dev. Biol. 324, 236–244 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Clay, M. R. & Halloran, M. C. Rho activation is apically restricted by Arhgap1 in neural crest cells and drives epithelial-to-mesenchymal transition. Development 140, 3198–3209 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Holmes, W. R., Carlsson, A. E. & Edelstein-Keshet, L. Regimes of wave type patterning driven by refractory actin feedback: transition from static polarization to dynamic wave behaviour. Phys. Biol. 9, 046005 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Ryan, G. L., Petroccia, H. M., Watanabe, N. & Vavylonis, D. Excitable actin dynamics in lamellipodial protrusion and retraction. Biophys. J. 102, 1493–1502 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Woolner, S., Miller, A. L. & Bement, W. M. Imaging the cytoskeleton in live Xenopus laevis embryos. Methods Mol. Biol. 586, 23–39 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Reyes, C. C. et al. Anillin regulates cell-cell junction integrity by organizing junctional accumulation of Rho-GTP and actomyosin. Curr. Biol. 24, 1263–1270 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Futatsumori-Sugai, M. et al. Utilization of Arg-elution method for FLAG-tag based chromatography. Protein Expr. Purif. 67, 148–155 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Many thanks to S. Maslakova for laboratory space and to B. Dlouhy-Massengale and S. Yang for technical assistance. This work was supported by the National Institutes of Health (GM52932 to W.M.B.) and the National Science Foundation (NSF MCB-0917887 and MCB-1041200 to G.v.D.) and by NIH instrumentation grant 1S10RR026729-01 (K. Eliceiri PI).

Author information

Authors and Affiliations

Authors

Contributions

W.M.B. and A.L.M. performed frog experiments; G.v.D. and W.M.B. performed starfish experiments. A.B.G. and M.L. developed the model, performed simulations, and conducted data analyses. A.M.M., A.M.K., M.E.L., A.E.G., C.P. and K.-C.S. generated and tested reagents. W.M.B., G.v.D. and A.B.G. wrote the manuscript.

Corresponding authors

Correspondence to William M. Bement, Andrew B. Goryachev or George von Dassow.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Cortical waves of actin assembly and disassembly in activated frog eggs and embryos.

(a) Surface view of frog blastomeres expressing GFP-lifeact to label F-actin. Top: single frame; cortical F-actin consists of irregularly sized patches throughout cortex, which, as illustrated in the kymograph made from the outlined region (bottom) travel continually across the surface. Bottom: In the kymograph, F-actin waves create slanted bands with semi-regular spacing. (b) High magnification surface view of activated frog egg expressing GFP-UtrCH. Top: single frame; cortical F-actin consists of irregularly sized patches, which, as illustrated in the kymograph made from the outlined region (bottom) travel continually across the surface. Bottom: In the kymograph, F-actin waves create slanted bands with semi-regular spacing. (c) High magnification surface view of full-grown (prophase 1-arrest) frog oocyte expressing GFP-UtrCH. Top: single frame; cortical F-actin is relatively uniform with no obvious patches and no apparent waving behavior as illustrated in the kymograph made from the outlined region (bottom). Bottom: In the kymograph, cortical F-actin is quite stable. (d) Single frame from time-series of frog embryo co-expressing GFP-Lifeact and mCherry-UtrCH, single channels with pseudocoloring as shown in swatch (pure Lifeact would be yellow; pure UtrCH would be blue; equal blend of each by intensity would be gray). Diagonal band on left panel is the zone for kymograph in Fig. 1d; boxed regions 1 and 2 blown up 2x in d′ show wave fronts colliding. In each, the wave fronts are initially yellowish, turn bluish after collision, then fade. Images are representative of at least 10 (a), at least 30 (b) or 3 (c, d)independent experiments, respectively.

Supplementary Figure 2 Cortical Rho activity waves and Rho-Ect2-dependence of cortical F-actin waves in activated frog eggs and frog embryos.

(a) Time-lapse sequence from activated frog egg expressing GFP-UtrCH to label F-actin and injected with C3 exotransferase to inhibit Rho (6 s intervals). No cortical F-actin waves are present but actin assembly occurs nonetheless as shown by comet like movement of F-actin-associated invagination. (b) Frog embryo expressing GFP-UtrCH and then microinjected with dominant negative Ect2 (left blastomere) or, as a control, nothing (right blastomere); top-still frames, bottom, kymograph. Dominant negative Ect2 eliminates the F-actin waves in the dominant negative Ect2 expressing cell. (c) Frog embryo microinjected with recombinant GFP-rGBD protein to label active Rho. Single frame (top) and kymograph (bottom), raw (left) and subtracted data (right, t0–t−3), highlighting rising Rho activity. The kymograph demonstrates that what otherwise looks like mundane inhomogeneity in the still image actually reflects regular waves of Rho activity; these are more evident in the processed half of both the image and kymograph. (d) Activated frog egg expressing GFP-anillin. Single frame (top) and kymograph (bottom), raw (left) and subtracted data (right, t0–t−3), highlights waves of cortical anillin recruitment. (e) Time-lapse sequence from activated frog egg expressing 3XGFP-rGBD to label active Rho and overexpressing Ect2. Top: Each panel is 20 s after previous panel. While the Rho wave patterns change rapidly every 20 s, note the repetition evident every 100 s (compare panels as columns). Bottom: inverted, black and white representation of dominant wave pattern in each column. Images are representative of 3 (a, b, d), 4 (c) and 6(e) independent experiments, respectively.

Supplementary Figure 3 Cortical Rho activity waves in starfish blastomeres.

(a) Image from time-lapse movie showing waves of cortical Rho activity within ingressing zygote furrows. (b, b′) Top: Images from time-lapse movie showing cortical Rho activity waves at equator of dividing blastomeres. Bottom: kymographs taken from blastomeres above. Waves persist during much of furrow ingression. See Supplementary Video 22 . (c) Top: Image from time-lapse movie showing cortical Rho activity at equator of four different blastomeres. Bottom: kymographs taken from blastomeres above. Waves are present during ingression. See Supplementary Video 23 . (d) Top: Image from time-lapse movie showing cortical Rho activity in later, smaller blastomeres than those in a-c. Rho activity at equator is more continuous. Bottom: Kymographs taken from blastomeres indicated above. Rho activity is largely continuous rather than wave-like. See Supplementary Video 24 . Images representative of 4 independent experiments.

Supplementary Figure 4 Reconstruction of excitable dynamics.

(a) Projection of full wavelength onto plane of normalized concentrations (Rho, actin) generates a closed loop parameterized by phase (−π,π) and colour-coded. Loop computed using activator-inhibitor model introduced here. Domain boundaries of high/low values of Rho and F-actin used to reconstruct kinetic relationships in panels b,c denoted by straight-line segments. (b) Left panel: activator-inhibitor model prediction (Rho—activator, F-actin—inhibitor) for dependence of d[Rho]/dt on [Rho] based on panel a. Parameterization by phase shown for comparison with a. Right panel: d[Rho]/dt versus [Rho] from images (see Methods). Data shown as mean ± s.d. Note qualitative agreement between data and model prediction. (c) Left panel: activator-inhibitor model prediction (Rho—activator, F-actin—inhibitor) for dependence of d[actin]/dt on [actin] based on panel a. Right panel: d[actin]/dt versus [actin] from images. Note qualitative agreement between data and model prediction. Results are mean + s.d.; n = 900 cycles. (d) Phase of excitable dynamics reconstructed from Rho time series (top panel) measured at cortex. Morlet transform of time series generates amplitude and phase functions for every time point and candidate temporary period of dynamics. Amplitude maxima (yellow dashed line) show how most likely value of period T depends on time (here, T = 80 s). T = 80 s line is projected onto the Morlet phase function and a single time-dependent value of phase (bottom panel) is read along that line (yellow dashed line). (e) Rho wave attenuation in Ect2-overexpressing starfish oocytes treated with 10 μM Jasplakinolide during first meiosis; kymographs made from strips indicated in e′, numbered by order of anaphase entry. Gap in kymographs shows time of drug addition. Jasplakinolide induces slow accumulation of excess cortical actin but does not completely abolish actin turnover (not shown). Before drug addition, oocytes 1–3 exhibited prominent waves; without treatment, waves typically continue about half the time between metaphases I and II; in three of these cells, jasplakinolide rapidly reduces wave amplitude (the fourth may not have experienced the same concentration of drug). e′ displays wave patterns just before and shortly after drug addition. Images are representative of 3 independent experiments.

Supplementary Figure 5 Cortical F-actin antagonizes Rho activation.

(a) Time-lapse series showing active Rho (malachite) and F-actin (copper) in merge or as single channels in starfish oocyte overexpressing Ect2 and subject to global treatment with latrunculin. Shortly after latrunculin application the entire cortical F-actin network shatters, an event accompanied by a transient and global increase of Rho activity. Arrowhead indicates site of meiotic spindle. (b) Time-lapse series showing active Rho (malachite) and F-actin (copper) in merge or as single channels in normal (that is, non-Ect2 overexpressing) starfish oocyte subject to local F-actin disassembly at site indicated by pipet tip schematic. As in cells overexpressing Ect2 (Fig. 5g, h) local F-actin disassembly is accompanied by a burst of Rho activation. Hollow arrowhead indicates site of meiotic spindle; filled arrowheads indicate bursts of Rho activity. (c) Time-lapse series showing active Rho (malachite) and F-actin (copper) in normal starfish blastomere subject to local F-actin disassembly at site indicated by pipet tip schematic. Loss of cortical F-actin from the equator (white rectangle) produces a burst of Rho activity just as it does elsewhere on cell cortex. (c′′) Images of equator taken from time-lapse movie of cell above showing bursts of Rho activation following F-actin disruption within nascent furrow. Images are representative of 6 (a) and 3 (b, c) independent experiments, respectively.

Supplementary Figure 6 Computational model of excitable cortical dynamics.

(a) A set of parameters used for model simulations has been chosen to fit the temporal period and dominant wavelength of excitable wave pattern as well as the time shift between Rho and F-actin signals observed in starfish oocytes overexpressing Ect2. (b) The model predicts that the increase in Ect2 activity results in the change of the wave pattern that can be quantified by the activity ratio, which is defined as la/λ, the fraction of the wavelength λ where the value of normalized Rho signal is above 50% of the average over one wave period. Top panel: At the Ect2 activity generating the wave pattern typical of meiotic starfish oocyte interphase ([Ect2]0 = 1 μM), activity ratio is 0.3. Bottom panel: Increase in Ect2 activity ([Ect2]0 = 2 μM) results in the wave pattern with broad wave crests and narrow refractory zones; activity ratio is 0.6. Average level of normalized Rho signal is indicated by dashed line. (c) The model predicts that the wave pattern observed in meiotic starfish oocytes during polar body emission can be explained by an increase in Ect2 activity. Top panel: Activity ratio computed from the experimental data (see Methods for details). The peak in activity ratio corresponds to polar body emission. Bottom panel: Activity ratio computed from the results of simulation with changing activity of Ect2. Data are shown as mean ± s.d.

Supplementary information

Supplementary Information

Supplementary Information (PDF 6121 kb)

Surface view of cleaving blastomere in mid-cleavage stage frog embryo (one of 32 + cells) expressing GFP-Utr, single optical plane, 2.4 s/frame at 30 fps (time in seconds).

Corresponds to Fig. 1a. F-actin foci come and go rapidly, transiently increasing in density to near-uniformity shortly before cytokinetic furrow formation, whereafter the density declines again. (MOV 29153 kb)

High-magnification surface view of activated frog egg expressing GFP-Utr, single optical plane, 4 s/frame at 30 fps (time in min:sec).

Corresponds to Fig. 1b, without colour coding. Kymograph scrolling beneath frame sequence made by averaging across the rectangle indicated. This sequence spans two apparent M-phases in 46 min, after each of which F-actin density transiently increases to near uniformity. Between these pulses, waves continually traverse the surface with apparently random direction changes; cable networks are frequently apparent between the margins of fading wave fronts. (MOV 47034 kb)

Cleaving frog blastomere expressing both GFP-Utr (blue) and Lifeact-mCherry (yellow); single optical plane, 12 s/frame at 30 fps (time in min:sec).

Corresponds to Fig. 1d. Throughout, leading wave fronts are yellowish, whereas trailing edges, and cable networks left in the wake of passing waves, are white or bluish. Note, as shown in Fig. 1d, that GFP-Utr and Lifeact-mCherry both label the same structures–that is, there is no pure colour in this sequence, all structures contain some blue and some yellow. (MOV 25611 kb)

Surface view of activated frog egg expressing 3xGFP-rGBD, average projection of 4 1 micron sections, 10 s/frame at 15 fps (time in min:sec).

Corresponds to Fig. 2c. 20-min sequence plays twice, the second time alongside a median-filtered rearward subtraction (current time minus three frames previous). This highlights the subtle waves traveling across a background of low-level cortical Rho activity. (MOV 19874 kb)

Surface view of activated frog egg expressing 3xGFP-rGBD plus wild-type Xenopus Ect2, average projection of 4 1 micron sections, 10 s/frame at 15 fps (time in min:sec).

Corresponds to Fig. 2d. 20-min sequence plays twice, the second time alongside a median-filtered rearward subtraction (current time minus three frames previous). Compared to normal cells (Fig. 2c, video 4) waves of Rho activity are far more pronounced. Not only is the amplitude greater, waves appear to occur in quasi-repeating patterns, and individual wave fronts continue further across the surface before dissipating or colliding with other waves. (MOV 20228 kb)

Medial focal slabs of two starfish oocytes expressing GFP-rGBD, one with extra wild-type S. purpuratus Ect2.

Both sequences cover first meiosis with polar body emission. First sequence (normal oocyte) is a max. projection of 8 1 micron sections, second sequence (Ect2 over-expressing) is a max. projection of 8 1.5 micron sections; both are 20 s/frame at 15 fps (time in min:sec). Correspond to Fig. 3a, b. In the normal oocyte, a broad coherent wave of Rho activity initiates at the vegetal pole, passes toward the animal pole, and ”breaks” around the meiotic spindle, coalescing into the cytokinetic zone that pinches off the polar body. With extra Ect2, the single coherent wave breaks up into brighter, more persistent wavelets, which continue throughout and after polar body emission. Second sequence plays twice, the second time with the first sequence inset for comparison. (MOV 17544 kb)

Surface view of Rho waves in starfish oocyte expressing GFP-rGBD and wild-type S. purpuratus Ect2, max. projection of 11 1 micron sections, 12 s/frame at 15 fps (time in min:sec).

Corresponds to Fig. 3c. This sequence covers second meiosis; first polar body has been successfully extruded already. Waves start just before second polar body emission, show a global increase in intensity, then settle into a regime with prominent, well-spaced fronts that travel tens of microns with consistent direction. As highlighted by insets in Fig. 3c, approximately repeating patterns emerge post-meiotically. (MOV 10204 kb)

High-magnification surface view of Rho waves in normal starfish oocyte expressing GFP-rGBD, max. projection of 5 0.6 micron sections, 6 s/frame at 30 fps (time in min:sec).

Corresponds to Fig. 3d. This sequence covers first meiosis in a slightly compressed oocyte. Likely due to the compression, polar body emission fails in this case; nevertheless, the sequence of events is otherwise normal: Rho activity begins at the end of M-phase at the vegetal pole, thus sweeping in from the left edge in this view. The macro-wave of Rho activation can be seen to consist of many short-lived low-amplitude wavelets that scatter like chop in a sea breeze. While the cytokinetic zone is more persistent, flickers reveal that it too consists of a flock of similar wavelets. (MOV 8664 kb)

One of four cells in a normal starfish embryo expressing both GFP-rGBD (malachite) and mCherry-Utr (copper), max. projection of 14 0.6 micron sections, 17 s/frame at 15 fps (time in min:sec).

Corresponds to Fig. 4a. Cytokinetic Rho activity begins with a transient, global but non-uniform pulse, rapidly focusing at the equator. Wavelets similar in size to those observed in oocytes populate the early furrow. F-actin enrichment does not overlap perfectly with Rho activity; rather, F-actin enrichment develops in the wake of Rho activity waves. (MOV 3611 kb)

Starfish oocyte expressing both GFP-rGBD (malachite) and mCherry-Utr (copper), plus wild-type S. purpuratus Ect2.

Projection of 14 0.8 micron sections, 17 s/frame at 20 fps (time in min:sec). Corresponds to Fig. 4c. In this nearly-animal-pole view, the site of meiosis is just out of view beyond the bottom of the frame. F-actin waves consistently follow, rather than overlap with, Rho waves, and Rho waves move into F-actin-poor zones. (MOV 35489 kb)

Activated frog egg expressing GFP-rGBD (malachite) and mCherry-Utr (copper), plus wild-type Xenopus Ect2, average projection of 4 1 micron sections, 10 s/frame at 20 fps (time in min:sec).

Corresponds to Fig. 4d. Rho activity waves and F-actin assembly waves have similar forms but at successive times; consequently, there is little overlap between wave peaks. Thus, throughout this sequence very little yellow (indicating colocalization) intercedes between malachite and copper. (MOV 15382 kb)

Starfish zygote expressing GFP-rGBD (malachite) and mCherry-Utr (copper), plus wild type S. purpuratus Ect2, max. projection of 5 0.5 micron sections, 6 s/frame at 20 fps (time in min:sec).

Corresponds to Fig. 4e. This sequence begins in M-phase, with a relatively uniform F-actin carpet dominated by microvilli, and low-amplitude Rho activity waves. At anaphase Rho waves rapidly build to a near-uniform peak as microvilli give way to a finer-textured F-actin cortical layer, which in turn quickly breaks into waves as the cytokinetic zone develops. As furrow ingression proceeds, Rho/F-actin waves continue in tropical and polar regions. Little yellow is evident between malachite and copper fronts, but distinct dark areas separate advancing malachite front from trailing edges of copper waves. (MOV 27306 kb)

Reconstruction of excitable phase on the cortex of starfish oocyte overexpressing Ect2 permits to reveal spiral wave cores.

Phase (right panel) is colour-coded as in Fig. 5. Arrowhead points to a spiral wave core that persists for nearly 59 min. Corresponding Rho activity signal (GFP-rGBD) is shown for comparison (right panel). Note that no spiral wave is readily distinguishable in the Rho activity pattern by naked eye. Corresponds to Fig. 5d–f. Frame rate is 41.3 s. (MOV 1189 kb)

Ect2-expressing starfish oocytes (malachite = GFP-rGBD, copper = mCherry-Utr) exposed to a pipette filled with agarose impregnated with 10 μM Latrunculin B in seawater.

Max. projection of 15 1.5 micron sections, 20 s/frame at 15 fps (time in min:sec). Corresponds to Fig. 5g. In the oocyte nearest the drug source, the cortical F-actin rind shatters and melts on the side facing the pipette, accompanied by a surge of Rho activation. On the other side of the oocyte, waves transiently switch to a high-amplitude, long-period regime before the cortical F-actin layer shreds and ultimately collapses there too. In contrast, the oocyte further from the drug source never undergoes complete F-actin disassembly, and, following an initial burst, sustains the high-amplitude long-period wave regime for nearly 10 min. Note that because oocytes contain a fixed amount of the Utrophin-based probe for F-actin, even complete disassembly does not change the average fluorescence in the cell. (MOV 5959 kb)

Ect2-expressing starfish oocyte (malachite = GFP-rGBD, copper = mCherry-Utr) exposed to a pipette filled with agarose impregnated with 1 μm Latrunculin B in seawater.

Max. projection of 19 1 micron sections, 18 s/frame at 15 fps (time in min:sec). Corresponds to Fig. 5h. This oocyte was post-meiotic, and exhibited Ect2-enhanced waves typical for this stage (similar to Fig. 4c), when the pipette was brought close to the right edge. At the applied dose, F-actin never completely disassembled, and indeed new assembly continued throughout the sequence. However, the period and amplitude of Rho/F-actin waves increased dramatically, first near the pipette and then around the entire cell, except in the patch of cortex attached to the coverslip, which apparently experiences lower drug concentration such that the original wavelength remains. The high-amplitude, long-period regime continued for 30 min before onset of zygotic M-phase put a stop to excitability. (MOV 7596 kb)

Starfish oocytes expressing GFP-rGBD, wild-type S. purpuratus Ect2, and Δ90-Cyclin B, treated with Roscovitine.

Max. projection of 11 2 micron sections, 19 s/frame at 30 fps (time in min:sec after Roscovitine treatment). Corresponds to Fig. 6e. A group of 12 oocytes were held tightly packed and slightly compressed between a slide and coverslip; 40 μM Roscovitine was added by perfusing the chamber; the perfusing stream ran SE-NW, and oocytes on the left and lower right were directly exposed to the stream, while the other were partially occluded (note that Z-series did not reach maximum diameter: oocytes on the left side were tightly packed). Roscovitine elicits excitability in the solvent-exposed cells within 5 min, culminating in C-phase-like burst of global Rho activation, followed by sustained waves over 2 + h. Buried oocytes respond later but similarly, with a longer build-up from wave initiation to the global burst of Rho activity. (MOV 43377 kb)

Fertilized starfish oocyte expressing GFP-rGBD and wild-type S. purpuratus Ect2, cut before 1st meiosis into nucleated and anucleate halves.

Max. projection of 14 1 micron sections, 20 s/frame at 20 fps (time in min:sec). Corresponds to Fig. 7a. Upper half contains sperm entry point and meiotic spindle; lower half is anucleate and lacks centrosomes. Both halves become synchronously excitable for 1st meiosis, then 2nd meiosis. This oocyte apparently received a modest dose of Ect2, such that excitability persists only for a short time around meiotic divisions, which both complete successfully in the nucleated half. (MOV 11162 kb)

Fertilized starfish oocyte expressing GFP-rGBD (no extra Ect2) and treated with 5 μM nocodazole before 1st meiosis.

Max. projection of 14 0.6 micron sections, 16 s/frame at 20 fps (time in min:sec). Corresponds to Fig. 7e. Sperm entry point is visible to the lower left, meiotic spindle to the upper right. Vegetal-to-animal ”macro-waves” of Rho activation proceed as normal, except that instead of culminating in a cytokinetic zone they reach the animal pole and dissipate. There is no zone of Rho suppression around the site of meiosis, as there normally would be; indeed, the very animal pole exhibits peak Rho activity, and even persistent activity between divisions. (MOV 18549 kb)

Simulation of the excitable dynamics on the cortex of starfish oocyte undergoing meiosis.

Rho activity and F-actin density are colour-coded as in Fig. 8. Note a striking change in the wave pattern during the increase in global Ect2 activity that represents the polar body extrusion phase (see Methods for details). Compare with Supplementary Fig. 6d. Corresponds to Fig. 8a–c. (MOV 1201 kb)

Simulation of the furrow formation in mitotic starfish blastomere.

Rho activity and F-actin density are colour-coded as in Fig. 8. During the simulation spatial distribution of cortical Ect2 is focussed towards the equator to imitate the microtubule-dependent translocation (see Methods for details). Compare with Figs 3e, 4a. Corresponds to Fig. 8d, e. (MOV 1356 kb)

GFP-rGBD- and Ect2-expressing starfish embryo treated with 40 μM nocodazole during cytokinesis.

Max. projection of 8 1 micron sections, 20 s/frame at 20 fps (time in min:sec after nocodazole treatment). Corresponds to Fig. 8h. A binucleate cell that had failed first cleavage was chosen, because at the time of treatment it displays a) prominent cytokinetic Rho zones, b) a spindle-distant zone to the left populated by distinct waves, and c) spindle-proximal zones that lack waves. Nocodazole treatment abolishes the cytokinetic Rho zones and low-level waves repopulate the entire surface as furrows regress; waves disappear after 30 min as next M-phase commences. (MOV 7557 kb)

GFP-rGBD in a normal cleaving blastomere from a 16-cell starfish embryo.

Max. projection of 42 0.5 micron sections, 12 s/frame at 15 fps. Corresponds to Supplementary Fig. 3b. A narrow strip around the equator was imaged so as to capture one-third of the entire furrow circumference as it ingresses. Animation leaves kymograph (as in Supplementary Fig. 3b) in its wake. Blotches of Rho activity at each time point actually represent waves within the furrow. (MOV 874 kb)

GFP-rGBD in a normal 64-cell starfish embryo, four cells.

Max. projection of 16 0.6 micron sections, 9 s/frame at 15 fps. Corresponds to Supplementary Fig. 3c. At this stage, furrow zones are coherent but still clearly exhibit waves during zone formation and initial ingression. (MOV 2451 kb)

GFP-rGBD in a normal starfish embryo, 128 + cells.

Max. projection of 12 0.8 micron sections, 14 s/frame at 15 fps. Corresponds to Supplementary Fig. 3d. As cell size decreases throughout embryogenesis, waves are less apparent in cleavage furrows. Rho activity zones are initially inhomogeneous, but rapidly cohere as ingression proceeds. (MOV 4969 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Bement, W., Leda, M., Moe, A. et al. Activator–inhibitor coupling between Rho signalling and actin assembly makes the cell cortex an excitable medium. Nat Cell Biol 17, 1471–1483 (2015). https://doi.org/10.1038/ncb3251

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3251

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing