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Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability

Nature Cell Biology volume 22, pages 791–802 (2020)Cite this article

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Abstract

Tissue remodelling during Drosophila embryogenesis is notably driven by epithelial cell contractility. This behaviour arises from the Rho1–Rok-induced pulsatile accumulation of non-muscle myosin II pulling on actin filaments of the medioapical cortex. While recent studies have highlighted the mechanisms governing the emergence of Rho1–Rok–myosin II pulsatility, little is known about how F-actin organization influences this process. Here, we show that the medioapical cortex consists of two entangled F-actin subpopulations. One exhibits pulsatile dynamics of actin polymerization in a Rho1-dependent manner. The other forms a persistent and homogeneous network independent of Rho1. We identify the formin Frl (also known as Fmnl) as a critical nucleator of the persistent network, since modulating its level in mutants or by overexpression decreases or increases the network density. Absence of this network yields sparse connectivity affecting the homogeneous force transmission to the cell boundaries. This reduces the propagation range of contractile forces and results in tissue-scale morphogenetic defects.

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Fig. 1: Spatiotemporal dynamics of medioapical F-actin.
Fig. 2: Rho1 pathway inhibition reveals two differentially regulated medioapical F-actin subpopulations (GBE).
Fig. 3: Rho1 pathway inhibition reveals two differentially regulated medioapical F-actin subpopulations (DC).
Fig. 4: The formin Frl promotes the persistent F-actin network assembly.
Fig. 5: Frl antagonizes Rho1-induced medial pulsed contractility.
Fig. 6: Cellular- and tissue-scale effects of Frl loss or gain of function.
Fig. 7: The persistent network promotes the propagation of MyoII-induced contractile forces.

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Data availability

All data, such as raw images, supporting the findings of this study are available from the corresponding author upon reasonable request. Source data for Figs. 1–7 and Extended Data Figs. 1–4 are presented with the paper.

Code availability

Codes and plugins used in this study can be retrieved as referenced in the Methods section of the manuscript. All other custom codes are available from the corresponding author upon reasonable request.

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Acknowledgements

We are grateful to J. Mihály (Biological Research Centre, HAS, Szeged, Hungary) and A. Jenny (Albert Einstein College of Medicine, The Bronx, NY, USA) for providing fly stocks. We thank members of the Lecuit and Lenne groups for stimulating discussions and comments during the course of this project. We also thank FlyBase for maintaining databases and the Bloomington Drosophila Stock Center for providing fly stocks. The experiments were performed using the PiCSL-FBI core facility (IBDM, Marseille, France), a member of the France-BioImaging national research infrastructure supported by the French National Research Agency (ANR-10-INBS-04-01, “Investissements d’Avenir”). B.D. was supported by the ERC Biomecamorph (grant no. 323027) and Fondation Bettencourt Schueller. R.C. and J.-M.P. were supported by the CNRS. H.A. was supported by the ANR MechaResp (ANR-17-CE13-0032). T.L. was supported by the CNRS, followed by the Collège de France.

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Authors and Affiliations

  1. Aix Marseille Université, CNRS, IBDM-UMR7288, Turing Center for Living Systems, Marseille, France

    Benoit Dehapiot, Raphaël Clément, Hervé Alégot, Jean-Marc Philippe & Thomas Lecuit

  2. Institute of Genetics, Biological Research Centre, HAS, Szeged, Hungary

    Gabriella Gazsó-Gerhát

  3. Doctoral School of Biology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary

    Gabriella Gazsó-Gerhát

  4. Collège de France, Paris, France

    Thomas Lecuit

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Contributions

B.D. and T.L. conceived the project. B.D. performed experiments and quantifications and developed analytical methods. R.C. designed the numerical model and performed the simulations. H.A. performed the experiments presented in Supplementary Fig. 3a–c and Fig. 6l (FrlOE). G.G.-G. isolated the frl59/59 null allele. J.-M.P. created all the fluorescent constructs. B.D., R.C. and T.L. discussed the data and wrote the manuscript.

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Correspondence to Thomas Lecuit.

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Extended data

Extended Data Fig. 1 Quantifying the medio-apical F-Actin dynamics.

a, Comparison between live (eGFP::UtrCH) and fixed (Phalloidin) F-Actin localization in ectodermal (GBE) and amnioserosa cells (DC). b, Automatic cell segmentation procedure used to define medio-apical ROIs for quantification (see Methods). Briefly, cell boundaries are detected on the lower junctional plane using a watershed algorithm. The segmented cells are then identified and tracked over time to define ROIs. Finally, these ROIs are shrunk of a few pixels to discard the junctional signal and the medial fluorescence intensities are measured on the max. proj. of the Z-series. c, Presentation of the background subtraction procedure (see Methods). The background is evaluated on the lower Z-planes and subtracted from the max. proj. of the Z-series before quantification. Removing the background is critical to properly measure the total amount of fluorescence in the ever-changing apical cell surface (see time shift when comparing the medial F-Actin levels with or without background subtraction). d, To quantify the levels of pulsed contractility, we processed single cell profiles using a high-pass Butterworth IIR filter (see Methods). This filter is used to remove low frequency components and have been adjusted to fit the temporality of pulsatility in GBE and DC. Results in a,c,d have been systematically observed in 20 independent experiments. Scale bars size is directly indicated on the pictures. Statistical source data are provided in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Spatio-temporal tracking of pulses and numerical modeling.

a, Description of the method used to quantify to pulsatile Rho1 activity without cell segmentation (see Methods and Supplementary Movie 16). Clusters of AniRBD::eGFP signal are detected using a DBSCAN algorithm. These clusters are then converted into surface ROIs using convex hulls and overlapping ROIs tracked over time to follow individual pulses. Filters such as min./max. area or min./max. duration are applied to reduce tracking mistakes. The AniRBD::eGFP pulse amplitude measurements are performed considering the maximum of total fluorescence intensity for each track. b, MyoII pulses are automatically detected in time following the derivatives of high-pass filtered total medial MyoII levels (see Methods). Pulse temporal landmarks have been defined as follow: ti: initial time; tdmax: max derivative; tmax: max amplitude; tdmin: min derivative; tf: final time. c, MyoII pulses are automatically detected in space by monitoring the centre of mass of the medial sqh::mKate2 signal over time (see Methods and Supplementary Movie 22). The pulse centre used for the KLT analysis is defined by averaging the position of the recorded centre of mass between tdmax and tdmin of the pulse. This time interval corresponds to the period during which the apical surface contract during a pulse. d, Line plots: Averaged speed towards the pulse according to pulse temporal landmarks (see above) for different distance bins (see legend). The black arrows show the time of maximum speed. e, Schematics of the numerical model. The black circle depicts the actomyosin pulse, and arrows depict forces applied to boundary elements. Only a fraction of boundary elements is represented. Results in a are representative of 17 independent experiments. Results in b,c are representative of 16 independent experiments. Results in d are representative of 4 independent experiments. Scale bars size is directly indicated on the pictures. Statistical source data are provided in Source Data Extended Data Fig. 2.

Source data

Extended Data Fig. 3 Control engrailed-GAL4 overexpression and junctional actomyosin quantifications.

a, Distribution of measured nuclear NLS::RFP fluo. int. and selected threshold to define the control (<475) and engrailed-GAL4 induced NLS::RFP cells (>475). b, Box plots: mean medial F-Actin fluo. int. averaged per cell and over time (90 or 120 × 10 sec), normalized to the mean of controls. c, Box plots: cell averaged S.D. of high-pass filtered total medial F-Actin (left) fluo. int. and apical cell area (right), normalized to the mean of controls. d,e, Box plots: cell averaged mean junctional MyoII (left) and F-Actin (right) intensities in ectodermal (d) or amnioserosa cells (e), normalized to the mean of controls. Quantifications and statistical analysis in a-e were carried out using n = the total number of cells gathered from multiple embryos, as indicated below graphs (e = embryos, c = cells). Box plots in b,c,d,e: as described in Fig. 1 legend. Statistics in b,c,d,e: two-sided Mann–Whitney test, NS: p > 5E-2, *: p < 5E-2, **: p < 5E-3, ***: p < 5E-4, ****: p < 5E-5. Statistical source data are provided in Source Data Extended Data Fig. 3.

Source data

Extended Data Fig. 4 Junctional dynamics in Frl loss or gain of function during GBE.

a, Display of the shrinking and extending AJs in ectodermal cells during GBE, defined respectively as the disappearing and appearing junctions throughout movies duration. b, Line plots: averaged AJs length ± S.D. over time depicting shrinking (before reference time t = 0 min) and extending (after reference time t = 0 min) junctions. c, Line plots: one junction example of shrinking (top) and extending (bottom) AJs and the linear fit method used to extract the shrinking and extending rate. d, Box plots: junction shrinking and extending rate in WT vs frl59/59 (left) or WT vs FrlOE (right) ectodermal cells. e, Diagrams showing the reference angles used to quantify the shrinking (top) and extending (bottom) junction orientation. f, Box plots: junction orientation angle in WT vs frl59/59 (left) or WT vs FrlOE (right) ectodermal cells. Results in a-f are representative of 5 (2 control, 3 frl59/59) and 5 (3 control, 2 FrlOE) independent experiments. Quantifications and statistical analysis in b,d,f were carried out using n = the total number of junctions gathered from multiple embryos, as indicated below graphs (e = embryos, j = junctions). Box plots in d,f: as described in Fig. 1 legend. Statistics in d,f: two-sided Mann–Whitney test, NS: p > 5E-2, *: p < 5E-2, **: p < 5E-3, ***: p < 5E-4, ****: p < 5E-5. Statistical source data are provided in Source Data Extended Data Fig. 4.

Source data

Supplementary information

Reporting Summary

Supplementary Table 1

Fly genotypes and crosses

Supplementary Video 1

F-actin dynamics in ectodermal cells (GBE). Live ×100 imaging of F-actin (eGFP::UtrCH) in ectodermal cells during GBE. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 3 s.

Supplementary Video 2

F-actin dynamics in amnioserosa cells (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 3 s.

Supplementary Video 3

Medial F-actin turnover (GBE + DC). High temporal resolution live ×100 imaging of F-actin (eGFP::UtrCH) in ectodermal cells during GBE (left panel) and in amnioserosa cells during DC (right panel). The video represents a max. proj. of the 2 most apical z-planes, spaced by 0.33 µm and acquired every 1 s.

Supplementary Video 4

Medial MyoII and F-actin dynamics (GBE). Live ×100 imaging of MyoII (Sqh::mCherry, left panel) and F-actin (eGFP::UtrCH, right panel) in ectodermal cells during GBE. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 3 s.

Supplementary Video 5

Medial MyoII and F-actin dynamics (DC). Live ×100 imaging of MyoII (Sqh::mCherry, left panel) and F-actin (eGFP::UtrCH, right panel) in amnioserosa cells during DC. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 3 s.

Supplementary Video 6

Rho1 pathway inhibition (GBE). Live ×100 imaging of MyoII (Sqh::mCherry, top panels) and F-actin (eGFP::UtrCH, bottom panels) in ectodermal cells during GBE. Left panels: control embryo (water injected), middle panels: C3 transferase (Rho1 inhibitor) injected embryo, right panels: RhoGEF2−/− embryo. The videos represent a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 3 s.

Supplementary Video 7

Rho1 pathway inhibition (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. White outlines: control cells; yellow outline: Rho1N19 (Rho1 dominant-negative form) expressing cell. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 8

Rok kinase inhibition (DC). Live ×100 imaging of MyoII (Sqh::mCherry, top panel) and F-actin (eGFP::UtrCH, bottom panel) in amnioserosa cells during DC. Left panels: control embryo (water injected), right panels: H-1152 (Rok inhibitor) injected embryo. The videos represent a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 9

Frl loss of function (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. Left panel: control embryo, right panel: frlshRNA-expressing embryo. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 10

Frl loss or gain of function (GBE). Live ×100 imaging of F-actin (eGFP::UtrCH) in ectodermal cells during GBE. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 2 most apical z-planes, spaced by 0.33 µm and acquired every 2 s.

Supplementary Video 11

Frl loss or gain of function (DC). Live ×10 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 2 most apical z-planes, spaced by 0.33 µm and acquired every 2 s.

Supplementary Video 12

MyoII and F-actin dynamics in Frl loss or gain of function (GBE). Live ×100 imaging of MyoII (Sqh::mKate2, top panel) and F-actin (eGFP::UtrCH, bottom panel) in ectodermal cells during GBE. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 6 s.

Supplementary Video 13

MyoII and F-actin dynamics in Frl loss or gain of function (DC). Live ×100 imaging of MyoII (Sqh::mKate2, top panel) and F-actin (eGFP::UtrCH, bottom panel) in amnioserosa cells during DC. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 14

Frl gain of function (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. White outlines: control cells, yellow outline: FrlOE (overexpression) cell. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 15

Rho1GTP dynamics in Frl loss or gain of function (DC). Live ×100 imaging of Rho1GTP (AniRBD::eGFP) in amnioserosa cells during DC. Left panel: control embryo; middle panel: Frl shRNA expressing embryo; right panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 16

Automatic Rho1GTP pulse tracking (DC). Live ×100 imaging of Rho1GTP (AniRBD::eGFP) in amnioserosa cells during DC, showing the method used to automatically track Rho1GTP pulses without cell segmentation. Left panel: tracked ROIs, right panel: individual pulses detected using DBScan clustering. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 17

Epithelial dynamics in Frl loss or gain of function (GBE). Live ×40 imaging of F-actin (eGFP::UtrCH) in ectodermal cells during GBE. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The yellow cell outlines represent the results of cell segmentation and the white squares mark the localization of T1 events. The video represents 1 z-plane, acquired every 20 s.

Supplementary Video 18

Germband extension in Frl loss or gain of function (GBE). DIC live imaging of embryos undergoing GBE. Top panel: control embryo; middle panel: frl59/59 (null mutant) embryo; bottom panel: FrlOE (overexpression) embryo. The video represents 1 z-plane, acquired every 30 s.

Supplementary Video 19

Apical cell surface deformations in Frl loss or gain of function (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. Left panel: control embryo; middle panel: frl59/59 (null mutant) embryo; right panel: FrlOE (overexpression) embryo. The inserted images represent the results of cell segmentation. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 10 s.

Supplementary Video 20

Dorsal closure in Frl loss or gain of function (DC). Live ×10 imaging of F-Actin (eGFP::UtrCH) of embryos undergoing DC. Top panel: control embryo; middle panel: Frl shRNA expressing embryo; bottom panel: FrlOE (overexpression) embryo. The video represents a max. proj. of the 10 z-planes, spaced by 5 µm and acquired every 10 min.

Supplementary Video 21

Contractile event dynamics in Frl loss of function (DC). Live ×100 imaging of F-actin (eGFP::UtrCH) in amnioserosa cells during DC. Left panel: control embryo, right panel: frl59/59 (null mutant) embryo. The video represents a max. proj. of the 2 most apical z-planes, spaced by 0.33 µm and acquired every 2 s.

Supplementary Video 22

Automated pulse and KLT tracking (DC). Live ×100 imaging of MyoII (Sqh::mKate2, left panel) and F-actin (eGFP::UtrCH, right panel) in amnioserosa cells during DC. Left panel: automated MyoII pulse tracking in space, the white crosses represent the medial MyoII centre of mass. Right panel: F-actin KLT-tracked particles, the colour code represents the speed of tracked particles in µm s–1. The video represents a max. proj. of the 4 most apical z-planes, spaced by 0.33 µm and acquired every 5 s.

Supplementary Video 23

Numerical model. Representative simulations for different values of 𝜆 (ten examples per condition). The pulse is represented by the inner circle and the green segments indicate that a boundary element is connected to the pulse. The pulse position is randomly chosen in the different examples.

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Dehapiot, B., Clément, R., Alégot, H. et al. Assembly of a persistent apical actin network by the formin Frl/Fmnl tunes epithelial cell deformability. Nat Cell Biol 22, 791–802 (2020). https://doi.org/10.1038/s41556-020-0524-x

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