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A self-organized biomechanical network drives shape changes during tissue morphogenesis

Abstract

Tissue morphogenesis is orchestrated by cell shape changes. Forces required to power these changes are generated by non-muscle myosin II (MyoII) motor proteins pulling filamentous actin (F-actin). Actomyosin networks undergo cycles of assembly and disassembly (pulses)1,2 to cause cell deformations alternating with steps of stabilization to result in irreversible shape changes3,4,5,6. Although this ratchet-like behaviour operates in a variety of contexts, the underlying mechanisms remain unclear. Here we investigate the role of MyoII regulation through the conserved Rho1–Rok pathway7 during Drosophila melanogaster germband extension. This morphogenetic process is powered by cell intercalation, which involves the shrinkage of junctions in the dorsal–ventral axis (vertical junctions) followed by junction extension in the anterior–posterior axis8. While polarized flows of medial–apical MyoII pulses deform vertical junctions, MyoII enrichment on these junctions (planar polarity) stabilizes them6. We identify two critical properties of MyoII dynamics that underlie stability and pulsatility: exchange kinetics governed by phosphorylation–dephosphorylation cycles of the MyoII regulatory light chain; and advection due to contraction of the motors on F-actin networks. Spatial control over MyoII exchange kinetics establishes two stable regimes of high and low dissociation rates, resulting in MyoII planar polarity. Pulsatility emerges at intermediate dissociation rates, enabling convergent advection of MyoII and its upstream regulators Rho1 GTP, Rok and MyoII phosphatase. Notably, pulsatility is not an outcome of an upstream Rho1 pacemaker. Rather, it is a self-organized system that involves positive and negative biomechanical feedback between MyoII advection and dissociation rates.

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Figure 1: Spatial control over RLC phospho-cycles is required for MyoII polarity.
Figure 2: MyoII pulses with its regulators Rok, MBS and Rho1 GTP.
Figure 3: Pulsatility of Rho1 GTP, Rok and MBS requires MyoII activity.
Figure 4: Pulsatility is an emergent behaviour of a biomechanical network.

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Acknowledgements

We thank M. Rao for useful discussions, and the Lecuit and Lenne groups and the Labex INFORM (ANR-11-LABX-0054) for support and discussions. We are grateful to M. Krahn, A. Martin, V. Mirouse, J. Treisman and J. Zallen for the gift of flies. This work was supported by the ANR Archiplast and the ERC (Biomecamorph #323027) and HFSP (T.L. and E.M.). A.M. was supported by Ministère de l'Éducation nationale and the Association pour la Recherche contre le Cancer (ARC). We acknowledge the France-BioImaging/PICsL infrastructure (ANR-10-INSB-04-01).

Author information

Authors and Affiliations

Authors

Contributions

This project originated from discussions between T.L. and E.M., based on a computational model developed by E.M. A.M. and T.L. planned the project, discussed the data with input from E.M., and wrote the paper. A.M. did all the experiments and analysis. J.-M.P. made the fly constructs and helped with molecular biology.

Corresponding author

Correspondence to Thomas Lecuit.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 RNAi specifically downregulates MyoII phosphatase resulting in cellular and tissue-level defects.

a, Gene expression data of Mypt-75d measured from quantitative PCR in water- and dsRNA-injected embryos using two different Taqman probes. P values calculated with unpaired t-test. b, Distribution of percentage of GBE in water and Mypt dsRNA-injected embryos. c, Box plot of time taken for junction shrinkage in water and Mypt dsRNA-injected embryos. d, MyoII distribution in control (left) and MBS shRNA-expressing embryo (right). e, Average planar polarity of MyoII in these conditions. P values calculated with Mann–Whitney U-test. Scale bar, 5 μm.

Extended Data Figure 2 RLC phospho-cycling determines quantitative levels of MyoII at cell junctions.

a, Distribution of MyoII in embryos co-expressing wild-type (WT) RLC–GFP with wild-type RLC–mCherry (top) and phosphomimetic RLC–GFP with wild-type RLC–mCherry (bottom) injected with water (left) or Y27632 (right). b, Mean intensity of MyoII in mCherry (top) and GFP (bottom) channels in these conditions. P values calculated with Student’s t-test. c, Distribution of MyoII in control embryos or in embryos expressing constitutively active MBS (CA-MBS). d, Average junctional intensity of MyoII in these conditions. P values calculated with Student’s t-test. e, Average intensities of MyoII on transverse junctions in various conditions. P values calculated with Mann–Whitney U-test. Scale bar, 5 μm.

Extended Data Figure 3 Proportions and cycling of phosphorylated RLC regulates MyoII turnover and mechanical tension.

a, b, Mean fluorescence recovery time traces (normalized as described in methods) of RLC–GFP from vertical (red) and transverse junctions (blue) in wild-type (a) and Mypt-75d RNAi (b) embryos. The dotted lines are fits derived from the equation described in the methods. c, Table shows the goodness of fit (R2) and the respective mobile fraction from fitted curves in a and b. d, Plot shows the mobile fraction of RLC in different conditions. Values from each junction (open circles) and the average from all junctions (open rectangle) are shown. e, Cartoon showing scission of junctions and their elastic recoil in response f, Vertical and transverse junctions before (0 s) and after (5 s) laser ablation in different conditions. g, Recoil velocities of vertical and transverse junctions in different conditions. Values from each junction (open circles) and the average from all junctions (open rectangle) are shown. P values calculated with Mann–Whitney U-test. Scale bar, 1 μm.

Extended Data Figure 4 Wild-type Rok is planar polarized and pulsatile during GBE.

a, Average planar polarity of MBS and MyoII. b, Distribution of wild-type Rok (RokWT)–GFP without (left) and with (right) RLC–mCherry (top). Scale bar, 5 μm. c, Average enrichment of RokWT–GFP relative to MyoII enrichment at junctions categorized by their angles (bottom). d, Time snapshots of RokWT–GFP embryos injected with water (top) or 10 mM Y27632 (bottom). Scale bar, 1 μm.

Extended Data Figure 5 Rho1 GTP is required for MyoII activation.

a, Representative images of AniRBD–GFP (left) and RLC–mCherry (right) in water (top) and C3 exoenzyme (bottom) injected embryos. Scale bar, 5 μm. b, AniRBD–GFP and RLC–mCherry mean junctional intensities in water and C3 exoenzyme injected embryos. P values calculated by Student's t-test. c, Mean medial intensity time trace of AniRBD–GFP (left) and RLC–mCherry (right) in C3 exoenzyme injected embryos compared to a representative water injected embryo. Error bars represent s.d. d, Time snapshots showing medial pool of RLC–GFP in control and embryo expressing constitutively active Rho (RhoV14). Scale bar, 1 μm. e, Mean medial intensity time traces of RLC–GFP in different conditions and stages (Error bars: Standard deviation) f, Average pulse amplitudes of RLC–mCherry and kinase-dead Rok (RokKD)–GFP in control and RhoV14-expressing embryos g, h, Average duration and frequency of MyoII pulses in control and RhoV14-expressing embryos. P values calculated by Mann–Whitney U test.

Extended Data Figure 6 Non-linear MyoII accumulation from stochastic Rho1 activation.

a, b, Mean medial intensity time traces of RokKD–GFP (a) and AniRBD–GFP (b) in various conditions. Error bars indicated s.d. c, Average rate of change (first derivative) of AniRBD–GFP, RokKD–GFP and RLC–GFP medial intensities during pulse assembly. d, Temporal cross-correlation analysis of medial intensity time traces of RLC–mCherry with AniRBD–GFP during early GBE (stage 5a) and GBE (stage 7). e, Distribution of centres of masses of MyoII medial intensity at the time of pulse coalescence.

Extended Data Figure 7 MyoII regulatory pathway components are advected.

a, Time snapshot from medial pool of embryos expressing either RokKD–Venus (top) or RokKDΔRBD–Venus (bottom). b, Mean pulse amplitudes of RokKD–Venus and RokKDΔRBD–Venus. c, Pairwise correlation of RokKD– GFP, RokKDΔRBD–Venus and AniRBD–GFP pulse amplitudes with the respective RLC–mCherry pulse amplitudes d, Time snapshot from medial pool of embryos expressing either cytosolic GFP (top) or α-catenin–YFP (bottom). e, Mean medial intensity time traces of RLC–mCherry, cytosolic GFP and α-catenin–YFP during GBE. Scale bar, 1 μm.

Extended Data Figure 8 RLC dephosphorylation is required for pulse disassembly.

ac, Mean MyoII pulse duration (a), amplitude (b) and frequency (c) in various conditions. P values calculated with Mann–Whitney U-test. d, Average advection velocity of medial MyoII and RokKD in wild-type and mypt dsRNA-injected embryos. P values calculated with Mann–Whitney U-test. e, Average divergence values from many cells of embryos expressing wild-type RLC–GFP (left), phosphomimetic (EE) RLC–GFP (middle) and phosphomimetic RLC–GFP injected with Y27632 (40 mM) (right). f, Plot showing average divergence values as a function of cell radius in these conditions. P values calculated with one-sample t-test.

Extended Data Figure 9 Non-constant turnover and advection results in MyoII depletion during pulse disassembly.

a, Behaviour of MyoII (green) for different ratios of recruitment + advection/dissociation (black) (dashed line, ratio >1; dotted line, ratio = 1; dot–dash line, ratio <1; solid line: non-constant ratio starting from >1 and crossing over to <1). b, e, Medial intensity time traces of embryo co-expressing RLC–mCherry and RLC–GFP. The star shows the time of photo-bleach either at the onset of pulse assembly (b) or pulse disassembly (e). c, f, Time traces show the ratio of intensities from the red and the green channels (black). d, g, Fluorescence recovery curves obtained from the red/green ratio. The curves are normalized as described in the methods. h, Average of fluorescence recovery curves from many different experiments. Error bars, s.e.m. where n = number of pulses. The dotted curve corresponds to the fit derived from the equation described in the methods. Table shows the goodness of fit (R2) and the respective mobile fraction during pulse assembly and disassembly. i, Average divergence values of MyoII during pulse assembly (left) and disassembly (right). j, Plot shows average divergence values as a function of cell radius during pulse assembly and disassembly. P values calculated with one-sample t-test.

Extended Data Figure 10 A qualitative phase diagram of MyoII dynamics.

The phase diagram summarizes MyoII behaviour (stable or oscillatory) in the parameter space of MyoII dissociation and MyoII activity in different experimental conditions.

Supplementary information

Actomyosin pulse assembly and disassembly

Time lapse images show apical surface sum-z projection of embryos co-expressing UtrABD::GFP (green) and RLC::mCherry (magenta). Scale bar is 5 microns. (MOV 1571 kb)

Rok and MyoII phosphatase are pulsatile

Time lapse images show apical surface sum z-projection of embryos co-expressing RokKD::GFP (green) and RLC::Cherry (magenta) (top) and MBS::GFP (green) and RLC::Cherry (magenta) (bottom). Scale bar is 5 microns. (MOV 3965 kb)

Rho1GTP is required for MyoII activation

Time lapse images show apical surface sum z-projection of embryos expressing RLC::GFP in wild type embryos (Early GBE/Stage5a) and in embryos expressing Rho-CA(Early GBE/ Stage5a and GBE/ Stage7). Scale bar is 5 microns (MOV 5215 kb)

Rho1GTP is pulsatile

Time lapse images show apical surface sum z-projection of embryos co-expressing AniRBD::GFP (MOV 2321 kb)

MyoII activity is required for pulsatility of upstream regulators

Time lapse images show apical surface sum z-projection of embryos expressing UtrABD::GFP, AniRBD::GFP, Rok::GFP and MBS::GFP and injected with H1152/Y27632 10mM. Scale bar is 5 microns. (MOV 3286 kb)

PIV of medial MyoII

Time lapse images show subcellular region with superimposed vector fields obtained from PIV on apical medial particles of RLCWT::GFP. (MOV 925 kb)

Convergent advection is required for pulse assembly

Time lapse images show apical surface sum-z projection of embryos co-expressing UtrABD::GFP and RLC::mCherry and injected with either DMSO (top) or CytochalasinD 1mg/ml (bottom). Scale bar is 5 microns. (MOV 8278 kb)

RLC phospho-cycling is required for advection and pulse disassembly

Time lapse images show sum z-projection of WT-RLC::GFP embryos injected with water (left) or mypt dsRNA (middle) and EE-RLC::GFP expressing embryos injected with 40mM Y27632. Scale bar is 5 microns. (MOV 3196 kb)

Concentration of actomyosin networks slows down advection

Time lapse images show apical surface sum-z projection of embryos co-expressing UtrABD::GFP and RLC::mCherry in the background of Formin-CA over-expression. Scale bar is 5 microns. (MOV 1906 kb)

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Munjal, A., Philippe, JM., Munro, E. et al. A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524, 351–355 (2015). https://doi.org/10.1038/nature14603

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