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Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows

Nature volume 545, pages 103–107 (2017)Cite this article

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Abstract

During epithelial cytokinesis, the remodelling of adhesive cell–cell contacts between the dividing cell and its neighbours has profound implications for the integrity, arrangement and morphogenesis of proliferative tissues1,2,3,4,5,6,7. In both vertebrates and invertebrates, this remodelling requires the activity of non-muscle myosin II (MyoII) in the interphasic cells neighbouring the dividing cell1,3,5. However, the mechanisms that coordinate cytokinesis and MyoII activity in the neighbours are unknown. Here we show that in the Drosophila notum epithelium, each cell division is associated with a mechanosensing and transmission event that controls MyoII dynamics in neighbouring cells. We find that the ring pulling forces promote local junction elongation, which results in local E-cadherin dilution at the ingressing adherens junction. In turn, the reduction in E-cadherin concentration and the contractility of the neighbouring cells promote self-organized actomyosin flows, ultimately leading to accumulation of MyoII at the base of the ingressing junction. Although force transduction has been extensively studied in the context of adherens junction reinforcement to stabilize adhesive cell–cell contacts8, we propose an alternative mechanosensing mechanism that coordinates actomyosin dynamics between epithelial cells and sustains the remodelling of the adherens junction in response to mechanical forces.

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Figure 1: Contractile ring forces trigger MyoII accumulation in the neighbours.
Figure 2: Cortex detachment and E-cad decrease precede MyoII accumulation.
Figure 3: Lowering E-cad concentration at the ingressing AJ promotes MyoII accumulation.
Figure 4: Actomyosin flows drive the response of the neighbours.

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Acknowledgements

We thank M. Affolter, S. Bogdan, M. Fuller, J. Großhans, A. Martin, J. Mihaly, H. Oda, B. Sanson, D. St Johnston, J. Treisman, J. Zallen, VDRC, TRiP, Kyoto and Bloomington Stock Centres and DSHB for reagents; J. Prost, L. Szpiro for inputs; M. Thery, L. Blanchoin, Emilie Barou, H. Ennomanix, K. Cockburn and V. Greco for data and inputs; the Developmental Biology Unit imaging platform; J.-F. Joanny, F. Graner, A. Villedieu and R.-M. Mège for comments; P. Recho for help with simulations; ANR-MaxForce, ERC (TiMoprh, 340784), ARC (SL220130607097), ANR-DEEP (11-LBX-0044, ANR-10-IDEX-0001-02) and PSL grants for funding; FCT (SFRH/BD/51700/2011) and FRM (FDT20150531972) fellowships to D.P. and Wellcome Trust (110326/Z/15/Z), Trinity College and the Bettencourt-Schueller Foundation fellowships to E.H.

Author information

Author notes

  1. Sophie Herszterg

    Present address: The Francis Crick Institute, 1 Brill Place, London, NW1 1BF, UK

  2. Edouard Hannezo and Sophie Herszterg: These authors contributed equally to this work.

Authors and Affiliations

  1. Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934, Paris Cedex 05, F-75248, France

    Diana Pinheiro, Sophie Herszterg, Floris Bosveld, Isabelle Gaugue, Maria Balakireva, Zhimin Wang, Inês Cristo, Stéphane U. Rigaud, Olga Markova & Yohanns Bellaïche

  2. Sorbonne Universités, UPMC Univ Paris 06, CNRS, CNRS UMR 3215, INSERM U934, F-75005, France

    Diana Pinheiro, Sophie Herszterg, Floris Bosveld, Isabelle Gaugue, Maria Balakireva, Zhimin Wang, Inês Cristo, Stéphane U. Rigaud, Olga Markova & Yohanns Bellaïche

  3. Graduate Program in Areas of Basic and Applied Biology, Abel Salazar Biomedical Sciences Institute, University of Porto, Portugal

    Diana Pinheiro

  4. Department of Physics, Cavendish Laboratory, J. J. Thomson Avenue, University of Cambridge, Cambridge, CB3 0HE, UK

    Edouard Hannezo

  5. The Wellcome Trust/Cancer Research UK Gurdon Institute, Tennis Court Rd, University of Cambridge, Cambridge, CB2 1QN, UK

    Edouard Hannezo

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Contributions

D.P., E.H., S.H. and Y.B. designed the project. I.G., Z.W. and M.B. produced reagents. D.P., S.H., F.B. and I.C. performed live imaging experiments and genetics. D.P. performed fixed tissue imaging experiments. E.H. and S.U.R. developed methods and scripts for data analysis. D.P. and E.H. analysed the data. E.H. and O.M. developed theoretical models. E.H. performed simulations. D.P., E.H. and Y.B. wrote the manuscript.

Corresponding author

Correspondence to Yohanns Bellaïche.

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The authors declare no competing financial interests.

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Reviewer Information Nature thanks G. Gay, T. Harris and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Estimation of the pulling forces produced during contractile ring constriction in wild-type, pnutRNAi, rokRNAi and aniRNAi dividing cells.

a, Top view of a PH–GFP neighbour facing a PH–ChFP dividing cell during cytokinesis. Insets highlight the tight apposition of both cell membranes throughout cytokinesis. n = 12 cells (8 pupae). b, Top view and 3D reconstruction of a PH–GFP neighbour facing a PH–ChFP dividing cell (see Supplementary Video 1). n = 11 cells (7 pupae). c, PH–GFP and MyoII–3 × mKate2 distribution during cytokinesis. Insets highlight the progressive membrane deformation observed during cytokinesis, as well as MyoII accumulation in the neighbours (filled arrowheads). Orange and yellow dashed lines correspond to the orthogonal view of the dividing cell and its neighbours, respectively XZ and YZ planes. White dashed line in the YZ plane outlines the contractile ring throughout cytokinesis; asterisks indicate daughter cells. n = 18 cells (6 pupae). d, PH–GFP cell neighbouring a dividing cell in a MyoII–3 × mKate2 tissue. YZ view generated along the yellow box. Arrowheads as in c. n = 15 cells (5 pupae). e, E-cad–GFP and MyoII–mChFP localization upon contractile ring laser ablation (n = 48 ablations, 4 pupae). Laser ablation was performed before MyoII–mChFP accumulation in the neighbours (t = 0 s; orange box shows ablated region). Open arrowheads highlight absence of MyoII–mChFP accumulation in the neighbours. f, Rate of contractile ring constriction in wild-type, pnutRNAi, rokRNAi and aniRNAi dividing cells (8, 6, 11 and 4 pupae, respectively). g, i, k, m, Estimation of pulling forces produced by the contractile ring (labelled by MyoII–mChFP) on the AJ (marked by E-cad–GFP) by laser ablation in wild-type (g, n = 80 cells, 4 pupae), pnutRNAi (i, n = 37 cells, 3 pupae), rokRNAi (k, n = 54 cells, 5 pupae) and aniRNAi (m, n = 39 cells, 3 pupae) dividing cells (t = 0 s; orange box shows ablated region). Dots indicate pnutRNAi, rokRNAi and aniRNAi expressing cells, marked by the absence of cytosolic GFP. Dashed arrows indicate the dividing cells relaxation upon ring ablation. h, j, l, n, Recoil velocity upon contractile ring laser ablation versus the amount of ring constriction at the time of the ablation in wild-type (h, j, l, n), pnutRNAi (j), rokRNAi (l) and aniRNAi (n) dividing cells. In pnutRNAi, rokRNAi and aniRNAi dividing cells, the recoil velocity upon ring laser ablation is on average lower than in wild-type dividing cells (see o), and it scales less with the amount of contractile ring constriction at the time of the ablation (4, 3, 5 and 3 pupae, respectively). o, AJ recoil velocity upon contractile ring laser ablation in wild-type, pnutRNAi, rokRNAi and aniRNAi dividing cells (4, 3, 5 and 3 pupae, respectively). p, Normalized MyoII accumulation at 80% of the initial cell diameter in cells neighbouring wild-type, pnutRNAi, rokRNAi and aniRNAi dividing cells (5, 4, 11 and 4 pupae, respectively). Note that for pnutRNAi and aniRNAi dividing cells, we included wild-type and pnutRNAi or aniRNAi neighbours in our analysis, as when facing a wild-type dividing cell both pnutRNAi or aniRNAi neighbours accumulate MyoII to normal levels (Extended Data Fig. 5p–r). For rokRNAi dividing cells, we analysed wild-type neighbours exclusively, as Rok function is also essential in the neighbours (Extended Data Fig. 5p–r). n denotes number of cells throughout. **P < 0.01, ****P < 0.0001, Kruskal–Wallis test. A re-plot of the dataset in o and p is provided in Fig. 1e. Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 2 Crb, α-cat and Dlg distribution during cytokinesis.

a, d, g, Schematic representation of the localization of E-cad and the supra-apical marker Crb (a), α-cat (d) or the septate junction marker Dlg (g) along the apical–basal axis of epithelial cells. b, e, h, Top view of E-cad–3 × mKate2 or E-cad–3 × GFP and Crb–GFP (b; n = 47 cells, 2 pupae), α-cat–GFP (e; n = 58 cells, 2 pupae) or Dlg–mTagRFP (h; 85 cells, 8 pupae) distribution in the dividing cell and its neighbours during cytokinesis. Insets highlight that although the E-cad–3 × mKate2 or E-cad–3 × GFP signals progressively decrease at the ingressing membrane, both Crb–GFP and Dlg–mTagRFP signals are still present in the supra-apical membrane and at the septate junctions, respectively. Conversely, E-cad–3 × mKate2 and α-cat–GFP signals decrease concomitantly at the ingressing AJ. Crb–GFP, Dlg–mTagRFP and E-cad–3 × mKate2 or E-cad–3 × GFP were projected from two different apical–basal confocal sections, which correspond to the planes of maximum intensity of Crb–GFP or Dlg–mTagRFP and the AJ, respectively. Yellow dashed lines used to generate the side views in c, f and i. c, f, i, Side view (YZ) of E-cad–3 × mKate2 or E-cad–3 × GFP and Crb–GFP (c; n = 47 cells, 2 pupae), α-cat–GFP (f; n = 58 cells, 2 pupae) or Dlg–mTagRFP (i; 85 cells, 8 pupae) distribution in the dividing cell and its neighbours at the onset of cytokinesis. Green arrowheads denote Crb–GFP (c), α-cat–GFP (f) or Dlg–mTagRFP (i), while red arrowheads indicate E-cad–3 × mKate2 (c, f) or E-cad–3 × GFP (i) localization along the apical–basal axis of epithelial cells. Scale bars, 5 μm.

Extended Data Figure 3 Contraction of the detached cortical MyoII is not sufficient to sustain MyoII accumulation in the neighbours.

a, βH-spectrin–GFP (βH-spec–GFP) and MyoII–3 × mKate2 distribution upon contractile ring constriction. Asterisk denotes co-localization of MyoII–3 × mKate2 and βH-spec–GFP away from the ingressing AJ. n = 103 cells (4 pupae). b, Normalized MyoII intensity of the detached cortical MyoII and at 80% of the initial cell diameter in wild-type dividing cells (4 pupae). c, Schematic representation of the theoretical model describing cortex detachment in the neighbours. The AJs are highlighted in green, while the actomyosin cortex is shown in the red dashed line and in further detail in the insets. CR denotes the contractile ring. Double arrow indicates separation of the actomyosin cortex from the AJ—cortex detachment. Dashed black lines indicate the angle formed by the ingressing AJ. To probe the potential role of cortex detachment for MyoII accumulation in the neighbours, we analysed theoretically the detachment event. The cortex is a contractile layer lining the AJ, thus if it becomes curved, either because of an external deformation (that is, contractile ring constriction in the dividing cell) or a pressure difference between two cells (as observed during blebbing), this produces inward forces perpendicular to the membrane that need to be balanced by adhesion to prevent cortex detachment. The biophysical role of E-cad in this membrane–cortex interaction has been studied in vitro39, but to a lesser extent in vivo. Following the classical active gel theory10,40,41, we model the cortex as a thin viscous layer, of thickness h. γ is the local tension in the cortex, χ is the contractility due to MyoII motor power-stroke, and η is the cortical viscosity. Thus, along the direction x parallel to the membrane, the tension γ is the sum of active and viscous contributions γ = h(χ + η∂xv), with v the local velocity in the layer, under the lubrication regime40. If we consider a cortex connected to an AJ that can be curved with local curvature , in the quasi-static regime of the ingressing AJ during cytokinesis39, the cortex will detach if the normal forces γκ are greater than the maximal adhesion force density fc, which is proportional to the adhesion strength between the cortex and the membrane, as well as the local concentration c(x) of linker proteins. This is analogous to the de Gennes criteria for the unbinding of adhesive vesicles42. Therefore, for constant tension and adhesion strength, one expects the cortex to detach once a well-defined curvature κc, imposed by the contractile ring, is reached, such that κc = fc/γ. Thus, the model predicts that the critical curvature of cortical detachment can be modulated in vivo: the maximal curvature at detachment should increase with either an increase in membrane–cortex attachment, or a decrease in cortical tension (and vice versa). We tested this model by manipulating experimentally either cortical tension or membrane–cortex attachment (d–f). As curvature cannot be robustly defined in the ingressing AJ given the length scales examined, we alternatively measured the angle θ between the two sides of the ingressing junction (c). With d0 being the characteristic width of the ingressing region, this angle is related to curvature via κ ∝ (π − θ)/d0. To manipulate the adhesion force, we analysed cortex detachment in moeRNAi neighbours, as well as in E-cad heterozygous background (E-cad1x). Moesin is the only ERM protein in flies and it has an essential role in membrane–cortex attachment, since it directly binds both F-actin and the membrane43. As predicted theoretically, upon moeRNAi or in the E-cad1x mutant condition, cortex detachment occurs at higher angles than in wild-type neighbours; thus, the initially detached cortical MyoII is localized further away from the ingressing membrane during contractile ring constriction (d, e and h). Similarly, increasing the neighbours’ contractility by generating either sds22RNAi neighbours, which increases phospho-MyoII and phospho-Moe at the AJs44, or mbsRNAi neighbours, which globally increases phospho-MyoII by blocking the activity of the MyoII phosphatase45, results in cortex detachment at higher angles (d, e). Moreover, the angle of cortex detachment in these experimental conditions anti-correlates with the amount of constriction at detachment (f). Altogether, these data suggest that a balance between the adhesion force, cortex contractility and local membrane curvature regulates cortex detachment, in response to the pulling forces generated in the dividing cell. d, E-cad–GFP and MyoII–mChFP localization at cortex detachment in wild-type (n = 83 cells, 11 pupae), E-cad1x heterozygous mutant (n = 17 cells, 2 pupae), moeRNAi (n = 18 cells, 6 pupae), sds22RNAi (n = 19 cells, 5 pupae) and mbsRNAi (n = 25 cells, 6 pupae) neighbours. Dots indicate moeRNAi, sds22RNAi or mbsRNAi cells, marked by the absence of cytosolic GFP. Asterisks denote separation of MyoII–mChFP and E-cad–GFP signals at the ingressing region. e, Angle of cortex detachment in wild-type, E-cad1x, moeRNAi, sds22RNAi and mbsRNAi neighbours (11, 2, 6, 5 and 6 pupae, respectively). For all conditions, except E-cad1x, the dividing cell is wild-type. Note that the rate of constriction is similar for wild-type and E-cad1x cells and no detectable defects were observed during cytokinesis. f, Anti-correlation between the angle of cortex detachment (in e) and the amount of contractile ring constriction at detachment in wild-type, E-cad1x, moeRNAi, sds22RNAi and mbsRNAi neighbours (11, 2, 6, 5, 6 pupae, respectively). Slope is −1.97 ± 0.15 (R2 = 0.98). P < 0.001, F-test for a slope different from 0. g, E-cad–GFP and MyoII–mChFP localization in a pnutRNAi dividing cell and its neighbours during cytokinesis. Filled arrowheads indicate transient MyoII–mChFP accumulation in the neighbours; open arrowheads denote reduced MyoII–mChFP accumulation in the neighbours. n = 10 out of 20 cells (5 pupae). h, E-cad–GFP and MyoII–mChFP localization in the dividing cell and its moeRNAi neighbour. In g and h, dots denote pnutRNAi (g) and moeRNAi (h) cells, marked by the absence of cytosolic GFP, and asterisks denote separation of MyoII–mChFP and E-cad–GFP signals at the ingressing AJ. At t = 208 s, arrowhead indicates the transient accumulation of MyoII–mChFP at the detached cortex; at t = 340 s and 400 s, arrowheads indicate the re-localization of the MyoII–mChFP accumulation near the boundary between high and low E-cad–GFP (see Supplementary Video 4c). n = 18 cells (6 pupae). i, Percentage of wild-type and moeRNAi neighbours (11 and 20 pupae, respectively), where the detached cortex and the E-cad boundary are superimposed. j, Percentage of wild-type and moeRNAi neighbours (7 and 17 pupae, respectively), where the detached cortex either coalesces with the MyoII accumulation positioned at the E-cad boundary, or disassembles during cytokinesis (see Supplementary Video 4d). n denotes number of cells throughout. n/n indicates number of cells/total number of cells. *P < 0.05, ***P < 0.001, ****P < 0.0001. Student’s t-test (b) and Kruskal–Wallis test (e). Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 4 Decrease of E-cad concentration at the ingressing AJ is not impaired after Dynamin, Rab11 or Sec5 loss-of-function.

a, E-cad–GFP dividing cell facing an E-cad–3 × mTagRFP neighbour. Yellow open arrowheads denote concomitant decrease of E-cad–GFP and E-cad–3 × mTagRFP at the ingressing AJ. n = 17 cells (5 pupae). b, E-cad–GFP localization in the dividing cell and its neighbour expressing a dominant-negative temperature-sensitive dynamin allele, shits (dots, marked by absence of cytosolic GFP) at 29 °C. n = 21 cells (3 pupae). c, d, E-cad–GFP localization in a YFP–Rab11DN dominant-negative (c; n = 22 cells, 3 pupae) or sec5 (d; n = 9 cells, 4 pupae) dividing cell and its neighbours. Dots indicate YFP–Rab11DN (c) or sec5 (d) cells, marked by YFP expression or by the absence of nls–GFP. Open arrowheads in b–d denote E-cad–GFP decrease at the ingressing AJ. e–g, Integrated density (IntDen) of E-cad–GFP at the ingressing AJ at the onset of AJ elongation (t30% constriction) and upon full contractile ring constriction (tfinal) for wild-type dividing cells facing either wild-type (e) or rokRNAi neighbours (f), and pnutRNAi dividing cells (g; 5, 7 and 6 pupae, respectively). h, Schematic representation of the E-cad dilution model. In this model, local membrane elongation, due to contractile ring (CR) constriction, progressively dilutes E-cad along the ingressing AJ (highlighted in the lighter green line used at the ingressing junction). i, j, Width of ingressing region normalized by total curvilinear AJ length (i) and height of the ingressing region (μm, j) as a function of the amount of constriction for wild-type dividing cells facing wild-type or rokRNAi neighbours and pnutRNAi dividing cells (5, 7 and 6 pupae, respectively). Insets represent the width and height of the ingressing region (E-cad–GFP, green; MyoII–mChFP, red, also shown in Fig. 2a). For wild-type dividing cells facing wild-type neighbours, the normalized width of the ingressing AJ is small (average 0.12) and decreases during constriction, whereas the height increases, thereby indicating that AJ elongation is a rather local process. Similarly, in wild-type dividing cells facing rokRNAi neighbours, the normalized width remains small during constriction (average 0.26) and the height increases, indicating that the elongation of the AJ is also mainly local. Contrary to this, in cells neighbouring pnutRNAi dividing cells, the normalized width of the pulled region is much wider (average 0.49) and the height is much smaller and remains constant during constriction. This argues for a more global elongation of the ingressing AJ. k, Normalized total AJ elongation upon full contractile ring constriction of wild-type dividing cells (tfinal) facing wild-type or rokRNAi neighbours and pnutRNAi dividing cells (5, 7 and 6 pupae, respectively). l, Normalized local E-cad–GFP intensity at the ingressing AJ versus the normalized total AJ elongation in wild-type dividing cells (5 pupae), and the corresponding theoretical integration (line corresponds to best-fit parameters measured by E-cad–GFP FRAP, shaded regions correspond to 1 s.d. confidence interval). The coefficient of determination and the s.d. of the residuals is respectively R2 = 0.82 and S = 0.12. n denotes number of cells throughout. NS, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001. Paired Student’s t-test (e–g) and Kruskal–Wallis test (k). Data are mean ± s.e.m. (e–g, i–k) or ± s.d. (l). Scale bars, 5 μm.

Source data

Extended Data Figure 5 Local AJ elongation is sufficient to trigger a decrease of E-cad concentration at the ingressing AJ.

a, b, Photoactivation of E-cad–3 × PAGFP at the AJ shared between the dividing cell and its neighbour (a; n = 19 cells, 5 pupae) or at the tip of the ingressing AJ (b; n = 25 cells, 6 pupae) in a MyoII–3 × mKate2 tissue (t = 0 s, orange boxes denote photoactivated regions). White brackets indicate reduction of photoactivated E-cad–3 × PAGFP signal at the ingressing AJ. Yellow filled arrowheads indicate that at the base of the ingressing AJ, the photoactivated E-cad–3 × PAGFP signal remains high. c, d, E-cad–GFP distribution in the dividing cell and its rokRNAi neighbours (dots, marked by the absence of cytosolic GFP). Kymograph in d along the yellow box. Yellow open arrowheads denote reduced E-cad–GFP signal at the ingressing AJ. n = 20 cells (7 pupae). e, Normalized local E-cad intensity at ingressing AJ (solid line), and normalized total AJ elongation (dashed line) versus the amount of constriction in wild-type dividing cells facing rokRNAi neighbours (7 pupae). f, Numerical integration of E-cad levels on a locally elongating AJ, as measured in wild-type dividing cells facing rokRNAi neighbours. g–i, E-cad–GFP and MyoII–mChFP distribution in a wild-type dividing cell facing a rokRNAi and a wild-type neighbour. Kymographs in h and i generated along the yellow and orange lines, respectively. White open arrowheads denote reduced MyoII–mChFP accumulation in the neighbours; yellow filled arrowheads denote absence of E-cad–GFP decrease at the ingressing AJ; yellow open arrowheads denote decrease of E-cad–GFP at the ingressing AJ. In 15% of cases (n = 20, 7 pupae), the contractile ring is positioned off-centre. In these cases, junction elongation on the wild-type side is small and no decrease in E-cad is observed (h). Furthermore, MyoII does not accumulate in this wild-type neighbour. As shown above, in the rokRNAi side, the junction elongates and E-cad decreases (c–f, i). This further argues that junction elongation is an important factor in the local decrease in E-cad signal. j, m, Normalized local (j) or total E-cad–GFP (m) intensity at the ingressing AJ versus the normalized total AJ elongation in wild-type dividing cells facing rokRNAi neighbours (j; 7 pupae), or pnutRNAi dividing cells (m; 6 pupae) and the corresponding theoretical integrations, respectively (line corresponds to the best-fit parameters measured by E-cad–GFP FRAP, shaded regions correspond to 1 s.d. confidence interval). The coefficient of determination and s.d. of the residuals are respectively R2 = 0.69, S = 0.14 (j) and R2 = 0.73, S = 0.03 (m). k, l, E-cad–GFP distribution in a pnutRNAi dividing cell and its neighbours. Kymograph in l along the yellow line. Yellow arrowheads as in g. In 14 out of the 28 cells analysed (6 pupae), cells neighbouring pnutRNAi dividing cells show a transient decrease in E-cad–GFP levels at the ingressing AJ. In all cases, these reductions in E-cad levels occur during earlier stages of contractile ring constriction. All cells shown express pnutRNAi, marked by the absence of cytosolic GFP. n, o, E-cad–GFP and MyoII–mChFP distribution in an aniRNAi dividing cell and its neighbours (dots, marked by absence of cytosolic GFP). Kymograph in o generated along the yellow line. Arrowheads as in g. aniRNAi dividing cells exert lower pulling forces (see Fig. 1e and Extended Data Fig. 1m–o) on the neighbour membranes during constriction and induce a less pronounced decrease in E-cad signal along the ingressing AJ. Note that in comparison to the upper junction, the lower junction exhibits a partial reduction of E-cad levels. Accordingly, a small MyoII accumulation can be observed. n = 17 cells (3 pupae). p, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 32 cells, 5 pupae), pnutRNAi (n = 32 cells, 4 pupae), rokRNAi (n = 29 cells, 11 pupae) and aniRNAi (n = 20 cells, 4 pupae) neighbours facing a wild-type dividing cell. Dots denote pnutRNAi, rokRNAi and aniRNAi cells, marked by the absence of cytosolic GFP. White filled and open arrowheads indicate MyoII–mChFP accumulation and reduced accumulation in the neighbours, respectively. Note that the phenotype of rokRNAi neighbouring cells is specific, as pnutRNAi and aniRNAi neighbours facing a wild-type dividing cell still accumulate MyoII similarly to wild-type neighbours (see r). q, Rate of contractile ring constriction in wild-type dividing cells facing wild-type, pnutRNAi, rokRNAi and aniRNAi neighbours (5, 4, 11 and 4, respectively). Although Rok abrogates MyoII accumulation in the neighbours, it doesn’t affect the rate of constriction in the dividing cell (see r). r, Normalized MyoII accumulation at 80% of the initial cell diameter in wild-type, pnutRNAi, rokRNAi and aniRNAi neighbours (5, 4, 11 and 4 pupae, respectively) facing wild-type dividing cells. n denotes number of cells throughout. ****P < 0.0001, Kruskal–Wallis test. Data are mean ± s.e.m. (e, q, r) or ± s.d. (j, m). Scale bars, 5 μm.

Source data

Extended Data Figure 6 Lowering E-cad concentration in cells neighbouring a pnut dividing cell rescues MyoII accumulation.

a, E-cad (dashed outline), wild-type and pnut (dots) cells marked by 0, 1 and 2 copies of E-cad–3 × GFP, respectively. The residual E-cad levels inside the E-cad hypomorphic mutant cells (E-cadhyp) were assessed by antibody staining. White full and dashed arrows denote E-cad staining and decreased staining at the AJ, respectively. n = 79 clones (11 fixed pupae). b, Representation of the experimental setup in Fig. 3i, in which a pnut dividing cell, marked by 2 copies of E-cad–3 × GFP, is facing a wild-type neighbour and an E-cadhyp neighbour, marked by 1 and 0 copies of E-cad–3 × GFP, respectively. c, Percentage of cytokinesis failure of pnut dividing cells facing either wild-type or E-cad neighbours (53 or 35 pupae, respectively). In contrast to previous findings3, our data show that the cytokinesis failure of pnut dividing cells is not rescued by reducing E-cad levels in the neighbours. d, Rate of contractile ring constriction in wild-type and pnut dividing cells facing either wild-type or E-cad neighbours (6, 11, 15 and 15 pupae, respectively). In agreement with c, the rate of constriction is similar in pnut dividing cells facing either wild-type or E-cad neighbours. e, Normalized AJ elongation at the onset of AJ elongation (t30% constriction), at 80% of the initial cell diameter (timing in which MyoII accumulation in the neighbours was measured) and upon full contractile ring constriction (tfinal) of wild-type and pnut dividing cells neighboured by wild-type or E-cad neighbours (6, 11, 15 and 15 pupae, respectively). f, Representation of the experimental setup in g, in which an E-cadhyp pnut double-mutant dividing cell, marked by 0 copies of E-cad–3 × GFP, is facing a wild-type neighbour, marked by E-cad–3 × GFP. g, MyoII–3 × mKate2 distribution in a wild-type neighbouring cell, marked by E-cad–3 × GFP expression, facing an E-cad pnut double-mutant dividing cell (dots), marked by the absence of E-cad–3 × GFP. White arrowheads denote MyoII–3 × mKate2 accumulation in the neighbour. Yellow brackets indicate reduced E-cad–3 × GFP signal at the ingressing AJ. Orange arrowhead indicates cytokinesis failure, evident by the detachment of the midbody from the dividing cell membranes. n = 26 cells (10 pupae). h, Percentage of cytokinesis failure of E-cad pnut double-mutant dividing cells facing wild-type or E-cad pnut neighbours (11 or 10 pupae, respectively). These findings further support that the cytokinesis defects observed in pnut mutant cells (c) are intrinsic to the dividing cell. i, Rate of contractile ring constriction in wild-type or E-cad pnut double-mutant dividing cells facing wild-type neighbours (6 or 10 pupae, respectively). j, Normalized MyoII accumulation at 80% of the initial cell diameter in wild-type cells neighbouring wild-type or E-cad pnut double-mutant dividing cells (6 or 10 pupae, respectively). k, Ratio of E-cad–3 × GFP intensity at the tip versus the base of the ingressing AJ in wild-type cells neighbouring wild-type or E-cad pnut double-mutant dividing cells (6 or 10 pupae, respectively). n denotes number of cells throughout. n/n indicates the number of cells that failed cytokinesis/total number of cells. *P < 0.05, ****P < 0.0001, ANOVA (d) or Mann–Whitney U-test (i–k). Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 7 Analysis of the role of Vinc, Rho and several F-actin nucleators in MyoII accumulation in the neighbours.

a, Structure of vincΔ3 and GFP–Vinc alleles generated by CRISPR/Cas9-mediated homologous recombination, showing position of GFP tag insert relative to vinc coding sequence. M1, methionine 1; P2, proline 2. b, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 38 cells, 2 pupae) and vinc (n = 44 cells, 2 pupae) neighbours. Filled arrowheads denote MyoII–mChFP accumulation in the neighbours. c, Rate of contractile ring constriction in wild-type and vinc dividing cells (2 and 2 pupae, respectively). d, Normalized MyoII accumulation at 80% of the initial cell diameter for wild-type and vinc neighbours (2 and 2 pupae, respectively). e, GFP–Vinc and MyoII–3 × mKate2 localization during cytokinesis. During constriction, GFP–Vinc is reduced along the ingressing AJ and does not accumulate with MyoII–3 × mKate2 at the base of the ingressing membrane. Greyscale inset shows GFP–Vinc localization upon full contractile ring constriction. n = 46 cells (3 pupae). f, Cytokinesis failure in rokRNAi, rho and dia dividing cells. n/n indicates the number of cells that failed cytokinesis/total number of cells (10, 10 and 7 pupae, respectively). The percentage of cytokinesis failure in rho and dia dividing cells is higher than that observed in rokRNAi dividing cells. However, loss of Rho or Dia function in the neighbours only delays MyoII accumulation in the neighbours, whereas rokRNAi in the neighbours strongly impairs it (g–j). g, i, E-cad–GFP and MyoII–mChFP localization in a rho (g; n = 30 cells, 10 pupae) or a dia (i; n = 26 cells, 7 pupae) mutant cell neighbouring a wild-type dividing cell (dots, marked by the absence of nls–GFP). Open arrowheads denote decreased MyoII–mChFP accumulation from mid-constriction until 80% of the initial cell diameter and MyoII–mChFP accumulation following midbody formation, respectively. h, j, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation in wild-type (n = 53 cells, 9 pupae), rokRNAi (n = 29 cells, 11 pupae) and rho (h; n = 30 cells, 10 pupae) or dia (j; n = 26 cells, 7 pupae) neighbouring cells facing wild-type dividing cells. k, n, Dia–eGFP (k; n = 33 cells, 9 pupae) or Arp3–GFP neighbours (n; n = 30 cells, 2 pupae) facing a dividing cell in a MyoII–3 × mKate2 tissue. Greyscale insets denote Dia–eGFP (k) or Arp3–GFP (n) localization upon full contractile ring constriction. Both Dia–eGFP and Arp3–GFP are uniformly localized along the ingressing region throughout constriction in the dividing cell. l, E-cad–GFP and MyoII–mChFP localization in wild-type and arpc1 mutant neighbours (dots, marked by the absence of nls–GFP) facing a wild-type dividing cell (n = 27 cells, 7 pupae) at 80% of the initial cell diameter. Arrowheads as in b. m, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation for wild-type (n = 25 cells, 5 pupae) and arpc1 (n = 27 cells, 7 pupae) neighbours facing wild-type dividing cells. o, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 96 cells, 12 pupae), fhos (n = 36 cells, 3 pupae), capu (n = 29 cells, 4 pupae), daam (n = 26 cells, 5 pupae), form3RNAi (n = 29 cells, 7 pupae), frlRNAi (n = 35 cells, 13 pupae), ena (n = 25 cells, 8 pupae) and chic (n = 27 cells, 6 pupae) neighbours. Since Capu and fhos loss-of-function mutants are viable, MyoII accumulation was analysed in capu or fhos tissues. Notably, the rate of contractile constriction in both fhos and capu dividing cells is similar to wild-type cells and no detectable defects were observed during cytokinesis (p). For all other conditions, the dividing cell is wild-type. Dots denote daam, form3RNAi, frlRNAi, ena and chic mutant cells, marked by the absence of His2a–GFP, cytosolic GFP or nls–GFP. Filled and open arrowheads denote MyoII–mChFP accumulation and reduced accumulation in the neighbours, respectively. p, Rate of contractile ring constriction in wild-type, fhos or capu dividing cells (2, 3 or 4 pupae, respectively). q, Normalized MyoII accumulation at 80% of the initial cell diameter for wild-type, fhos, capu, daam, form3RNAi, frlRNAi, ena and chic neighbours (12, 3, 4, 5, 7, 13, 8 or 6 pupae respectively). r, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation for wild-type (n = 57 cells, 10 pupae), chic (n = 27 cells, 6 pupae) or rokRNAi (n = 29 cells, 11 pupae) neighbours facing wild-type dividing cells. n denotes number of cells. *P < 0.05, ****P < 0.0001, Mann–Whitney U-test (c), Student’s t-test (d), ANOVA (m, q), Kruskal–Wallis test (h, j, p, r). Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 8 Modelling the neighbouring cell’s response.

a, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 25 cells, 5 pupae), dia chic (also known as profilin) double-mutant (n = 28 cells, 7 pupae), dia arpc1 double-mutant (n = 29 cells, 7 pupae) and dia arpc1 chic triple-mutant (n = 28 cells, 4 pupae) cells neighbouring wild-type dividing cells at 80% of the initial cell diameter and 1 min after midbody formation. Dots denote dia chic, dia arpc1 or dia arpc1 chic cells, marked by the absence of nls–GFP. Filled and open arrowheads indicate MyoII–mChFP accumulation and reduced accumulation in the neighbours, respectively. As single Formin, Arp2/3 or Enabled mutants show at most a delay in accumulation of MyoII (see Extended Data Fig. 7), we tested whether a drastic reduction of F-actin nucleation might impair the neighbours’ response. Loss of arpc1 function does not impair MyoII accumulation in the neighbours (see Extended Data Fig. 7l, m), but the dia aprc1 double mutant has a stronger phenotype than dia alone, especially upon midbody formation (see Extended Data Fig. 7i, j). However, loss of both dia and aprc1 function does not fully abrogate MyoII accumulation. We therefore repeated these experiments in a sensitized background by reducing the overall levels of F-actin using a chic mutant, which has a general role in actin polymerization in epithelial cells46. While reducing chic function in a dia mutant background modestly enhances the phenotype of dia loss of function, we found that loss of Dia, Arp2/3 and Profilin function (dia arpc1 chic triple-mutant cells) strongly affects MyoII accumulation both at 80% of the initial cell diameter and after midbody formation (see b). These results argue that either Dia or the Arp2/3 complex can generate the necessary F-actin network to promote MyoII accumulation, and that only a marked reduction in F-actin polymerization using the triple-mutant combination can efficiently abrogate MyoII accumulation in the neighbours. Together, these analyses argue against the role of a single specific Actin nucleator or the involvement of a specific F-actin network in promoting MyoII accumulation in the neighbours. b, Normalized MyoII accumulation at 80% of the initial cell diameter and 1 min after midbody formation for wild-type, dia single-, dia chic double-, dia arpc1 double- and dia arpc1 chic triple-mutant cells neighbouring wild-type dividing cells (5, 7, 7, 7 and 4 pupae, respectively). *P < 0.05, **P < 0.01, ****P < 0.0001. Kruskal–Wallis test. c–h, One-dimensional numerical integration of the active gel equations. The friction coefficient is shown in grey and the steady-state MyoII concentration is in blue. c, For a decreased value of the friction coefficient at the centre of the integration box, MyoII shows two accumulations at both interfaces between high and low friction. d, e, For a decreased value of the friction coefficient at the edge of the simulation box, Myosin final intensity shows two possible solutions: accumulation at the boundary between the low and high friction domains (d), or accumulation at the low friction edge of the simulation box (e). f, g, Same one-dimensional numerical integration as in (d and e), but with either preferential polymerization in the high friction zone (f), or advancing low friction region to mirror AJ ingression during contractile ring constriction (g). In both cases, MyoII robustly accumulates at the interface between the high and low friction domains. h, Same one-dimensional numerical integration as in (d and e), but with a boundary condition of non-zero velocity at the tip (v(x = 0,t) = 0.1 thin lines, v(x = 0,t) = 0.2 thick lines). The dashed lines show v(x = 0,t) = 0 (d) for comparison. We show that the MyoII concentration profile (blue) is unaffected by this boundary condition, and the velocity profile (orange) becomes flatter for increasing boundary velocity (see Supplementary Note). i, Same numerical integration as in Fig. 4b with a three-dimensional representation of the steady-state MyoII concentration. j, k, Steady-state velocity of the actomyosin flows in the horizontal and vertical direction, respectively vx (j) and vy (k). l, Steady-state MyoII concentration for increasing protrusion lengths, at constant contractility (colour-code as in i). Actomyosin flows can only occur above a critical length threshold, and the amount of MyoII accumulation increases with the protrusion length. m, Steady-state MyoII concentration for increasing contractility, at constant protrusion length (colour-code as in i). Actomyosin flows can only occur above a critical contractility threshold, and the amount of the accumulation increases with contractility. n, Steady-state MyoII concentration for intermediate contractility, protrusion length and friction parameters as in (i), with a zero-flux boundary condition. Results are qualitatively similar to (i), with the peak of MyoII accumulation showing a slight shift downwards. o, Steady-state MyoII concentration with friction uniformly low everywhere (remaining parameters are similar to n). Under these conditions, MyoII accumulation can have multiple and random locations (right- and left-side vertices in this simulation), instead of a robust accumulation at the base of the protrusion. n denotes number of cells throughout. Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 9 Dynamics and apical-basal localization of the actomyosin flows in the neighbours.

a, b, Lifeact–Ruby expressing cells neighbouring a wild-type dividing cell in a MyoII–3 × GFP tissue. Insets highlight progressive accumulation of MyoII–3 × GFP and Lifeact–Ruby in the neighbours. Kymograph in b along the yellow box. n = 25 (18 pupae). c, Velocity of F-actin and MyoII flows in wild-type neighbours facing wild-type dividing cells (7 and 15 pupae, respectively). d, F-actin speckle probability density in wild-type neighbours facing wild-type dividing cells (4 pupae). Error bars denote s.e.m. of a multinomial distribution. e–g, Lifeact–Ruby expressing neighbour facing a dividing cell in a Dlg–GFP tissue. Top panel corresponds to apical plane; bottom panel corresponds to a more basal plane, at the level of the septate junctions (schematically represented in e). Kymograph in g along the yellow boxes. Insets and kymographs show that the Lifeact–Ruby flows observed in the ingressing junction during cytokinesis are restricted to the apical domain. Note that the bright dots visible in the basal kymograph correspond to Lifeact–Ruby aggregates, and not directional speckles (see Supplementary Video 7e). n = 8 cells (5 pupae). h, i, Rok–GFP and MyoII–3 × mKate2 distribution during cytokinesis. Kymograph in i generated along the yellow box. During cytokinesis, Rok–GFP is localized at the membrane/cortex and also flows with MyoII–3 × mKate2 during the ingression of the future daughter cell’s membranes (insets and kymograph). The localization of Rok is therefore in full agreement with previous findings reporting that this kinase can bind to the membrane in both vertebrates and Drosophila47,48. Moreover, Rok flows were already reported in germband cells and shown to depend on MyoII activity49. Overall, the constitutive nature of the ROCK kinase47, its known binding to the membrane and MyoII fully support the notion that self-organized actomyosin flows are triggered by the local decrease in E-cad concentration. n = 25 (3 pupae). j, k, Ratio of F-actin intensity (labelled by Lifeact–GFP) at the ingressing region versus the remaining AJs (j) and the ratio of F-actin intensity at the medial pool versus the AJs (k) in wild-type or rok cells neighbouring a wild-type dividing cell (7 or 7 pupae, respectively). Note that F-actin intensity within the ingressing region is similar to its intensity at the remaining AJs for both wild-type and rok neighbours, indicating that loss of Rok activity does not reduce the amount of F-actin within the ingressing region. l, E-cad–GFP and MyoII–mChFP localization in wild-type (n = 38 cells, 2 pupae) and Shroom (shr; n = 33 cells, 4 pupae) dividing cells, as well as its neighbour. Arrowheads denote MyoII–mChFP accumulation in the neighbours. To test further the role of Rok in F-actin polymerization, we analysed whether Shroom and fhos, two Rok effectors known to regulate F-actin polymerization48,50, would be required for MyoII accumulation in the neighbours. Neither the loss of fhos (Extended Data Fig. 7o–q) nor Shroom activity affects MyoII accumulation (see n), arguing against a role of Rok in F-actin polymerization. m, Rate of contractile ring constriction in wild-type and shr dividing cells (4 and 4 pupae, respectively). n, Normalized MyoII accumulation at 80% of the initial cell diameter for wild-type and shr neighbours (2 and 4 pupae, respectively). n denotes number of cells throughout. In c and d, n/n = number of speckles quantified/corresponding number of cells quantified. Mann–Whitney U-test (c, j, m) and Student’s t-test (k, n). Data are mean ± s.e.m. Scale bars, 5 μm.

Source data

Extended Data Figure 10 Actomyosin flows in wild-type and ani, moe, E-cadanddia mutant conditions.

a–d, Wild-type (a, b; n = 25 cells, 4 pupae) or aniRNAi (c, d; n = 46 cells, 8 pupae) dividing and neighbouring cells expressing Lifeact–Ruby in a MyoII–3 × GFP tissue. Kymographs in b and d along the yellow boxes in a and c, respectively. Insets and respective kymograph in (a, b) highlight progressive accumulation of MyoII–3 × GFP and Lifeact–Ruby in the neighbours cells, via actomyosin flows, while in (c, d) it denotes a reduction in the actomyosin flows observed in cells neighbouring an aniRNAi dividing cell, as well as the diminished accumulation of MyoII–3 × GFP and Lifeact–Ruby at the base of the ingressing AJ (see Fig. 1d, e, Supplementary Video 2b and Fig. 4k, l). Note that in c and d, all cells express aniRNAi cells, marked by Lifeact–Ruby. To visualize F-actin dynamics exclusively in the neighbours in a–d, we photobleached the dividing cell before contractile ring constriction. e–h, Lifeact–Ruby-expressing wild-type (e, f; n = 27 cells, 5 pupae) or moeRNAi (g, h; n = 30 cells, 10 pupae) cells neighbouring a wild-type dividing cell in a MyoII–3 × GFP tissue. Kymographs in f and h along the yellow boxes in e and g, respectively. Insets and kymographs highlight the progressive accumulation of MyoII–3 × GFP and Lifeact–Ruby in both the wild-type and moeRNAi neighbours, via actomyosin flows. Dots denote moeRNAi cells, marked by Lifeact–Ruby. In agreement with our observation that MyoII accumulation occurs in moeRNAi neighbouring cells (Extended Data Fig. 3h and Supplementary Videos 4c, d), the F-actin flows are similar in velocity and amount to those observed in wild-type neighbouring cells (see m, n). i–l, E-cad (i, j, n = 16 cells, 5 pupae) or dia (k, l, n = 19 cells, 3 pupae) cells, marked by Lifeact–GFP, neighbouring a wild-type dividing cell in a MyoII–3 × mKate2 tissue. Dots denote E-cad (i) or dia (k) mutant cells. Kymographs in j and l generated along the yellow boxes in i and k, respectively. E-cad neighbours accumulate actomyosin at the base of the ingressing AJ (see Fig. 3j) and exhibit actomyosin flows similar to wild-type neighbours (see m and n). By contrast, dia neighbours exhibit delayed accumulation of MyoII (Extended Data Fig. 7i, j). Accordingly, in this context the F-actin flows are slower than in wild-type neighbours (see Fig. 4o, p). m, Percentage of wild-type, moeRNAi and E-cad neighbouring cells (12, 10, 5 pupae, respectively) exhibiting no flows, intermittent flows (<3 detectable speckles), or sustained flows (≥3 detectable speckles) when facing wild-type dividing cells. n, Actin flow velocity for wild-type, moeRNAi and E-cad neighbours facing wild-type dividing cells. n/n indicates number of speckles/number of cells (12, 9 and 5 pupae, respectively). Kruskal–Wallis test. Scale bars, 5 μm.

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Supplementary information

Supplementary Information

This file contains a Supplementary Theory Note and Supplementary Tables 1-2. (PDF 775 kb)

Supplementary Data

This file contains the supplementary codes 1-3: Supplementary Code 1 - E-Cad dilution, numerical integration of the dynamics of E-Cadherin on an elongating junction (written for Mathematica); Supplementary Code 2 - Actomyosin dynamics in one-dimension, numerical integration of the dynamics of actomyosin on a junction with variable friction in one-dimension (written for Mathematica); Supplementary Code 3 - Actomyosin dynamics in two-dimensions, numerical integration of the dynamics of actomyosin on a domain with variable friction in two-dimensions (written for Freefem++). (ZIP 2273 kb)

3D organisation of the dividing and neighbouring cell membranes

3D segmentation of a PH–GFP expressing cell neighbouring a PH–ChFP dividing cell upon co-ingression of both cells’ membranes during cytokinesis. White arrow: indicates the ingression of the PH–GFP neighbour in between the dividing cell membranes (see Extended Data Fig. 1a, b). Scale bar: 5µm (MP4 3588 kb)

Lowering contractile ring pulling forces in the dividing cell affects Myosin II accumulation in the neighbours

a, E-cad–GFP and MyoII–mChFP localization in the dividing cell and its neighbours upon contractile ring laser ablation after MyoII–mChFP accumulation. Dashed box: ablated region. Time was set to 0s at the ablation time (see Fig. 1c). b, E-cad–GFP and MyoII–mChFP localization in cells neighbouring wt, pnutRNAi, rokRNAi and aniRNAi dividing cells (see Fig. 1d–e and Extended Data Fig. 1p). White arrows: MyoII–mChFP accumulation in the neighbouring cells. White open arrows: absence or decrease of MyoII–mChFP accumulation in the neighbouring cells. D, dividing cell; N, neighbouring cell. Scale bars: 5µm (MP4 9052 kb)

Myosin II accumulation in the neighbouring cells is preceded by cortex detachment and a decrease of E-Cad concentration at the ingressing AJ

E-cad–GFP and MyoII–mChFP localization in the dividing cell and its neighbours. Time was set to 0s at cytokinesis onset. White asterisks: separation of the MyoII–mChFP and the E-cad–GFP signals at the ingressing AJ. Yellow arrows: decrease of E-cad–GFP signal at the ingressing AJs. White arrows: MyoII–mChFP accumulation in the neighbouring cells (see Fig. 2a, b). Scale bar: 5µm (MP4 4009 kb)

Cortex detachment is not sufficient to sustain MyoII accumulation in the neighbouring cells.

a, E-cad–GFP and MyoII–mChFP localization upon laser ablation of the initially detached cortical MyoII. Orange box: ablated region at t = 0 s. White arrows: MyoII–mChFP accumulation in the neighbouring cells upon laser ablation of the initially detached cortex (see Fig. 2d). b, E-cad–GFP and MyoII–mChFP localization in cells neighbouring pnutRNAi dividing cells. White asterisks: separation of cortical MyoII–mChFP from the ingressing AJs (see Extended Data Fig. 3g). c, d, E-cad–GFP and MyoII–mChFP dynamics in moeRNAi neighbouring cells (marked by the lack of cytosolic GFP; highlighted in (d) with white dots) during cytokinesis. c, White asterisk: separation of MyoII–mChFP from the ingressing AJ. Yellow open arrow: decrease of E-cad–GFP signal at the ingressing AJ. White arrow: MyoII–mChFP accumulation in the neighbouring cells (see Extended Data Fig. 3h). d, White dashed boxes: inset regions. White arrowheads: detached cortical MyoII–mChFP coalesces with the MyoII–mChFP accumulation localized at the boundary of the high-low E-cad–GFP signal. Open arrowheads: detached cortical MyoII–mChFP disassembles, while MyoII accumulates at the base of the ingressing AJ (see Extended Data Fig. 3i, j). Scale bars: 5µm (MP4 12909 kb)

Photoactivated E-cadherin is diluted along the ingressing membrane during cytokinesis.

MyoII–3XmKate2 and a photoactivatable version of E-cad, E-cad–3XPAGFP during cytokinesis. The ingressing AJ is photoactivated at the rim of the contractile ring at the interface between the dividing cell and its neighbour (dashed box). Time is set to 0s at the time of E-cad–3XPAGFP photoactivation (see Extended Data Fig. 5b). Scale bar: 5µm (MP4 2080 kb)

Lowering E-Cad concentration at the ingressing AJ triggers MyoII accumulation in cells neighbouring a pnut dividing cell

E-cad–3XGFP and MyoII–3XmKate2 distribution in a pnut mutant dividing cell, marked by 2 copies of E-cad–3XGFP, and its E-cad mutant neighbour, marked by the absence of E-cad–3XGFP. White dashed line: indicates the boundary with E-cad mutant cells. White asterisk: separation of the MyoII–3XmKate2 and the E-cad–3XGFP signals at the ingressing AJ. Bracket: decrease of E-cad–3XGFP signal at the ingressing AJ. White arrow: MyoII–3XmKate2 accumulation in the neighbouring cells (see Fig. 3i–k). Scale bar: 5µm (MP4 3982 kb)

Actomyosin flows in the neighbouring cells

a, MyoII–RFP dividing cell facing a MyoII–GFP neighbouring cell upon photobleaching of the MyoII–GFP accumulation in the neighbours. Orange dashed box: photobleached region at t = 0 s. Arrowheads: MyoII–GFP speckles moving along the ingressing AJ and accumulating at its base (see Fig. 4d, e). b, MyoII–Dendra2 photoconversion at the rim of the contractile ring during cytokinesis. Red dashed box: photoconverted region at t = 0 s. Arrowhead: photoconverted MyoII–Dendra2 speckle moving with the ingressing AJ. White arrow: accumulation of photoconverted MyoII–Dendra2 in the neighbouring cells (see Fig. 4f). c, d, Lifeact–GFP (c) or Lifeact–Ruby (d) expressing neighbours facing a dividing cell in a MyoII–3XmKate2 (c) or MyoII–3XGFP (d) tissue. Dashed Box: region shown in the insets (d). White Arrows: MyoII and Lifeact accumulation in the neighbours (see respectively (c) Fig. 4g, h and (d) Extended Data Fig. 9a, b). e, Lifeact–Ruby expressing cell neighbouring a dividing cell in a Dlg–GFP tissue at the level of the AJs (Apical panel) and of the Septate Junctions (Basal panel – 2µm below the apical plane). F-Actin flows are observed apically, at level of the AJs (arrow) and not more basally, at the level of the Septate junctions (open arrow; see Extended Data Fig. 9e–g). Scale bars: 5µm (MP4 24322 kb)

Actomyosin flows are reduced in cells neighbouring a pnut dividing cell and in rok neighbouring cells facing a wt dividing cell

a, pnutRNAi dividing cell and its neighbours in a Lifeact–Ruby and MyoII–3XGFP tissue. pnutRNAi cells express Lifeact–Ruby. White asterisks: separation of the MyoII–3XGFP signal from the tip of the ingressing AJ, highlighted by the contractile ring. White open arrows: reduced Lifeact–Ruby and MyoII–3XGFP flows in cells facing a pnutRNAi dividing cell (see Fig. 4i–l). Note that to clearly visualize F-actin dynamics exclusively in the neighbours, we photobleached Lifeact–Ruby in the dividing cell prior to cytokinesis onset. b, rok neighbour, marked by Lifeact–GFP expression (white dots), facing a wt dividing cell in a MyoII–3XmKate2 tissue. White open arrows: reduced Lifeact–GFP flows in rok neighbours (see Fig. 4m–p). Scale bars: 5µm (MP4 9334 kb)

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Pinheiro, D., Hannezo, E., Herszterg, S. et al. Transmission of cytokinesis forces via E-cadherin dilution and actomyosin flows. Nature 545, 103–107 (2017). https://doi.org/10.1038/nature22041

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