An actin-based viscoplastic lock ensures progressive body-axis elongation (original) (raw)

Data availability

Source Data for Figs. 1d, 2c–f, 3c–e, 4d, e and Extended Data Figs. 1b, d, k, 3b, c, 4c, d, 5d, e, l, 7a, b, 9f, as well as numbers of replicates and P values (where applicable) for all figures are provided in the online version of the paper.

Code availability

All MATLAB scripts used for the present analysis are available upon reasonable request.

Change history

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

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Acknowledgements

The authors thank A. Spang, S. Grill, Y. Bellaïche and R. Voituriez for critical comments on the manuscript and M. Gettings for improving the English. We also thank the Caenorhabditis Genetics Center (funded by the NIH Office of Research Infrastructure Programs P40 OD010440) and National BioResource Project at Tokyo Women's Medical University for strains, the IBPS Imaging Facility for advice. This work was supported by the Agence Nationale pour la Recherche, the European Research Council (grant no. 294744), Israel–France Maïmonide exchange program grants, and installation funds from the Centre National de la Recherche Scientifique (CNRS) and University Pierre et Marie Curie (UPMC) to M.L. A.L. was supported by a fellowship from the Fondation pour la Recherche Médicale (FDT201805005501). This work was also made possible by institutional funds from the CNRS, University of Strasbourg and UPMC, the grant ANR-10-LABX-0030-INRT, which is a French State fund managed by the Agence Nationale de la Recherche under the framework programme Investissements d’Avenir labelled ANR-10-IDEX-0002-02 to the IGBMC. The work of P.M. and E.B. was partly supported by the Agence Nationale de la Recherche (contract ANR-11-EQPX-0029 Morphoscope2), the work of S.O. was partly supported by the National Institutes of Health (grant AR048615).

Author information

Author notes

  1. These authors contributed equally: Alicia Lardennois, Gabriella Pásti

Authors and Affiliations

  1. CNRS UMR7622, Institut de Biologie Paris–Seine (IBPS), Sorbonne Université, Paris, France
    Alicia Lardennois, Teresa Ferraro, Flora Llense & Michel Labouesse
  2. IGBMC –CNRS UMR 7104, INSERM U964, Development and Stem Cells Department, Université de Strasbourg, Illkirch, France
    Gabriella Pásti, Julien Pontabry, David Rodriguez, Samantha Kim, Christelle Gally & Michel Labouesse
  3. INSERM U1182 – CNRS/ UMR7645, Laboratoire d’Optique et Biosciences, Ecole Polytechnique, Paris, France
    Pierre Mahou & Emmanuel Beaurepaire
  4. RS2D, Mundolsheim, France
    Julien Pontabry
  5. Departments of Pathology and Cell Biology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA, USA
    Shoichiro Ono

Authors

  1. Alicia Lardennois
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  2. Gabriella Pásti
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  3. Teresa Ferraro
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  4. Flora Llense
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  5. Pierre Mahou
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  6. Julien Pontabry
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  7. David Rodriguez
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  8. Samantha Kim
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  9. Shoichiro Ono
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  10. Emmanuel Beaurepaire
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  11. Christelle Gally
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  12. Michel Labouesse
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Contributions

M.L. conceived the project. A.L. performed most experiments with initial contributions from G.P. T.F. conceived the modelling, F.L. generated the FHOD-1 variants and the screen reported in Supplementary Table 3, P.M. and E.B. helped with TIRF–SIM imaging, T.F. and J.P. performed image analysis, C.G. shared data from a related screen, S.K. helped with the spc-1(ra409) mini-RNAi screen, D.R. provided technical assistance and S.O. provided the outcrossed gsnl-1 and viln-1 mutants. M.L. wrote the manuscript, and all authors commented and proofread it (except S.K., who was an intern), A.L. assembled figures, T.F. conceived and wrote the supplementary mathematical modelling material and F.L. prepared the Methods section.

Corresponding author

Correspondence toMichel Labouesse.

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Peer review information Nature thanks Edwin Munro, Bruce Vogel and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Fig. 1 Genes required to maintain embryonic elongation.

a, RNAi screen in a pak-1 mutant identified spc-1 as an enhancer (Supplementary Table 1). b, DIC images and quantification of newly hatched wild-type body length (n = 38 embryos), pak-1(tm403) (n = 32 embryos), spc-1(RNAi) (n = 26 embryos) and spc-1(RNAi) pak-1(tm403) (n = 36 embryos). Scale bars, 25 µm (WT and pak-1), 10 µm (spc-1 and spc-1 pak-1). Data represent mean values ± s.d. Two-sided paired _t_-test. c, A yeast two-hybrid screen using the PAK-1 N-terminal domain as a bait identified the SPC-1 SH3 domain as a prey (orange background) (Supplementary Table 2). d–j, Loss of the proteins GIT-1 and PIX-1, acting upstream of PAK-1 in the mechanotransduction pathway promoted by muscle contractions, in the absence of spc-1 also triggers a retraction phenotype. d–j, Elongation curves (d) and terminal phenotypes of wild type (n = 12 embryos), pak-1(tm403) (e; n = 11 embryos), git-1(tm1962) (f; n = 10 embryos), pix-1(gk416) (g; n = 10 embryos), spc-1(RNAi) pak-1(tm403) (h; n = 9 embryos), spc-1(RNAi) git-1(tm1962) (i; n = 11 embryos), spc-1(RNAi) pix-1(tm416) (j; n = 8 embryos). Data represent mean ± s.e.m. k–n, Elongation curves (k) and DIC pictures showing the terminal phenotypes of unc-112(RNAi) embryos (l; n = 14) and unc-112(RNAi) pak-1(tm403) (m; n = 8 embryos). n, Terminal phenotype of unc-112(RNAi) spc-1(ra409) obtained by inducing unc-112(RNAi) in the strain ML2436 bearing a rescuing extrachromosomal spc-1::gfp array and looking for embryos having lost the array; we could only obtain a few embryos of the desired phenotype despite numerous repeats (n = 4 embryos), all of which had the phenotype illustrated here, which is similar to that of spc-1(ra409) alone. Data represent mean ± s.e.m. Scale bars in e–j, l–n, 17 µm. *P < 0.05; **P < 0.001; ***P < 0.0001.

Source data

Extended Data Fig. 2 PAK-1and SPC-1 colocalize with actin filaments.

a, b, Distribution of PAK-1::mKate (a; n = 20 embryos) and SPC-1::GFP (b; n = 13 embryos) in a late embryo. Enlarged images of PAK-1 and SPC-1 showing a filamentous distribution in the dorsoventral epidermis similar to actin filaments. c, Fluorescence images of PAK-1::mKate (red) and SPC-1::GFP (green) (n = 20 embryos). The panel shows the colocalization image for the most-apical focal planes (top image), and full XZ (green panel) and YZ (red panel) projections. The level of co-localization is high based on Pearson’s correlation coefficient (0.7–0.9, n = 20 embryos). The highest level of colocalization is detected at the apical cortex. d, Fluorescence images of _Plin-26::_VAB-10(ABD)::mKate (red) and SPC-1::GFP (green) (n = 8 embryos). The panel shows the colocalization image for the most-apical focal planes (top image), and full XZ (green panel) and YZ (red panel) projections. The level of colocalization is high based on Pearson’s correlation coefficient (0.7–0.9, n = 8 embryos). The colocalization is detected almost exclusively at the apical cortex. The gene lin-26 drives expression in the epidermis; VAB-10(ABD) corresponds to the two actin-binding domains (calponin homology) of the protein VAB-10. Scale bar, 10 µm.

Extended Data Fig. 3 Actin-filament continuity and orientation at three elongation stages.

a, d–i, Epidermal actin filaments visualized with the Pdpy-7::LifeAct::GFP reporter construct in wild type (a), pak-1(tm403) (d), spc-1(RNAi) (e), spc-1(RNAi) pak-1(tm403) (f), unc-112(RNAi) (g), fhod-1(tm2363) (h) and fhod-1(tm2363) spc-1(RNAi) (i) at mid-elongation (twofold equivalent) stage. Yellow rectangle, ROI. Scale bar, 10 µm. ROI after binarization (green) and major axis detection (red) (a, top middle, d–i, bottom), based on three steps of image treatment for continuity and orientation analysis (a, right). Actin continuity: distribution of actin segments based on their length (a, bottom middle). b, Quantification of actin-filament continuity; the graph represents the length (in pixels) along the circumferential axis of actin filaments in early, mid and late (corresponding to 1.7-fold, 2-fold and 3-fold equivalent stages in a wild-type embryo, respectively) embryos of wild-type (early n = 12, mid n = 19, late n = 16), pak-1(tm403) (early n = 16, mid n = 21, late n = 16), spc-1(RNAi) (early n = 15, mid n = 21, late n = 20), spc-1(RNAi) pak-1(tm403) (early n = 12, mid n = 17, late n = 26), unc-112(RNAi) (early n = 8, mid n = 13, late n = 12), fhod-1(tm2363) (early n = 12, mid n = 14, late n = 10), fhod-1(tm2363); spc-1(RNAi) (early n = 7, mid n = 11, late n = 8), spc-1(ra409) pak-1(tm403) (mid n = 14, late n = 20) and unc-112(RNAi) ; spc-1(ra409) pak-1(tm403) (early n = 8, mid n = 15, late n = 19) genotypes. c, Actin-filament orientation based on FFT and binarization. Wild-type (early n = 12, mid n = 18, late n = 14), pak-1(tm403) (early n = 16, mid n = 20, late n = 16), spc-1(RNAi) (early n = 14, mid n = 18, late n = 18), spc-1(RNAi) pak-1(tm403) (early n = 12, mid n = 18, late n = 21), unc-112(RNAi) (early n = 8, mid n = 13, late n = 12), fhod-1(tm2363) (early n = 12, mid n = 14, late n = 10), fhod-1(tm2363); spc-1(RNAi) (early n = 7, mid n = 11, late n = 8), spc-1(ra409) pak-1(tm403) (mid n = 14, late n = 19) and unc-112(RNAi) spc-1(ra409) pak-1(tm403) (early n = 8, mid n = 15, late n = 19) genotypes. Note that the characteristics of actin filaments in spc-1(RNAi) pak-1(tm403) embryos differ mostly at the equivalent of the twofold stage when muscles become active. At earlier and later stages, spc-1(RNAi) embryos and spc-1(RNAi) pak-1(tm403) embryos become similar. Each graph represents median values, 25th and 75th percentiles. The whiskers extend to the most extreme data points not considered outliers. Two-sided paired _t_-test. *P < 0.05; **P < 0.001; ***P < 0.0001; n.s, not significant.

Source data

Extended Data Fig. 4 Changes in embryo diameter during elongation.

a, b, Fluorescence micrographs of embryos expressing the Pepid::Lifeact::GFP construct in the epidermis at three elongation stages early, middle and late (corresponding to 1.7-fold, 2-fold and 3-fold equivalent stages in a wild-type embryo, respectively) for wild-type (a) and spc-1(RNAi) pak-1(tm403) embryos (b). Scale bar, 10 µm. The Pepid promoter corresponds to Pdpy-7. The yellow lines correspond to the segments used to measure the dorsoventral width of the V1 seam cell. c, d, Quantification of the average V1 cell circumferential width normalized to the initial width during elongation (c), and of the average dorsoventral circumferential width at the level of the V1 seam cell (d), which was calculated using the measured embryo length and V1 cell width, taking into consideration the conservation of the total embryo volume, in wild-type (early n = 38, mid n = 10, late n = 14), pak-1(tm403) (early n = 26, mid n = 8, late n = 20), spc-1(RNAi) (early n = 24, mid n = 26, late n = 18), spc-1(RNAi) pak-1(tm403) (early n = 22, mid n = 30, late n = 38), unc-112(RNAi) (early n = 8, mid n = 9, late n = 8), and unc-112(RNAi) spc-1(ra409) pak-1(tm403) (early n = 7, mid n = 12, late n = 17) embryos. Error bars, s.e.m. A notable feature of spc-1(RNAi) pak-1(tm403) embryos is that the circumferential dimension of the seam cells decreased much more than that of their dorsoventral cells, which most probably reflects the actin-filament integrity defects combined with a _F_seam force largely unchanged.

Source data

Extended Data Fig. 5 Bending and severing of actin bundles during muscle contractions.

a, b, Kymographs of the regions boxed in yellow in Fig. 3a, b after spinning-disc time-lapse imaging of epidermal actin filaments (Pdpy-7::LifeAct::GFP reporter) in wild-type (a) and spc-1(RNAi) pak-1(tm403) (b) embryos at mid-elongation (twofold equivalent) stage. Scale bar, 5 µm. c, Principle of the RNAi screen performed to identify proteins mediating actin remodelling; the recipient strain carried a rescuing, but frequently lost, spc-1(+) transgene (green). d, Quantification of L1 hatchling length after downregulation or mutation of the indicated gene_s_; the presence of the spc-1::gfp transgene is denoted +. Control worms fed on L4440 bacteria. e–k, Elongation curves (e) and DIC images showing the terminal phenotypes of pak-1(tm403) (f; n = 11 embryos), gsnl-1(tm2730); pak-1(tm403) (g; n = 9 embryos), viln-1(ok2413); pak-1(tm403) (h; n = 9 embryos), gsnl-1(tm2730); spc-1(RNAi) pak-1(tm403) (i; n = 5 embryos), viln-1(ok2413); spc-1(RNAi) pak-1(tm403) (j; n = 11 embryos) and spc-1(RNAi) pak-1(tm403) (k; n = 9 embryos). Pink box in e, period of muscle activity. Data represent mean ± s.e.m. Scale bar, 25 µm. l, Quantification of the L1 hatchling body length of wild type (n = 65 hatchlings), gsnl-1(tm2730) (n = 52 hatchlings), viln-1(ok2413) (n = 43 hatchlings), viln-1(ok2413); gsnl-1(tm2730) (n = 41 hatchlings), pak-1(tm403) (n = 47 hatchlings), gsnl-1(tm2730); pak-1(tm403) (n = 51 hatchlings), viln-1(ok2413); pak-1(tm403) (n = 70 hatchlings), viln-1(ok2413); gsnl-1(tm2730); pak-1(RNAi) (n = 35), spc-1(RNAi) (n = 27 hatchlings) and viln-1(ok2413); gsnl-1(tm2730); spc-1(RNAi) (n = 41 hatchlings). Data represent mean ± s.d. Two side paired _t_-test. *P < 0.05; **P < 0.001; ***P < 0.0001; n.s, not significant.

Source data

Extended Data Fig. 6 Time-dependent length of a Kelvin–Voigt model in different conditions.

a, A generic Kelvin–Voigt system exposed to a constant force _F_epid, and its predicted elongation change for _F_seam = 0.85 and four different values of _α_DV based on the equation \({F}_{epid}={F}_{seam}\;{\alpha }_{DV}\). b, A similar system exposed to two forces, _F_epid and an oscillating force Fmuscles, and predicted elongation change using _F_epid = 0.85 and Fmuscles with an amplitude equal to 1 and the behaviour depicted in the blue-boxed inset. For simplicity, we will refer to the amplitude of Fmuscles as _F_muscles. As the pulsatile force induces both compression and stretching (see Fig. 1c), its net input on elongation is transient and the system oscillates around the maximal value reached without Fmuscles. In all other panels (except in a), Fmuscles was set as a periodic function with positive and negative steps of duration 6 s modulated by a cosine function, alternating with periods of null value of duration 15 s (b, inset). c, A Kelvin–Voigt system with mechanical plasticity introduced according to equations (1), (4), (6) and (7) in the Supplementary Information, and predicted elongation change using _F_epid = 0.85, _F_c = 0, _F_muscles = 3 and four distinct values of the plasticity factor β, or using _F_epid = 0.85, _F_c = 0, β = 0.10 and four distinct values of _F_muscles. d, A Kelvin–Voigt system in which the plasticity is defective (β = 0), and in which there is actin tearing according to equation (7) in the Supplementary Information, inducing a progressive reduction of _F_epid, and predicted elongation change with an initial value of _F_epid = 0.85, the tearing factor γ = 0.15 and _F_muscles = 3; the inset outlined in blue shows the behaviour of _α_DV(t) over time. In a–d, the elastic constant of the spring is k = 1, the initial resting length has the value λ(t = 0) = 1, and the viscosity is η = 10. e, Result of the fit for the following genotypes: WT, unc-112(−) alone spc-1(−) alone, spc-1(−) pak-1(−) double, unc-112(−); spc-1(−) pak(−) according to equations (1), (4), (9) to (11) in the Supplementary Information. The values of the parameters are specified in paragraphs 1.5 and 1.6 in the Supplementary Information. The shallow decrease in length for the curve of unc-112(−); spc-1(−) pak-1(−) after 150 min is due to a deformation of the embryos under the effect of unc-112 knockdown but not to retraction, which is why the fit has been evaluated on the first 150 min of the curve.

Extended Data Fig. 7 Comparable retraction phenotypes after the combined loss of SPC-1 and PAK-1 or SPC-1 and FHOD-1.

a, Principle of the retraction screen in a spc-1 mutant that identified fhod-1. b, DIC image of spc-1 deficient embryos after feeding on L4440 control (n = 21 hatchlings) or fhod-1(RNAi) (n = 25 hatchlings) bacteria, and quantification of spc-1(ra409) L1 hatchling body length after feeding. Data represent mean ± s.d. Two-sided paired _t_-test. c–j, Elongation curves and (d) corresponding DIC images showing the terminal phenotypes at hatching of wild type (d; n = 12 embryos), fhod-1(tm2363) (e; n = 10 embryos), fhod-1(RNAi) (f; n = 10 embryos), pak-1(tm403) (g; n = 11 embryos), fhod-1(RNAi) pak1(tm403) (h; n = 10 embryos), spc-1(RNAi) pak-1(tm403) (i; n = 8 embryos) and fhod-1(tm2363); spc-1(RNAi) (j; n = 9 embryos). Data in c represent mean ± s.e.m. Scale bar, 25 µm.

Source data

Extended Data Fig. 8 PAK-1 and FHOD-1 form aggregates in spc-1(RNAi) loss of function.

a, PAK-1::GFP localization in wild-type and spc-1(RNAi) embryos. Yellow box, area enlarged below the panel. Note the punctae in SPC-1 deficient embryos. b, FHOD-1 localization in wild-type and spc-1(RNAi) embryos. Note the aggregates (arrowheads). Note also that FHOD-1::GFP displayed a filamentous organization reminiscent of actin filaments. Scale bar, 10 µm.

Extended Data Fig. 9 Actin displacement ratio.

a–d, Spinning-disc microscopy tracking of actin filaments visualized with a Pdpy-7::Lifeact::GFP marker specifically expressed in the epidermis. Individual displacement tracks of wild-type (a), pak-1(tm403) (b), spc-1(RNAi) (c) and spc-1(RNAi) pak-1(tm403) (d) embryos at a stage equivalent to twofold in a wild-type embryo. Scale bar, 10 µm. e, Typical kymographs of the Lifeact::GFP–labelled actin filaments in wild-type and spc-1(RNAi) pak-1(tm403) embryos from which the tracks in a–d were derived. Time interval between two images is 0.41 s. Yellow dots correspond to landmarks for quantitative analysis. f, Quantification of the displacement duration in (N = number of embryos, n = number of contractions): wild type, N = 11, n = 51; pak-1(tm403), N = 11, n = 26; spc-1(RNAi), N = 11, n = 73; spc-1(RNAi) pak-1(tm403), N = 11, n = 89. Data represent median values, 25th and 75th percentiles. The whiskers extend to the most extreme data points not considered outliers. Two-sided paired _t_-test. *P < 0.05; **P < 0.001; ***P < 0.0001; n.s., not significant.

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Supplementary Information

This file contains details of Supplementary Mechanical Modelling and Supplementary References.

Reporting Summary

Supplementary Tables

This file contains Supplementary Tables 1-8.

Supplementary Data 1

Results of statistical tests: _P_-values for all statistical tests (even when not displayed on the figures).

Supplementary Data 2

Number of replicates: Number of analyzed embryos and independent repeats of all experiments.

Video 1

Embryonic elongation and retraction profiles Combined DIC timelapse video. Image acquisition was every 5 minutes in wild-type, pak-1(tm403), spc-1(RNAi), spc-1(RNAi) pak-1(tm403) embryos. Scale Bar, 10 µm.

Video 2

Muscle-dependence of the retraction profile Combined DIC timelapse video of unc-112(RNAi) and unc-112(RNAi); spc-1(RNAi) pak-1(tm403) embryos. Scale Bar,10 µm.

Video 3

Retraction profile of fhod-1; spc-1 defective embryos Combined DIC timelapse videos of spc-1(ra409) and fhod-1(RNAi); spc-1(ra409) embryos. Scale Bar, 10 µm.

Video 4

Epithelial actin displacement in mutants Fluorescence video showing the displacement of actin filaments labelled with Pdpy-7::lifeact::GFP in the epidermis in wild-type, pak-1(tm403), spc-1(RNAi) and spc-1(RNAi) pak-1(tm403) embryos. Time interval, 0.41 s. Scale Bar,10 µm.

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Lardennois, A., Pásti, G., Ferraro, T. et al. An actin-based viscoplastic lock ensures progressive body-axis elongation.Nature 573, 266–270 (2019). https://doi.org/10.1038/s41586-019-1509-4

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